The Polyandrous Queen Honey Bee: Biology and Apiculture 9815079131, 9789815079135

Citation preview

The Polyandrous Queen Honey Bee: Biology and Apiculture Authored by Lovleen Marwaha Department of Zoology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India

The Polyandrous Queen Honey Bee: Biology and Apiculture Author: Lovleen Marwaha ISBN (Online): 978-981-5079-12-8 ISBN (Print): 978-981-5079-13-5 ISBN (Paperback): 978-981-5079-14-2 © 2022, Bentham Books imprint. Published by Bentham Science Publishers Pte. Ltd. Singapore. All Rights Reserved. First published in 2022.

BSP-EB-PRO-9789815079128-TP-299-TC-14-PD-20230116

BENTHAM SCIENCE PUBLISHERS LTD.

End User License Agreement (for non-institutional, personal use) This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the ebook/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work. Bentham Science Publishers agrees to grant you a non-exclusive, non-transferable limited license to use the Work subject to and in accordance with the following terms and conditions. This License Agreement is for non-library, personal use only. For a library / institutional / multi user license in respect of the Work, please contact: [email protected].

Usage Rules: 1. All rights reserved: The Work is the subject of copyright and Bentham Science Publishers either owns the Work (and the copyright in it) or is licensed to distribute the Work. You shall not copy, reproduce, modify, remove, delete, augment, add to, publish, transmit, sell, resell, create derivative works from, or in any way exploit the Work or make the Work available for others to do any of the same, in any form or by any means, in whole or in part, in each case without the prior written permission of Bentham Science Publishers, unless stated otherwise in this License Agreement. 2. You may download a copy of the Work on one occasion to one personal computer (including tablet, laptop, desktop, or other such devices). You may make one back-up copy of the Work to avoid losing it. 3. The unauthorised use or distribution of copyrighted or other proprietary content is illegal and could subject you to liability for substantial money damages. You will be liable for any damage resulting from your misuse of the Work or any violation of this License Agreement, including any infringement by you of copyrights or proprietary rights.

Disclaimer: Bentham Science Publishers does not guarantee that the information in the Work is error-free, or warrant that it will meet your requirements or that access to the Work will be uninterrupted or error-free. The Work is provided "as is" without warranty of any kind, either express or implied or statutory, including, without limitation, implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the results and performance of the Work is assumed by you. No responsibility is assumed by Bentham Science Publishers, its staff, editors and/or authors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products instruction, advertisements or ideas contained in the Work.

Limitation of Liability: In no event will Bentham Science Publishers, its staff, editors and/or authors, be liable for any damages, including, without limitation, special, incidental and/or consequential damages and/or damages for lost data and/or profits arising out of (whether directly or indirectly) the use or inability to use the Work. The entire liability of Bentham Science Publishers shall be limited to the amount actually paid by you for the Work.

General: 1. Any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims) will be governed by and construed in accordance with the laws of Singapore. Each party agrees that the courts of the state of Singapore shall have exclusive jurisdiction to settle any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims). 2. Your rights under this License Agreement will automatically terminate without notice and without the

need for a court order if at any point you breach any terms of this License Agreement. In no event will any delay or failure by Bentham Science Publishers in enforcing your compliance with this License Agreement constitute a waiver of any of its rights. 3. You acknowledge that you have read this License Agreement, and agree to be bound by its terms and conditions. To the extent that any other terms and conditions presented on any website of Bentham Science Publishers conflict with, or are inconsistent with, the terms and conditions set out in this License Agreement, you acknowledge that the terms and conditions set out in this License Agreement shall prevail. Bentham Science Publishers Pte. Ltd. 80 Robinson Road #02-00 Singapore 068898 Singapore Email: [email protected]

BSP-EB-PRO-9789815079128-TP-299-TC-14-PD-20230116

CONTENTS PREFACE ................................................................................................................................................ i CHAPTER 1 THE QUEEN HONEY BEE: INTRODUCTION, DEVELOPMENT, PHEROMONES, MATING, AND ROLE IN THE COLONY ........................................................... 1.1. INTRODUCTION ................................................................................................................... 1.1.1. General Information about Colonial Organizations ...................................................... 1.1.2. Different Castes and Respective Duties ........................................................................ 1.1.3. General Differences in the Three Castes ...................................................................... 1.1.4. Ovarian State of Active Queen and Sterile Workers .................................................... 1.1.5. Queen Quality Influencing Factors ............................................................................... 1.1.6. Queen Failure ................................................................................................................ 1.2. DEVELOPMENT OF QUEEN .............................................................................................. 1.2.1. Queen Development: General Information ................................................................... 1.2.2. Development Post-Queen Egg Hatching ...................................................................... 1.2.3. Queen Larval Development .......................................................................................... 1.2.4. Role of Royal Jelly ........................................................................................................ 1.2.5. Plasticity of the Queen Development Phase ................................................................. 1.3. QUEEN PHEROMONES ....................................................................................................... 1.3.1: Queen Pheromones Composition .................................................................................. 1.3.2. Queen Mating Status Correlation with QMPs .............................................................. 1.3.3. Functions of Pheromones .............................................................................................. 1.4. MATING .................................................................................................................................. 1.4.1. Nuptial Flight ................................................................................................................ 1.4.2. DCA .............................................................................................................................. 1.4.3. Process of Mating and Post Mating .............................................................................. 1.5. ROLE OF QUEEN HONEY BEE .......................................................................................... REFERENCES ...............................................................................................................................

1 2 2 3 10 11 12 13 14 14 15 15 17 19 20 20 21 22 23 23 24 24 25 26

CHAPTER 2 THE QUEEN HONEY BEE DUTIES IN THE COMPOSITE COLONIES ........... 2.1. REPRODUCIBILITY OF QUEEN HONEY BEE ............................................................... 2.1.1. General Information on Queen Reproduction ............................................................... 2.1.2. Factors Influencing Queen Reproductive Characteristics ............................................. 2.1.3. Queen Making Decision in the Colony ......................................................................... 2.1.4. The Queen Elimination Procedure ................................................................................ 2.2. QUEEN PHEROMONES ....................................................................................................... 2.2.1. General Information about Queen Pheromones ............................................................ 2.2.2. Queen Mandibular Gland Pheromones ......................................................................... 2.2.3. Dufour’s Gland ............................................................................................................. 2.3. CONCLUSION ........................................................................................................................ REFERENCES ...............................................................................................................................

35 35 35 39 41 44 45 45 46 47 49 49

CHAPTER 3 THE QUEEN HONEY BEE MORPHOLOGY, DEVELOPMENT, AND REPRODUCTIVE SYSTEM ................................................................................................................. 3.1. INTRODUCTION ................................................................................................................... 3.2. MORPHOLOGY OF THE QUEEN ...................................................................................... 3.3. DEVELOPMENT OF THE QUEEN HONEY BEE ............................................................ 3.4. OVARIAN STRUCTURAL INTEGRITY (3A-B) ............................................................... REFERENCES ...............................................................................................................................

57 57 58 59 60 62

CHAPTER 4 ROYAL JELLY AS LARVAL FOOD FOR HONEY BEES .................................... 67 4.1. INTRODUCTION ................................................................................................................... 67

4.1.1. Information That Is General Regarding Royal Jelly ..................................................... 4.2. COMPOSITION ...................................................................................................................... 4.2.1. Sugar ............................................................................................................................. 4.2.2. Lipid .............................................................................................................................. 4.2.3. Protein ........................................................................................................................... 4.2.4. Phenols, Flavonoids And Free Amino Acids ................................................................ 4.2.5. Vitamins, Minerals And Other Bioactive Sustances ..................................................... 4.2.6. The Importance of Royal Jelly to the Developmental Process ..................................... 4.3. SECRETION OF ROYAL JELLY ........................................................................................ 4.4. DIET OF OTHER CASTES ................................................................................................... CONCLUSION .............................................................................................................................. REFERENCES ...............................................................................................................................

67 68 69 70 71 72 72 73 74 75 76 77

CHAPTER 5 QUALITY INFLUENCING FACTORS AND DISEASE RESISTANCE IN QUEEN OF APIS MELLIFERA (HYMENOPTERA: APIDAE) ....................................................... 5.1. INTRODUCTION ................................................................................................................... 5.2. STRONG QUEEN AND COLONY ....................................................................................... 5.3. QUEEN QUALITY AND ASSOCIATED FACTORS ......................................................... 5.3.1. Larval Selection Influence ............................................................................................ 5.3.2. Other Factors ................................................................................................................. 5.4. OVARIAN WEIGHT AND FUNCTIONALITY AS QUEEN QUALITY INDEXES ...... 5.5. QUALITY OF QUEEN ON THE BASICS OF SPERMATHECAE .................................. 5.6. DISEASES AND REPRODUCTIVITY OF QUEEN HONEY BEE .................................. CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

83 83 87 89 89 93 93 94 95 97 97

CHAPTER 6 DIFFERENTIAL PHEROMONE SECRETION BY FEMALE CASTES IN APIS MELLIFERA (HYMENOPTERA: APIDAE) ....................................................................................... 6.1. INTRODUCTION ................................................................................................................... 6.2. QUEEN PHEROMONES ....................................................................................................... 6.2.1. Queen Mandibular Gland ............................................................................................. 6.2.1.1. Composition of QMPs and Variation in Accordance of Mating Status .......... 6.2.1.2. Secretion of QMP ............................................................................................ 6.2.1.3. Colonial Regulation Imposed by QMP ........................................................... 6.2.1.4. On Retinue ....................................................................................................... 6.2.1.5. On Wax Secretion ............................................................................................ 6.2.1.6. On Ovarian Development ............................................................................... 6.2.2. Dufour's Gland ............................................................................................................. 6.2.3. Koschevnikov Gland .................................................................................................... 6.2.4. Tarsal Gland ................................................................................................................. 6.3. WORKERS' PHEROMONES ............................................................................................... Alarm pheromone ................................................................................................................... 6.3.1. Koschevnikov Gland .................................................................................................... 6.3.2. Mandibular Gland ........................................................................................................ 6.3.3. Brood Recognition Pheromones .................................................................................. 6.3.4. Nasonov Gland ............................................................................................................. 6.3.5. Dufour's Gland Pheromone .......................................................................................... 6.3.6. Footprint Pheromones .................................................................................................. 6.3.7. Forager Pheromone ...................................................................................................... 6.3.8. Other Pheromones ........................................................................................................ CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

111 111 121 121 122 124 125 126 128 128 128 129 129 130 130 130 131 131 131 132 132 132 133 133 133

CHAPTER 7 MANDIBULAR PHEROMONE TYPES, FUNCTIONS, SYNTHESIS, AND ASSOCIATED GENETIC ELEMENTS IN THE QUEEN HONEY BEE, APIS MELLIFERA .... 7.1. INTRODUCTION ................................................................................................................... 7.2. COMPOSITION OF QMP ..................................................................................................... 7.3. DIFFERENTIAL MANDIBULAR PHEROMONAL COMPOSITION IN QUEEN AND WORKER HONEY BEE ............................................................................................................... 7.4. SYNTHESIS OF QMPS .......................................................................................................... 7.5. EFFECT OF QMP ................................................................................................................... 7.5.1. On Mating and Swarm .................................................................................................. 7.5.2. On Retinue .................................................................................................................... 7.6. WORKERS' MANDIBULAR GLAND PHEROMONES ................................................... 7.7. GENE ASSOCIATED WITH MANDIBULAR GLAND .................................................... CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

138 138 141 145 145 148 148 150 154 156 157 157

CHAPTER 8 RETINUE BEHAVIOUR OF WORKER HONEY BEES ......................................... 8.1. INTRODUCTION ................................................................................................................... 8.2. PHEROMONES FOR ATTRACTION OF NEST MATES ................................................ 8.2.1. Factors For Inducing Variation in Retinue Behaviour .................................................. 8.2.2. Various Components of QMP and Retinue in Workers ................................................ 8.2.3. QMP Influence on Drone and Swarming ...................................................................... 8.2.4. Colonial Transfer of Queen Pheromones ...................................................................... CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

163 163 165 166 166 167 168 169 169

CHAPTER 9 INFLUENCE OF QUEEN PHEROMONES ON WORKER OVARIAN PCD IN APIS MELLIFERA (HYMENOPTERA: APIDAE) ............................................................................ 9.1. INTRODUCTION ................................................................................................................... 9.2. QUEEN PHEROMONES RELATED TO PCD IN WORKERS' OVARIES ................... 9.2.1. General Information ...................................................................................................... 9.2.1.1. General Ovarian Structure ............................................................................. 9.2.1.2. Larval Gonadial Development ........................................................................ 9.2.2. Main Pheromones Which Influence PCD ..................................................................... 9.2.3. Differential Pheromonal Synthesis in Queen and Workers .......................................... 9.3. QUEEN'S INFLUENCE ON PROGRAMMED CELL DEATH ........................................ 9.3.1. General Information ...................................................................................................... 9.3.2. Mechanism of PCD ....................................................................................................... 9.3.3. Detection of PCD in Workers' Ovaries ......................................................................... CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

175 175 176 176 179 180 180 182 184 184 185 187 187 188

CHAPTER 10 GENETIC INFLUENCE ON OVARIAN DEVELOPMENT PLASTICITY IN APIS MELLIFERA (HYMENOPTERA: APIDAE) ............................................................................ 10.1. INTRODUCTION ................................................................................................................. 10.2. DIFFERENTIAL GENE EXPRESSION IN QUEEN AND WORKERS ........................ 10.3. BRIEF DESCRIPTION OF VARIOUS GENETIC REGULATORS OF PCD .............. 10.3.1. Anarchy Gene ............................................................................................................. 10.3.2. Ark Gene ..................................................................................................................... 10.3.3. Miscellaneous Description of Various Genes Involved in Development ................... 10.3.4. Hormonal Effect on Gene Expression ........................................................................ CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

197 197 198 199 199 200 200 204 205 205

CHAPTER 11 DRONE DEVELOPMENT, BIOLOGY, AND AND INTERACTION WITH THE QUEEN IN APIS MELLIFERA ............................................................................................................. 11.1. INTRODUCTION ................................................................................................................. 11.2. GENERAL DEVELOPMENT ............................................................................................. 11.3. DIPLOID DRONES ............................................................................................................... 11.4. LIFE SPAN ............................................................................................................................. 11.5. DRONE NUMBER ................................................................................................................ 11.6. FLIGHT ACTIVITY ............................................................................................................. 11.7. DRIFT AND ORIENTATION OF DRONES' FLIGHT TO THE HIVE ........................ 11.8. ATTRACTION OF DRONES TO VIRGIN QUEEN ........................................................ 11.9. DRONE DEVELOPMENT ................................................................................................... 11.10. REPRODUCTIVE SYSTEM OF DRONES ..................................................................... 11.11:MATING ............................................................................................................................... 11.12. INFLUENTIAL FACTORS FOR DRONE QUALITY ................................................... 11.13. DRONE AGING .................................................................................................................. CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

211 211 211 212 214 215 217 218 219 220 221 223 224 224 225 225

CHAPTER 12 MATING AND REPRODUCTION IN QUEEN HONEY BEE .............................. 12.1. INTRODUCTION ................................................................................................................. 12.2. MATING AND FREQUENCY OF OCCURRENCE ........................................................ 12.3. FACTORS AFFECTING MATING .................................................................................... 12.4. PHEROMONES .................................................................................................................... 12.5. DRONES CONGREGATION AREAS ............................................................................... 12.6. DRONE ATTRACTION ....................................................................................................... CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

232 232 238 239 240 241 242 242 242

CHAPTER 13 SWARMING AND QUEEN HONEY BEE ............................................................... 13.1. INTRODUCTION ................................................................................................................. 13.2. FACTORS AFFECTING SWARMING ............................................................................. 13.2.1. Queen Age As Promoting Factor for Swarming ......................................................... 13.2.2. Reduced Queen Pheromones ...................................................................................... 13.2.3. Infection as Cause of Swarming ................................................................................. 13.3. ROLE OF QUEEN MANDIBULAR PHEROMONES IN SWARMING ........................ 13.4. OTHER QUEEN GLANDS PHEROMONES IN SWARMING ...................................... 13.5. MAJOR EVENTS IN SWARMING .................................................................................... 13.5.1. Pre-Swarming Phase ................................................................................................... 13.5.2. Exodus of Swarm ....................................................................................................... 13.5.3.Swarming ..................................................................................................................... 13.5.4. Migration During Swarming ....................................................................................... 13.6.TYPES OF SWARMING WORKERS ................................................................................. 13.7.AGE AND PHYSIOLOGY OF SWARMING WORKERS ............................................... 13.8. NATAL COLONY AFTER SWARMING ......................................................................... CONCLUSION ............................................................................................................................... REFERENCES ...............................................................................................................................

247 247 248 249 250 251 251 253 254 254 257 258 260 262 262 263 263 263

CHAPTER 14 REQUEEN PROCESS AND IMPORTANCE .......................................................... 14.1. INTRODUCTION ................................................................................................................. 14.2. QUEEN'S ROLE AND GENERAL DEVELOPMENT ..................................................... 14.3. METHOD OF QUEEN PRODUCTION ............................................................................. CONCLUSION ...............................................................................................................................

274 274 276 279 282

REFERENCES ............................................................................................................................... 283 SUBJECT INDEX .................................................................................................................................... 2

i

PREFACE Dear Readers, The book Polyandrous Queen Honey Bee is attributed to the predominant caste of honey bee colony. This specific book provides information about the development of a queen honey bee, associated genetic elements, her pheromone profile, life span, immunity, mating, reproduction, artificial method of bee rearing, swarming and the role of a queen in the colony. While working on apiculture, I was encouraged to share my experience and observations on bee farming. Further, the desire to compile the scattered information on a specific topic furthered the book writing process. Additionally, while visiting various apiaries and observing honey bee cultural practices including traditional and scientific practices, both served as great factors for compiling information in the form of a book. I am further planning to publish two more books dedicated to worker honey bees and drones. I would like to thank my parents, sister, friends, and my students, who have shown the required support and encouragement for the completion of this work. I personally thank the management of Lovely Professional University, Punjab, India for providing the required facilities for Apiculture. Regards,

CONSENT FOR PUBLICATION Not applicable.

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS Declared none.

Lovleen Marwaha Associate Professor Department of Zoology School of Bioengineering and Biosciences Lovely Professional University Punjab, India

Polyandrous Queen Honey Bee, 2022, 1-34

1

CHAPTER 1

The Queen Honey Bee: Introduction, Development, Pheromones, Mating, and Role in the Colony Abstract: Apis mellifera (2n=32), commonly known as the European honey bee or the Western honey bee, is a eusocial insect. Each honey bee colony is a composite unit of thousands of bees, with three different castes: a polyandrous reproductively active queen; thousands of workers; and a few hundred drones. The queen and the workers represent the female caste that develops from fertilized eggs, whereas the drones are male bees formed from unfertilized or fertilized eggs. In the case of the female honey bees, the phenomenon of polyphenism can be easily highlighted, which is the developmental plasticity of the same genomic contents to express differently as per environmental cues. During the queen larval developmental phase, the exclusive diet is royal jelly, which induces hyper-secretion of juvenile and ecdysone hormones that ultimately cause sequential activation of certain genetic elements, specifically after 3rd instar onward. For the worker honey bee larvae, initially, the diet includes royal jelly exclusively, followed by honey, pollen grains, and worker jelly, which collectively direct development toward the worker caste. Furthermore, for harmonious social interaction, the queen secretes certain volatile chemical bouquets including 9ODA(2E)-9-oxodecenoic acid), 9-HDA (9-hydroxy-(E)-2-decenoic acid), 10-HDA (10-hrdroxy-2-decenoic acid), HVA (4-hydroxy-3-methoxyphenylethanol), HOB (Methyl-p-hydroxybenzoate), 10-HDAA (10-hydroxydecanoic acid), OLA (oligolactide), methyl oleate, decyl decanoate, linolenic acid, coniferyl alcohol, cetyl alcohol, etc. The concerned pheromones facilitate the regulation of workers' behavior; workers' ovarian suppression; retinue control; overall worker’s development modulation; colonial product production; swarming tendency; pseudo-queen formation suppression; mating, etc. The queen honey bee is polyandrous, as she mates with many drones during the nuptial flight in 'Drone Congregation Areas (DCA)’, within about 2 weeks of her post-emergence. This chapter provides a comprehensive review of the polyandrous queen honey bee; her synchronous developmental phases; her pheromone dominance; her regulation and coordination of colonies; her mating preference and habits; and her role in a composite hive. Subsequent chapters provide an elaborative view of different aspects of the queen honey bees' life cycle.

Keywords: Developmental Plasticity, Polyphenism and DCA, Queen Honey Bee.

Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

2 Polyandrous Queen Honey Bee

Lovleen Marwaha

1.1. INTRODUCTION 1.1.1. General Information about Colonial Organizations The honey bee is a colonial insect, with overlapping adult progenies, a proper division of labor, and social interaction. The specific insect constructs the wax hive, with hexagonal cells for brood, honey, and pollen grain storage. In a honey bee colony, about 20,000–50,000 bees live harmoniously. Usually, three different morphological types of bees can be easily identified in a hive (Fig. 1a - e). Furthermore, these bee castes are anatomically, physiologically, reproductively, and functionally different. The honey bees further differentiate into two classes: the reproducible caste and the non-reproducible caste. The queen and drones are the only obligatorily reproducible bees, while workers are non-reproducible or facultatively reproducible, depending upon queen-less or queen-righted conditions. In other words, a honey bee colony is composed of a single fertile dominant queen, thousands of sterile workers, and a few hundred fertile drones.

Fig. (1a). Depicts the influence of the different genomic contents and the larval diets on the phenotype of the honey bee castes. The honey bee queen can lay unfertilized and fertilized eggs, which can develop into male and female castes within the honey bee colony.

The Queen honey bee lays two types of eggs; fertilized (2n = 32) and unfertilized (n = 16). The female castes develop from fertilized eggs, whereas the male caste can develop from unfertilized or fertilized eggs (Fig. 1a). The honey bee develops by a haplodiploid sex determination mechanism. In honey bees, the female larva (2n = 32) develops into a worker honey bee if fed on a diet composed of pollen,

Queen Honey Bee

Polyandrous Queen Honey Bee 3

nectar, and brood food, whereas if the female larva (2n = 32) is fed on royal jelly exclusively, then it develops into the queen honey bee. The development of the queen is influenced by the larval diet given to them for the first 72 hours. The royal jelly is a composite mixture of protein-rich material from the mandibular and hypo-pharyngeal glands of the workers. The specific workers' secretion is provided to the queen larva for the initial 6 larval developmental days, whereas in the case of the worker honey bee development, the worker larvae are fed on royal jelly for the first 48 hours, subsequently on worker jelly (for more clarity refer to Fig. (1j)) with a composition different from royal jelly (Wilson, 1976; Winston, 1987; Shi, et al., 2011).

Fig. (1b). A queen honey bee with a group of workers around her. Under normal circumstances, usually a single queen dominates the hive, and due to her pheromone secretion, she remains surrounded by a variable number of workers. The specific picture had been clicked just before oviposition, as the queen was inspecting different wax cells for egg laying, while workers were continuously attending to her for the attachment of laid eggs to the base of the hexagonal wax cells.

1.1.2. Different Castes and Respective Duties The honey bee colony comprises female castes (2n = 32), including monopolized polyandrous queens and facultative fertile worker honey bees, whereas the male

4 Polyandrous Queen Honey Bee

Lovleen Marwaha

caste (n/2n = 32) is represented by reproductively active drones only. The different castes perform different duties, which are • Queen Honey Bee (Fig. 1b - c) • Reproduction; therefore, colony strength modulation. • Pheromone Secretion; hence regulation of social organization of the colony, worker ovarian development suppression, domination in the colony, sex attraction for drones of the other colonies.

Fig. (1c). Diagram specifying the role of the queen within the colony, which includes colony strength modulation, retinue behavior in the workers, developmental regulation of the workers, and overall productivity of the colony.

• Worker Honey Bee (Fig. 1d) • Reproduction; usually occurs in the absence of the queen, thus in a facultative way. • Other functions: cleanliness of the hive, construction of hexagonal wax cells, brood rearing, secretion of royal jelly and worker jelly, swarming, honey processing, pollen processing, propolis preparation, retinue queen, drones' care, ventilation, temperature regulation, carcase disposing, foraging, water collection, and protection of the colony.

Queen Honey Bee

Polyandrous Queen Honey Bee 5

Fig. (1d). Forager worker bees of Apis mellifera, feeding on sugar crystals. While feeding honey bees, it has been reported that workers prefer sucrose solution to sugar crystals. Preference to liquid food is correlated with the natural habits of foragers.

Fig. (1e). Forager worker honey bees carry pollen-filled baskets to the colony. On the meta-thoracic legs of worker honey bees, special pollen baskets are present, which facilitate the quantitative transportation of pollen grains to the hive.

6 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (1f). A picture of a strong colony, reflecting the considerable strength of worker bees. This colony is headed by a strong queen, which can be detected by a good number of capped worker cells. Additionally, worker bees of different age groups can be seen while performing different duties.

Fig. (1g). Click on a queenless colony with poor colony strength and with drone eggs. In specific conditions, the colony was maintained on an artificial diet before rejoining. In the absence of the queen, the probability of pseudo-queen formation increases and they can lay unfertilized eggs or drone eggs.

Queen Honey Bee

Polyandrous Queen Honey Bee 7

Fig. (1h). An image shows the guard honey bees at the entrance of the hive for protection from robbers or other invaders. Usually a group of old worker honey bees remain at the entrance to check the bees entering into the hive. In honey-filled conditions, the colony becomes more prone to be attacked by robbery bees. Guard honey bees discourage such intruders from fulfilling their ill desires.

Fig. (1i). A click specifies a group of worker honey bees doing fanning at the entrance of the hive. The specific behaviour was captured on a day with a temperature range of 35–40 0C, when the hive was open for a short duration. Some of the workers were doing fanning on frames, while others were doing fanning at the entrance of the hive.

8 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (1j). A click from a strong colony with the worker larvae and capped worker pupae. The C-shaped white-coloured pupae of the different instars can be easily detected in the uncapped brood cells.

Fig. (1k). A click of the same hive section from another frame with uncapped brood cells, capped brood cells, drones, and worker honey bees in a section of hive. A few wax cells filled with unripe honey and pollen are left interdistributed among the brood cells. The specific act facilitates the brood rearing process of workers.

Queen Honey Bee

Polyandrous Queen Honey Bee 9

Fig. (1l). Click to depict uncapped and capped honey cells for the unripe and ripe honey storage, respectively. Consequently, the cells filled with pollen grains are also visible. Usually in the upper portion, honey bees store honey in the brood chamber. Further, if a queen excluder is used, then the colony uses frames of super chamber for the honey storage.

• Drone Honey Bee (Fig. 1e) * Mating; occurs during the nuptial flight. * Genetic Differentiation; adding the genetic diversity and quality improvement by contributing paternal genomic content.

Fig. (1m). A Drone Honey Bee of Apis mellifera within the hive. The number of drones is variable, which is dependent upon the availability of food in the hive and food sources outside the colony, temperature, the decision of workers, mating requirements, swarming season, etc.

10 Polyandrous Queen Honey Bee

Lovleen Marwaha

1.1.3. General Differences in the Three Castes Morphological differences between the queen, worker, and drone can be easily visualized even with the unaided human eye (Fig. 1b, d,e). The queen possesses a head and thorax structure similar to that of workers, but with a longer abdomen. Worker honey bees are comparatively smaller than other castes. Workers are reproductively sessile in queen-righted colonies and remain busy in other kinds of activities including wax cell construction, brood rearing, honey processing, water collection, regulating ventilation, wax cell construction, and foraging (Winston 1987; Rangel et al., 2016; 2019). For foraging tasks, the metathoracic legs of worker honey bees bear corbicula, which is structurally designed for quantitative pollen grain transport. Workers possess a barbed stinger with a sting, a characteristic, whereas the queen possesses a straight stinger with a sting multiple times. Furthermore, a worker's barbed stinger is attached to a poison sac, which is present at the end of the abdomen. Drones, the male caste of the honey bee colony, possess a comparatively larger head and thorax than both the female castes. Drone abdomen is thick and blunt, with a bullet-shaped appearance (Fig. 1e). Polyphenism is quite evident in the case of the female honey bee caste, as from the same genomic content, different larval diets induce the formation of two specific types of morphology: anatomy, physiological integrity, and functional characteristics. In the male caste, depending upon the development of unfertilized or fertilized eggs, the formation of haploid or diploid drones occurs, which differ morphologically and anatomically. A diploid drone formation occurs more frequently if the queen mates with drones of her own colony (a detailed description is given in the subsequent chapter). In addition to queens, workers, and drone honey bees, there could be another caste known as intermediate morph (IMs), with characteristics of either queen or worker honey bee, in a honey bee colony. Generally, inter-caste development occurs when queen rearing is initiated on 3–4 day-old worker larvae (Beetsma 1979, Dedej et al. 1998). Moreover, IM formation can be initiated when sugar is added to worker larvae's food (Asencot and Lensky 1976). In IMs, the shape of the head, mandible, stinger, and corbiculae resemble workers, whereas hair distribution in the thoracic and abdominal regions, along with pheromone composition, resembles that of a typical queen honey bee. IMs secrete similar volatile chemicals like those of the queen (Plettner et al. 1993; 1996; 1997; Moritz et al. 2003). Therefore, IMs' presence can provide a false illusion of the existence of the same volatile chemical as that of the queen (Hoffman et al., 2004).

Queen Honey Bee

Polyandrous Queen Honey Bee 11

In honey bee colonies, multiple queen residency is possible, by ablating the mandibles of queens to avoid inter-queen rivalry (Williams, 1987; Ruttner, 1976; Koeniger, et al., 2005a,b; Zheng, et al., 2009). 1.1.4. Ovarian State of Active Queen and Sterile Workers Queen The honey bee dominates the honey bee colony due to her sole right of reproduction in the female castes. For specific processes, she possesses an anatomically and functionally well-developed reproductive system, especially with adaptability for mating and oviposition. In worker bees, under the influence of queen pheromones, genetic suppression, and larval diet, degeneration of the ovarian system occurs by programmed cell death (PCD). In the case of the queen bee, royal jelly influences the secretion of juvenile and ecdysone, which concomitantly activates gene expression, which with aggregately activates the proper development of the reproductive system. In the honey bee queen, ovaries are localized in the abdominal cavity. The honey bee queen possesses meristic polytrophic ovaries made up of hundreds of ovarioles (Snodgrass 1956; Wilson 1976). The individual, the ovariole, is portioned into the terminal filament which accommodates oogonia. The next portion is the germarium, where formation of the follicle takes place, and the last potion is the vitellarium, which localizes the growing follicle comprising oocytes and nurse cell chamber (Cruz-Landim2009). Further, the concerned reproductive organ is less developed in a virgin queen than in a post-mated egg-laying queen. In the virgin queen, ovaries are morphologically different and comparatively smaller than in the mated queen(Shehata et al., 1981; Winston, 1987;Patricio and Cruz, 2002). It has been reported that the egg-laying queen possesses eight times larger ovaries than the virgin queen (Shehata et al., 1981). Over activation of ovarian development occurs after mating and especially during the egg-laying phase of the queen. Actually, in queens, subsequent to mating, there are patterns in gene expression patterns in different organs, including the brain and ovaries, which aggregately influence ovarian development, physiology, and behaviour of the concerned female caste (Richard and Tarpy, 2007; Kocher et al., 2008; Nino et al., 2013). Ovarian weight is influenced by the number and size of ovarioles, which in turn is dependent upon the number as well as developmental stages of eggs in the specific structure. Additionally, the number of eggs is correlated with the length of ovarioles, which is further specified by queen ovarian size and symmetry. All these developmental designs are ultimately influenced by a protein-rich larval diet (Dedej, et al., 1998; Hatch et al., 1999; Tarpy et al., 2000).

12 Polyandrous Queen Honey Bee

Lovleen Marwaha

The queen's ovarian functioning is influenced by several factors like genetic integrity, structural organization, physiological influence of hormones, the influence of maternal pheromones, temperature, rearing conditions, larval diet, and age before entering into the queen's development path. Shehata, et al. (1981) reported that during winter, there is a reduction in the egg-laying activity of adult queens and a further reduction in queen pupal ovarian development. The causative factors could be extreme temperature, reduced food availability, the queen's body physiology, and environmental cues influenced by genetic expression, which ultimately affect the reproduction potential of the queen. 1.1.5. Queen Quality Influencing Factors Queen Quality Influencing Factors: The quality of the queen is assessed on the basis of her fecundity, fertility, and retinue ability through the secretion of pheromones. The queen, with considerable reproductive potential, influences her colony significantly. Nelson and Smirl (1977) and Nelson and Gary (1983) reported that a honey bee colony headed by a stronger queen usually has proportionally significant colonial strength, produces more honey, and does a greater collection of pollen throughout the season than a colony headed by a lower quality queen. Various studies suggest that the queen's reproductive potential can be indicated by her body size, ovarian number, spermatheca diameter, stored sperm count, and sperm viability (Woyke, 1971; Dedej et al., 1998; Hatch et al., 1999; Tarpy et al., 2000; Tarpy and Mayer, 2009; Delaney et al., 2011; Tarpy et al., 2011; Tarpy et al., 2011; Rangel et al., 2016). Furthermore, it has been correlated that the queen body size influences her mating frequency, her reproductive organ size, and her sperm storage capacity (Eckert, 1934; Gilley et al., 2003; Jackson et al., 2011). According to Nelson and Gary (1983), honey production and colony strength are correlated with the queen body weight. The quality of a queen is assessed on the basis of her fecundity, fertility, and retinue ability through the secretion of pheromones. Her queen, with considerable reproductive potential, influences her colony significantly. Nelson and Smirl (1977) and Nelson and Gary, 1983, reported that a honey bee colony headed by a stronger queen usually has proportionally significant colonial strength, produces more honey and does greater collection of pollen throughout the season than a colony headed by a lower quality queen. Various studies suggest that the queen's reproductive potential can be indicated by her body size, ovarian number, spermatheca diameter, stored sperm count, and sperm viability (Woyke, 1971; Dedej et al., 1998; Hatch et al., 1999; Tarpy et al., 2000; Tarpy and Mayer, 2009; Delaney et al., 2011; Tarpy et al., 2011; Rangel et al., 2016). Furthermore, it has been correlated that the queen body size influences her mating frequency, her

Queen Honey Bee

Polyandrous Queen Honey Bee 13

reproductive organ size, and her sperm storage capacity (Eckert, 1934; Gilley et al., 2003; Jackson et al., 2011). According to Nelson and Gary (1983), honey production and colony strength are correlated with queen’s body weight. Queen quality is significantly influenced by larval age when development is diverted toward the queen, a specific caste. Woyke (1971) demonstrated that there is a negative correlation between queen grafting age and the size of spermathecae. Tarpy et al. (2011) and Rangel and Tarpy (2013) reported that queens reared from 0-day old worker larvae possess comparatively higher mating numbers and greater sperm storage capacity in comparison to queens raised from 2-day old worker larvae. 1.1.6. Queen Failure The honey bee is the only obligatorily reproductive active caste in the colony; therefore, she regulates the strength of the colony, agricultural productivity, and pollination services. The queen's life span generally extends from 1–3 years. With an increase in the age of the queen, there is a likelihood of supersure events in colonies (Szabo, 1993), and therefore, the apiarists generally replace the queen annually. For this queen replacement process, either a queen-less colony but with brood is allowed to rear its own queen, or a queen-less colony without brood is requeened artificially by an adult queen. Further, a queen-less colony, in either of the aforementioned conditions, can respond positively to an artificial re-queen if the newly introduced queen possesses great fertility and she secretes colonycompatible pheromones strongly. Sometimes, a queen-less colony exhibits a lower likelihood of artificial re-queening, which eventually results in the queen failing. Baer et al. (2016) concluded that the queen failure subsequent to the introduction of a young mated queen into a honey bee colony occurs due to the depletion of sperm storage in her spermathecae and to unsuccessful fertilization. Usually, a queen from a poor-brood colony possesses less than 3 million sperm in her sperm thecae, which indicates a poorly mated queen (Woyke, 1996). Sperm viability acts as a major criterion which adversely affects colony productivity and strength (Lodesani et al., 2004; Tarpy et al., 2012; Tarpy and Olivarez, 2014). Lower sperm viability can promote the queen to lay down eggs in small brood patches or to lay a drone layer, mainly of unfertilized eggs (Collins, 2000; Collins et al., 2004). Additionally, queen failure can result due to pesticide exposure, improper management practices, chemically coated pollen grain diet or other chemical impregnated plant food items, infection of her with certain parasites and pathogens and/or due to infestation with certain pests (Wallner, 1999; Frazier et al., 2008; Amiri et al., 2017). Pesticide exposure can reduce queen weight, reduce

14 Polyandrous Queen Honey Bee

Lovleen Marwaha

the number of ovarioles and store sperm viability (Haarmann, et al., 2002; Pettis, et al., 2004; Burley, et al., 2008; Chaimanee, et al., 2016; Rangel, et al., 2016). Pesticide exposure by improper management can increase the frequency of superfluence (Sandrock, et al., 2014; Traynor, et al., 2016;Tsvetkov, et al., 2017). Further, queen failure can result due to certain parasites and pathogens, which include deformed wing virus, a causative inducer of ovarian degeneration and Nosema infection, which increases the level of vitellogenin production, along with hyper expression of other immune genes (Alaux, et al., 2011; Gauthier, et al., 2011;Chaimanee, et al., 2014). Queen failing can be detected by poor brood pattern, which refers to empty pupal wax capped cells as an indication of not laying eggs well or improper care by worker honey bees (van Engelsdorp et al., 2013). Additionally, other factors for poor brood patterns include chalk brood disease, sac brood virus, Nosema spp., and pesticide exposure, which can affect brood viability (Brodschneider, et al., 2010; Wu, et al., 2011; van Engelsdorp, et al., 2013). Worker honey bees remove diseased or infected brood, which ultimately results in poor brood pattern formation (Spivak and Reuter, 1998; Habo and Harris, 2009). Transplanted experiments dealing with the shifting of queens from poor brood pattern colonies to good brood pattern colonies demonstrate the positive influence of the colony environment on brood pattern instead of exclusively the queen’s egg laying capacity as a causative factor. Furthermore, it has been reported that pesticide exposure influences the brood pattern formation in colonies. In a good colony environment, a good quality queen lays eggs in a better brood pattern (Lee, et al., 2019). 1.2. DEVELOPMENT OF QUEEN 1.2.1. Queen Development: General Information The honey bee is a holometabolous insect with all developmental phases including eggs, larvae, pupae, and adults. As explained earlier, the female caste, both queen and workers, develop from fertilized eggs, within 16 days and 21 days, respectively, whereas the male caste can develop from unfertilized or fertilized eggs within 24 days. Female larvae are totipotent for the first three days post hatching, or, in other words, female larvae can be developmentally directed toward either the queen type or worker type, within this specific time period. Female larvae (queen or worker) are fed on royal jelly exclusively for the first three larval days. During the first 4–9 days of larval growth, tremendous

Queen Honey Bee

Polyandrous Queen Honey Bee 15

developmental diversion occurs, as if any female larva is fed on royal jelly exclusively, queen development will be accomplished, whereas if any female larvae is fed on worker jelly during this phase, worker development will be accomplished. This developmental diversion highlighted the role of environmental cues (larval diet) in differential development. In this entire book, the primary focus is on the queen honey bee. Development of the queen honey bee begins with oocytes from germ cells in the ovaries. The oocytes subsequently develop into egg cells and nurse cells inside the queen's ovary (Guizeit et al. 1993). With further growth, the egg starts absorbing nutrients from nurse cells. Thereafter, egg-encircling follicle cells secrete the chorion layer over the egg cell, eventually completing the egg formation process (Fleig 1995). 1.2.2. Development Post-Queen Egg Hatching The mature eggs of honey bees are white in colour, with dimensions of 1.3-1.8 mm in length. The Queen honey bee can deposit eggs vertically at the bottom of the bee comb, which are attached by a worker honey bee to a wax cell (Fig. 1b). After the first 14 hours, a cleavage event takes place, resulting in the formation of a blastomere. Cleavage involves rapid mitotic division without cytoplasmic growth during the initial process. After about 10 hours, the blastomere starts dividing, eventually resulting in the creation of space at both ends of the egg. 35 hours afterward, the blastoderm becomes thick in the ventro-anterior regions, specifying the marking of gastrulation completion. After 49 hours, the head region becomes conspicuous. Nevertheless, body segmentation appears (Wisnston, 1987; Milne et al., 1988). Prior to larval hatching, approximately two hours before, there is liquid exudation from the egg surface, followed by slow dissolution of the chorion. 1.2.3. Queen Larval Development After about 72-76 hours of oviposition, C-shaped larvae become hatched in hexagonal wax cells (Collins 2004). During the larval phase, hexagonal wax cells are uncapped and filled with an ample amount of larval food, comprising royal jelly, worker jelly, honey, and pollen, depending upon the specific caste (Fig. 1f - g). In a queen honey bee, egg hatching requires three days, the larval phase requires about six days, and pupation needs seven days. Honey bee queen development comprises a total of five instars, which develop by moulting almost each day. For the queen larva, on the sixth day after hatching, worker honey bees seal the cells with wax capping. The larva pupates within wax cells. A mature

16 Polyandrous Queen Honey Bee

Lovleen Marwaha

larva undergoes ecidysis to be transformed into a pupa with an upright extended body in capped hexagonal wax cells. Subsequently, the pupa undergoes an ecdysis to develop into an adult whose eggs are laid in the wax cap. Pupae are referred to as capped brood, and for completion of the pupal phase, different time periods are required. In different castes, which include queens: 7 days, workers: 12 days, drones: 15 days (Fig. 1g).

Fig. (1n). Photograph depicting a section of a bee comb, with worker bees engaged in different duties specifically for brood rearing. Some workers can be seen while adding larval food for growing worker larvae, whereas some bees are preparing for capping wax cells enclosing late 5th instar worker larvae. The C-shaped white worker larvae enclosed in hexagonal wax cells can be easily identified with the unaided eye.

During pupation, the formation of head, eyes, antennae, mouth parts, thorax, legs, and abdomen takes place. Cuticles darken slowly with time, and the specific character can be used to predict the pupal stage. Eventually, in all honey bee castes, the pupal stage undergoes a final moult to emerge into the imago, which finally chews out cells. Development of different castes requires respectively 1516, 21 and 24 days (Fig. 1g) in the case of queens, workers, and drones (Winston 1987). In a queenright colony, queens are the only fertile caste with active ovaries (Ratnieks 1993), although sometimes a pseudo-queen can appear, but generally eggs laid by such queens are removed by other workers through the detection of coated pheromones on the egg surface.

Queen Honey Bee

Polyandrous Queen Honey Bee 17

Fig. (1o). Elucidation of development periods, assigned duties, and morphological features of different castes of honey bees. Polyphenism phenomenon can be easily captured from the above description as queen and workers develop from the same genomic content but with drastically different morphology, anatomy, physiology, development, reproduction, and life span. Developmental plasticity according to proper division of labour can also be correlated. Additionally, the role of royal jelly, an exclusive complete queen larval food, can also be considered in influencing the life span of specific castes.

1.2.4. Role of Royal Jelly Development of the queen occurs when the larva develops from a fertilized egg and is fed on royal jelly continuously, especially during the first 3–9 days. The specific topic has been considered for long as Stabe (1930) observed that the rate of growth of queens and workers is the same up to 48 hours, thereafter queen larvae become heavier (Tsnasyvourou and Benton, 1982). It has been reported that queen larvae consume, comparatively, 13% more food than worker larvae (Lambremont 1970). Food for worker larvae after the 3rd day is worker jelly. Electrophoretic analysis indicated that queen and worker jelly compositions are significantly different (Patel et al., 1960). Royalactin, a component of royal jelly, has been reported to influence the queen's development in a detectable manner. Royalactin depletion in royal jelly can promote development into the worker caste, whereas Royalactin addition can promote development into the queen caste. Royalactin induces specific effects by the epidermal growth factor receptor pathway, which enhances the secretion of juvenile hormone and further activates mitogen-activated protein kinase p70 and

18 Polyandrous Queen Honey Bee

Lovleen Marwaha

S6 kinase (Kamakura 2011). Royalactin induces alterations in gene expression and DNA methylation (Barchuk et al. 2007; Kucharski et al. 2008).

Fig. (1i). Specific click providing a view of a worker honey bee adding worker jelly to a developing worker larva. Furthermore, workers capped cells for synchronous organogenesis during the pupal phase can also be detected. Some of the unripe honey cells interspersed among the brood cells specify the brood rearing strategy of bees.

Fig. (1p). Unripe honey cells enclosed by pupal cells can be seen in a section of the hive. Further, click to highlight the working strategy of workers for brood rearing.

Queen Honey Bee

Polyandrous Queen Honey Bee 19

Fig. (1q). Similar click highlighting the presence of pollen grain filled wax cells encircled by capped brood cells.

1.2.5. Plasticity of the Queen Development Phase In honey bees, the female caste exhibits developmental plasticity, as the developmental pattern is influenced by the larval stage when it enters into the queen's developmental pathway (Barchuk et al., 2007; de Azevedo and Hartfelder, 2008). Differential developmental patterns result due to different larval diets and epigenetic modification of related genetic elements, which ultimately influence the phenotype, anatomy, physiology, development, ecdysis, role in the colony, and life span of female castes. Actually, different larval diets induce the expression of differential genes in different castes (Evans and Wheeler,1999). Workers can sense queen loss within 24 hours and thereafter start raising a new queen from fertilized eggs, if available (Hatch et al. 1999). In honey bees, fertilized eggs retain the potential to develop into either female caste depending upon the environment and diet given during initial larval development. It is considered that larval food acts as a major criterion which determines female caste formation. Moreover, other factors which influence the process include genetics and wax cell size (Winston 1987, Kucharski et al. 2008, Shi et al. 2011). It has been reported that if worker larva is transferred to a queen cell within the first three days of development, queen formation occurs as caste differentiation remains flexible during the initial larval phase, but if grafting occurs after 3 days of larval development, then caste development cannot take place in a reversible way (Weaver 1957;Woyke 1971; Rangel et al., 2016).

20 Polyandrous Queen Honey Bee

Lovleen Marwaha

1.3. QUEEN PHEROMONES 1.3.1: Queen Pheromones Composition The queen secretes certain volatile chemicals, which regulate the behavior of worker honey bees and increase the interaction between them. The Queen secretes various types of volatile chemicals through 15 different pheromone glands (Blum 1992). An exploration indicated Queen Mandibular Pheromones (QMPs) include 9-oxo-2(E)-decenoic acid (9-ODA), the two enantiomers of 9hydroxy-2(E-decenoic acid (9-HDA), as well as the two aromatic compounds methyl phydroxybenzoate (HOB) and 4-hydroxy-3-methoxyphenylethanol (HVA) (Slessor et al. 1988) (Table 1). Recent data reveals that queen honey bee secretes pheromones including 9-ODA(2E)-9-oxodecenoic acid), 9-HDA (9-hydroxy-()-2-decenoic acid), 10-HDA (10-hrdroxy-2-decenoic acid), HVA (4-hydroxy3-methoxyphenylethanol), HOB (Methyl-p-hydroxybenzoate), 10-HDAA (10hydroxydecanoic acid), OLA (oligolactide), methyl oleate, decyl decanoate, linolenic acid, coniferyl alcohol, cetyl alcohol etc,as elucidated by (Fig. 1l) (Keeling et al., 2003; Slessor et al., 2005; Maisonnasse et al., 2010;Rangel et al., 2016;) QMPs regulate the retinue behaviour in workers and act as sex attractant for drones (Gary 1962;Kaminski et al. 1990). QMPs induce an inhibitory influence on the secretion of juvenile hormone in worker honey bees and further affect the activation of the worker’s ovaries (Kaatz et al. 1992;Hoover et al. 2003). Table 1. Specifying Different Pheromones and the Functional Importance of the Same. QMPs

FUNCTIONs

9-oxo-(E)-2-decenoic acid, (+)- and (−)9-hydroxy (E)-2-decenoic acid, methyl Phydroxybenzoate, and 4-hydroxy-3 methoxy phenyl ethanol.

QMPs inhibit worker ovary activation Specific chemicals control functions like comb construction in worker honey bees Concerned volatiles increase resistance to starvation, further specific chemicals affect olfactory learning and memory in workers Specific pheromones inhibit the construction of drone and queen cells QMPs induce retinue behaviour Pheromones promote the stability of a swarm or possess “calming” influence.

Coniferyl alcohol, Methyl oleate, αLinolenic acid and Cetyl alcohol

These components are also vital for the retinue attraction of worker bees around the queen bee.

Queen Honey Bee

Polyandrous Queen Honey Bee 21

Fig. (1r). Diagram depicting different pheromones secreted by the queen honey bee for regulation, dominance, and co-ordination in the colony.

The queen mainly secretes 9-ODA, which is generally referred to as the “queen substance.” In case of worker honey bees, the main pheromones are 10-hydroxydecanoic acid (10-HDAA) and 10-hydroxy-2(E)-decenoic acid (10-HDA) (Plettner et al. 1993). Workers are not developmental irrevocable, as some of workers that start producing mandibular pheromones composite of 9-ODA, 10-HDAA and 10HDA, start reproducing as pseudo-queen (Simon et al. 2001). Pseudo-queens can secrete volatile chemicals similar to queen pheromone, therefore can elicit retinue behaviour and start proving their false dominant hierarchies in the colony (Crewe and Velthuis 1980; Plettner et al. 1993; Moritz et al. 2004). 1.3.2. Queen Mating Status Correlation with QMPs The newly emerged queen secretes pheromones similar to those of workers (Crewe 1982). With maturation and reproduction, there is a change in the composition of mandibular gland pheromones in queens (Slessor et al. 1990;

22 Polyandrous Queen Honey Bee

Lovleen Marwaha

Pankiw et al. 1996; Engels et al. 1997; Apsegaite and Skirkevicius 1995), due to a change in the physiology of specific castes (Rhodes et al. 2007) . Pankiw et al. (1996) reported that the volatile chemicals like 9-ODA and 9-HDA are less abundant in queens, but in queens, the specific pheromones are excessively secreted in queens, whereas Rhodes et al. (2007) observed that in the virgin queen, there is higher secretion of 9-ODA and 9-HDA than in mated queens of the same age. A few reports suggest that an unmated queen secretes pheromones in between workers and egg-laying mated queens (Apsegaite and Skirkevicius 1995, 1999; Plettner et al. 1997). Further chemical composition of pheromones from different queens, including pseudo-queen, drone layers, and reproductively active queens, indicated the presence of six mandibular gland compounds, 9-ODA, 9-HDA, 10-HDAA, 10HDA, HOB, and HVA. With an increase in reproductive capacity, there is a further rise in the amount of 9-HDA, 10HDAA, 10-HDA, and HVA but not of HOB (Slessor et al. 1990; Apsegaite and Skirkevicius, 1995 and Engels et al., 1997), whereas Plettner et al. (1997) reported that from virgin or drone laying queen to mated queen, there is an increase in the concentration of 9-ODA and HOB. In specific research works, selected queens could have variable reproductive potential and age that could be the possible reason for the change in the composition of mandibular pheromones. The ratios of 10HDAA/9-HDA and 9-ODA/(9-ODA+10-HDAA+10-HDA) indicate the fecundity of the queen (Plettner et al. 1993; Moritz et al., 2004). Rhodes et al. (2007) reported that the synthesis of HOB is influenced by the queen's reproductive capacity. Further, they observed that a 7-day-old virgin queen secretes a higher amount of 9-ODA than mated queens of the same age. The queen's reproductive value indicates a positive correlation between 9-HDA, 10-HDAA, 10HDA, and HVA, whereas 9-ODA possesses a negative correlation with the reproductive potential of a specific caste. A few other explorations indicated that the chemical composition of the queen's mandibular gland and Dufour’s gland varies with the insemination quantity and reproductive activeness of the queen (Richard et al., 2007; Kocher et al., 2008; Richard et al., 2011). 1.3.3. Functions of Pheromones The QMP's level is considered to specify the ovarian state and reproductive quality of the queen. Queens of superior quality and inferior quality secrete different QMPs, which are responsible for induction of differential behaviour in workers and the variable retinue response. QMPs are transferred from worker to

Queen Honey Bee

Polyandrous Queen Honey Bee 23

worker within a colony. Various pheromones induce different responses in workers, and the description is as follows: 9-ODA acts as a sex attractant for drones, especially during nuptial flight (Butler and Fairey 1964). Therefore, a specific pheromone is more important for virgins than mated queens. 9-ODA, 9-HDA, along with other QMP components, elicit the retinue behaviour in workers, inhibit workers' ovarian development and impose queen dominance in the colony (Butler and Fairey 1963; Slessor et al. 1988; Pettis et al. 1997; Keeling et al. 2001; Hoover et al. 2003; Strauss et al., 2008). The queen secretes various pheromones, known as queen retinue pheromones (QRP), from various glands, including the mandibular gland, Dufour’s gland, and tergal glands. QRP attracts workers to the queen to antennate, feed, and groom her. Through the secretion of queen pheromones, the queen gives a signal about her dominance and prevents to work behind a new queen (Keeling et al. 2003, Hoover et al. 2003, Le Conte and Hefetz 2008). Queen of superior quality and inferior quality secrete different QMPs, which are responsible for the induction of differential behaviour in workers and the induction of retinue in workers. HVA induces a reduction in the concentration of dopamine in the honey bee’s brain (Beggs et al., 2007). Dombroski et al. (2003) considered that HVA controls worker sterility as the specific pheromones can induce the suppression of worker ovaries. Further, HOB pheromones can influence mating in queen honey bees (Pankiw et al. 1996; Plettner et al. 1997). Worker honey bees can collectively decide to replace the queen in case of insufficient queen pheromonal secretion. Due to the depletion of stored sperm, queens can become drone-laying females during old age. In such a case, workers can raise a new queen, even in the presence of an older one. Older queens are superseded by young queens, which are raised by workers. 1.4. MATING 1.4.1. Nuptial Flight The honey bee queen is polyandrous as it mates with multiple drones in a single mating, which is known as nuptial flight. The specific flight generally occurs within 10 days of post-pupal hatching. (Taber, 1954; Taber and Wendel, 1958; Laidlaw et al., 1984). During the nuptial flight, the queen honey bee receives ample sperm, subsequent to mating with 10–20 drones, and she receives sperm in her spermatheca for her entire life span (Koeniger et al., 2014). Few reports specified that queen honey bees usually mate with 6–26 drones, with an average

24 Polyandrous Queen Honey Bee

Lovleen Marwaha

number corresponding to 12-14 (Estoup, et al., 1994; Neumann, et al., 1999;Tarpy, and Nielsen, 2002; Tarpy, et al., 2004; Kraus, et al., 2005; Delaney, et al., 2011). The Queen's honey bee is quite fertile, and her specific sperm storage is sufficient for her entire life. According to Snodgrass (1956), a single honey bee queen can lay up to 3,000 eggs daily. 1.4.2. DCA Mating usually occurs in drone congregation areas (DCA) (Jean-Prost, 1957; Ruttner, 1962; Zmarlicki, 1963; Ruttner, 1988;Schlüns, et al., 2005; Koeniger, et al., 2014)). In DCA, about 8000–15,000 drones can occur at a specific time (Bottcher, 1975; Koeniger and Koeniger, 2000; Koeniger, 2005). Before nuptial flight, the queen takes short flights of a few minutes (Fletscher and Tribe, 1977;Koeniger, 1986; Koeniger, and Koeniger, 2007). The queen mates with multiple drones during nuptial flight, which can occur on a single day or on multiple consecutive days (Roberts, 1944; Ruttner, 1954;Woyke, 1960; Woyke, 1964; Frank, et al., 2002;Schlüns, et al., 2005; Koeniger,and Koeniger, 2007). Further, the stored sperm quality and mating numbers act as major criteria for the execution of more flights and the initiation of the egg laying phase (Woyke, 1964; 1966; Schlüns, et al., 2005). Tarpy and Page (2000) concluded that a queen cannot regulate the number of mating times and mating frequency. Queen mating behaviour is influenced by several factors, including genetic content, physiology, temperature, wind, cloud, etc. (Alber, 1955; Oertel, 1956; Verbeek, 1976; Lensky and Demter, 1985;Koeniger, 1986). Additionally, drone presence during nuptial flights also acts as a critical factor in the decision of the number of mating and nuptial flights (Koeniger and Koeniger, 2007). 1.4.3. Process of Mating and Post Mating With multiple matings, there is an increase in genetic diversity so that colonies can withstand adverse biotic and abiotic conditions (Rueppell, et al., 2008). Additionally, multiple matings increase the amount of stored sperm, enhance the queen's attractiveness, and increase genetic diversity in the colony. Further specific genomic diversity in turn positively influences colonial division of labour, provides stability to colonies, increases communication among workers and reduced the incidence of diseases (Robinson, 1992; Jones, et al., 2004; Schlüns, et al., 2005; Richard, et al., 2007; Delaney, et al., 2011; Mattila, et al., 2014).

Queen Honey Bee

Polyandrous Queen Honey Bee 25

After mating, drones usually die due to the loss of the endophallus which remains in the sting chamber of the queen (Woyke, 1958; Koeniger, 1990). Further, successful mating can be assessed by spermatozoa count and patriline determination can be detected by microsatellite analysis (Woyke, 1960; 1964; Estoup, et al., 1994; Neumann, et al., 1999; Neumann, et al., 1999; Koeniger, and Koeniger, 2007). Stored sperm number and quality of sperm eventually influence the reproductive capability of the queen honey bee (Akyol, et al., 2008; AlLawati, et al., 2009;Delaney, et al., 2011; Tarpy, et al., 2012). The size of queen spermathecae also acts as an indicator of the reproductive potential of the queen, because the characteristics reflect the sperm storage capacity. Further, it has been reported that the size of spermathecae is dependent upon the rearing condition and the quality of diet provided to selected queen larvae (Tarpy, et al., 2000; Tarpy, 2012). If a queen is raised from young hatched larvae, then it possesses larger spermathecae (Hatch, et al., 1999; Tarpy, et al., 2000; Gilley, et al., 2003). Spermathecae of the superior quality queen can be about 1.2 mm (Carreck, et al., 2013; Hatjina, et al., 2014). According to Woyke, 1962, the threshold of sperm below 3 million, less than that, indicates an inadequately mated queen (Amiri, et al., 2017). Post mating, only 3-5% of each drone’s sperm actually migrate to queen spermathecae for storage (Woyke, et al., 1962). The post-mating queen starts laying eggs and the secretion of queen pheromones to dominate the colony. 1.5. ROLE OF QUEEN HONEY BEE The specific caste is the only obligatorily reproducible female caste in the colony. She possesses the sole right to influence the strength and productivity of the colony. Her strong pheromonal control can modulate the behaviour of thousands of nest residents. Her predominant duties are as follows: • Colonial strength regulation. • Caste ratio modulation. • Secretion of queen pheromones by different glands. • Product production is influenced by controlling the number of foragers. • Drone strength control by laying unfertilized/fertilized eggs. • Worker ovarian development suppression. • Workers overall development divergence during pre and post emergence.

26 Polyandrous Queen Honey Bee

Lovleen Marwaha

• Control over behaviour of the colony, including aggressiveness/ calmness • Swarming activation/suppression. • Disease resistance through the transmission of specific genetic elements to the next progeny. 1.6: Conclusion: To sum up, the polyandrous queen honey bee is a dominant bee, ruling over thousands of workers in that colony. The queen is responsible for harmonious social interaction, breeding and productivity of the colony. Complete colony collapse/ swarming can occur in her absence, if the colony does not have fertilized eggs or worker larvae below 4 days of hatching. REFERENCES Akyol, E, Yeninar, H & Kaftanoglu, O (2008) Live weight of queen honey bees (Apis mellifera L.) predicts reproductive characteristics. J Kans Entomol Soc, 81, 92-100. [http://dx.doi.org/10.2317/JKES-705.13.1] Alber, M (1955) Von der paarung der honigbiene. Z Bienenforsch, 3, 1-28. Al-Lawati, H, Kamp, G & Bienefeld, K (2009) Characteristics of the spermathecal contents of old and young honeybee queens. J Insect Physiol, 55, 117-22. [http://dx.doi.org/10.1016/j.jinsphys.2008.10.010] [PMID: 19027748] Alaux, C, Dantec, C, Parrinello, H & Le Conte, Y (2011) Nutrigenomics in honey bees: digital gene expression analysis of pollen’s nutritive effects on healthy and varroa-parasitized bees. BMC Genomics, 12, 496. [http://dx.doi.org/10.1186/1471-2164-12-496] [PMID: 21985689] Amiri, E, Strand, M, Rueppell, O & Tarpy, D (2017) Queen quality and the impact of honey bee diseases on queen health: potential for interactions between two major threats to colony health. Insects, 8, 48. [http://dx.doi.org/10.3390/insects8020048] [PMID: 28481294] Apðegaitë, V & Skirkevièius, A (1995) Quantitative and qualitative composition of extracts from virgin and mated honeybee queens (Apis mellifera L.). Pheromones, 5, 23-36. Asencot, M & Lensky, Y (1976) The effect of sugars and Juvenile Hormone on the differentiation of the female honeybee larvae ( L.) to queens. Life Sci, 18, 693-9. [http://dx.doi.org/10.1016/0024-3205(76)90180-6] [PMID: 1263752] Baer, B, Collins, J, Maalaps, K & Boer, SPA (2016) Sperm use economy of honeybee ( Apis mellifera ) queens. Ecol Evol, 6, 2877-85. [http://dx.doi.org/10.1002/ece3.2075] [PMID: 27217944] Barchuk, AR, Cristino, AS, Kucharski, R, Costa, LF, Simões, ZLP & Maleszka, R (2007) Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera. BMC Dev Biol, 7, 70. [http://dx.doi.org/10.1186/1471-213X-7-70] [PMID: 17577409] Beetsma, J (1979) The process of queen-worker differentiation in the honeybee. Bee World, 60, 24-39. [http://dx.doi.org/10.1080/0005772X.1979.11097727] Beggs, KT, Glendining, KA, Marechal, NM, Vergoz, V, Nakamura, I, Slessor, KN & Mercer, AR (2007) Queen pheromone modulates brain dopamine function in worker honey bees. Proc Natl Acad Sci USA, 104, 2460-4. [http://dx.doi.org/10.1073/pnas.0608224104] [PMID: 17287354] Blum, MS (1992) Honey bee pheromones in the hive and the honey bee, Dadant and Sons 385-9.

Queen Honey Bee

Polyandrous Queen Honey Bee 27

Böttcher, FK (1975) Beiträge zur Kenntnis des Paarungsfluges der Honigbiene. Apidologie (Celle), 6, 233-81. [http://dx.doi.org/10.1051/apido:19750304] Brodschneider, R & Crailsheim, K (2010) Nutrition and health in honey bees. Apidologie (Celle), 41, 278-94. [http://dx.doi.org/10.1051/apido/2010012] Burley, LM, Fell, RD & Saacke, RG (2008) Survival of honey bee (Hymenoptera: Apidae) spermatozoa incubated at room temperature from drones exposed to miticides. J Econ Entomol, 101, 1081-7. [http://dx.doi.org/10.1093/jee/101.4.1081] [PMID: 18767713] Butler, CG & Fairey, EM (1963) The role of the queen in preventing oogenesis in worker honeybees. J Apic Res, 2, 14-8. [http://dx.doi.org/10.1080/00218839.1963.11100051] Butler, CG & Simpson, J (1967) Pheromones of the queen honeybee (Apis mellifera L.) which enable her workers to follow her when swarming. Proc R Entomol Soc Lond, Ser A Gen Entomol, 42, 149-54. [). Oxford, UK: Blackwell Publishing Ltd.]. [http://dx.doi.org/10.1111/j.1365-3032.1967.tb00806.x] Carreck, NL, Andree, M, Brent, CS, Cox-Foster, D, Dade, HA, Ellis, JD, Hatjina, F & van Englesdorp, D (2013) Standard methods for Apis mellifera anatomy and dissection. J Apic Res, 52, 1-40. [http://dx.doi.org/10.3896/IBRA.1.52.4.03] Chaimanee, V, Evans, JD, Chen, Y, Jackson, C & Pettis, JS (2016) Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the organophosphate acaricide coumaphos. J Insect Physiol, 89, 1-8. [http://dx.doi.org/10.1016/j.jinsphys.2016.03.004] [PMID: 26979384] Collins, AM (2000) Relationship between semen quality and performance of instrumentally inseminated honey bee queens. Apidologie (Celle), 31, 421-9. [http://dx.doi.org/10.1051/apido:2000132] Collins, AM (2004) Variation in time of egg hatch by the honey bee, Apis mellifera (Hymenoptera: Apidae). Ann Entomol Soc Am, 97, 140-6. [http://dx.doi.org/10.1603/0013-8746(2004)097[0140:VITOEH]2.0.CO;2] Collins, AM, Williams, V & Evans, JD (2004) Sperm storage and antioxidative enzyme expression in the honey bee, Apis mellifera. Insect Mol Biol, 13, 141-6. [http://dx.doi.org/10.1111/j.0962-1075.2004.00469.x] [PMID: 15056361] Crewe, RM (1982) Compositional variability: the key to the social signals produced by honeybee mandibular glands. In: Breed, M.D., Mitchener, E.D., Evans, H.E., (Eds.), The biology of social insects, Westview, Boulder 318-22. Crewe, RM & Velthuis, HHW (1980) False queens: A consequence of mandibular gland signals in worker honeybees. Naturwissenschaften, 67, 467-9. [http://dx.doi.org/10.1007/BF00405650] Cruz-Landim, C (2009) Aparelho reprodutor feminino e ovogênese Abelhas: Morfologia e função de sistemas, Editora UNESP, São Paulo 15-55. [http://dx.doi.org/10.7476/9788539304301] de Azevedo, SV & Hartfelder, K (2008) The insulin signaling pathway in honey bee (Apis mellifera) caste development — differential expression of insulin-like peptides and insulin receptors in queen and worker larvae. J Insect Physiol, 54, 1064-71. [http://dx.doi.org/10.1016/j.jinsphys.2008.04.009] [PMID: 18513739] Dedej, S, Hartfelder, K, Aumeier, P, Rosenkranz, P & Engels, W (1998) Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee ( Apis mellifera carnica ). J Apic Res, 37, 183-90. [http://dx.doi.org/10.1080/00218839.1998.11100970]

28 Polyandrous Queen Honey Bee

Lovleen Marwaha

Delaney, DA, Keller, JJ, Caren, JR & Tarpy, DR (2011) The physical, insemination, and reproductive quality of honey bee queens (Apis mellifera L.). Apidologie (Celle), 42, 1-13. [http://dx.doi.org/10.1051/apido/2010027] Dombroski, TCD, Simões, ZLP & Bitondi, MMG (2003) Dietary dopamine causes ovary activation in queenless Apis mellifera workers. Apidologie (Celle), 34, 281-9. [http://dx.doi.org/10.1051/apido:2003024] Eckert, JE (1934) Studies in the number of ovarioles in queen honeybees in relation to body size. J Econ Entomol, 27, 629-35. [http://dx.doi.org/10.1093/jee/27.3.629] Engels, W, Rosenkranz, P, Adler, A, Taghizadeh, T, Lübke, G & Francke, W (1997) Mandibular gland volatiles and their ontogenetic patterns in queen honey bees, Apis mellifera carnica. J Insect Physiol, 43, 30713. [http://dx.doi.org/10.1016/S0022-1910(96)00110-2] [PMID: 12769892] Estoup, A, Solignac, M & Cornuet, JM (1994) Precise assessment of the number of patrilines and of genetic relatedness in honeybee colonies. Proc Biol Sci, 258, 1-7. [http://dx.doi.org/10.1098/rspb.1994.0133] Evans, JD & Wheeler, DE (1999) Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proc Natl Acad Sci USA, 96, 5575-80. [http://dx.doi.org/10.1073/pnas.96.10.5575] [PMID: 10318926] Fletcher, DJC & Tribe, GD (1977) b.—Natural emergency queen rearing by the African bee Apis mellifera adansonii and its relevance for successful queen production by beekeepers, II. In: Pretoria, South Africa, (Ed.), Proc Apimondia Inter Symp, 161-8. Fleig, R (1995) Role of the follicle cells for yolk uptake in ovarian follicles of the honey bee Apis mellifera L. (Hymenoptera : Apidae). Int J Insect Morphol Embryol, 24, 427-33. [http://dx.doi.org/10.1016/0020-7322(95)98841-Z] Frazier, M, Mullin, C, Frazier, J & Ashcraft, S (2008) What have pesticides got to do with it? Am Bee J, 148, 521-4. Franck, P, Solignac, M, Vautrin, D, Cornuet, JM, Koeniger, G & Koeniger, N (2002) Sperm competition and last-male precedence in the honeybee. Anim Behav, 64, 503-9. [http://dx.doi.org/10.1006/anbe.2002.3078] Gauthier, L, Ravallec, M, Tournaire, M, Cousserans, F, Bergoin, M, Dainat, B & de Miranda, JR (2011) Viruses associated with ovarian degeneration in Apis mellifera L. queens. PLoS One, 6e16217 [http://dx.doi.org/10.1371/journal.pone.0016217] [PMID: 21283547] Gary, NE (1962) Chemical mating attractants in the queen honey bee. Science, 136, 773-4. [http://dx.doi.org/10.1126/science.136.3518.773] [PMID: 17752107] Gilley, DC, Tarpy, DR & Land, BB (2003) Effect of queen quality on interactions between workers and dueling queens in honeybee ( Apis mellifera L.) colonies. Behav Ecol Sociobiol, 55, 190-6. [http://dx.doi.org/10.1007/s00265-003-0708-y] Gutzeit, HO, Zissler, D & Fleig, R (1993) Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles. Rouxs Arch Dev Biol, 202, 181-91. [http://dx.doi.org/10.1007/BF00365309] [PMID: 28305996] Haarmann, T, Spivak, M, Weaver, D, Weaver, B & Glenn, T (2002) Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations. J Econ Entomol, 95, 28-35. [http://dx.doi.org/10.1603/0022-0493-95.1.28] [PMID: 11942761] DeGrandi-Hoffman, G, Chambers, M, Hooper, JE & Schneider, SS (2004) Description of an intermorph between a worker and queen in African honey bees Apis mellifera scutellata (Hymenoptera: Apidae). Ann

Queen Honey Bee

Polyandrous Queen Honey Bee 29

Entomol Soc Am, 97, 1299-305. [http://dx.doi.org/10.1603/0013-8746(2004)097[1299:DOAIBA]2.0.CO;2] Harbo, JR & Harris, JW (2009) Responses to Varroa by honey bees with different levels of Varroa Sensitive Hygiene. J Apic Res, 48, 156-61. [http://dx.doi.org/10.3896/IBRA.1.48.3.02] Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Hatjina, F, Bieńkowska, M, Charistos, L, Chlebo, R, Costa, C, Dražić, MM, Filipi, J, Gregorc, A, Ivanova, EN, Kezić, N, Kopernicky, J, Kryger, P, Lodesani, M, Lokar, V, Mladenovic, M, Panasiuk, B, Petrov, PP, Rašic, S, Skerl, MIS, Vejsnæs, F & Wilde, J (2014) A review of methods used in some European countries for assessing the quality of honey bee queens through their physical characters and the performance of their colonies. J Apic Res, 53, 337-63. [http://dx.doi.org/10.3896/IBRA.1.53.3.02] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Jackson, JT, Tarpy, DR & Fahrbach, SE (2011) Histological estimates of ovariole number in honey bee queens, Apis mellifera, reveal lack of correlation with other queen quality measures. J Insect Sci, 11, 1-11. [http://dx.doi.org/10.1673/031.011.8201] [PMID: 21870968] (1957) OBSERVATIONS SUR LE VOL NUPTIAL DES REINES DABEILLES. C R Hebd Seances Acad Sci, 245, 2107-10. Jones, JC, Myerscough, MR, Graham, S & Oldroyd, BP (2004) Honey bee nest thermoregulation: diversity promotes stability. Science, 305, 402-4. [http://dx.doi.org/10.1126/science.1096340] [PMID: 15218093] Kaatz, HH, Hildebrandt, H & Engels, W (1992) Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees. J Comp Physiol B, 162, 588-92. [http://dx.doi.org/10.1007/BF00296638] Kamakura, M (2011) Royalactin induces queen differentiation in honeybees. Nature, 473, 478-83. [http://dx.doi.org/10.1038/nature10093] [PMID: 21516106] Kaminski, LA, Slessor, KN, Winston, ML, Hay, NW & Borden, JH (1990) Honeybee response to queen mandibular pheromone in laboratory bioassays. J Chem Ecol, 16, 841-50. [http://dx.doi.org/10.1007/BF01016494] [PMID: 24263599] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Koeniger, G, Koeniger, N, Ellis, J & Connor, LJ (2014) Mating biology of honey bees (Apis mellifera)Wicwas Press LLC. Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2008) Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera). BMC Genomics, 9, 232. [http://dx.doi.org/10.1186/1471-2164-9-232] [PMID: 18489784] Koeniger, G (1986) Reproduction and mating behaviour in Bee Genetics and Breeding, 255-80. [http://dx.doi.org/10.1016/B978-0-12-588920-9.50015-7] Koeniger, G (1990) The role of the mating sign in honey bees, Apis mellifera L.: does it hinder or promote multiple mating? Anim Behav, 39, 444-9. [http://dx.doi.org/10.1016/S0003-3472(05)80407-5] Koeniger, N & Koeniger, G (2000) Reproductive isolation among species of the genus Apis. Apidologie

30 Polyandrous Queen Honey Bee

Lovleen Marwaha

(Celle), 31, 313-39. [http://dx.doi.org/10.1051/apido:2000125] Koeniger, N & Koeniger, G (2007) Mating flight duration of Apis mellifera queens: As short as possible, as long as necessary. Apidologie (Celle), 38, 606-11. [http://dx.doi.org/10.1051/apido:2007060] Koeniger, N, Koeniger, G, Gries, M & Tingek, S (2005) Drone competition at drone congregation areas in four Apis species. Apidologie (Celle), 36, 211-21. [http://dx.doi.org/10.1051/apido:2005011] Koeniger, N, Koeniger, G & Pechhacker, H (2005) The nearer the better? Drones (Apis mellifera) prefer nearer drone congregation areas. Insectes Soc, 52, 31-5. b [http://dx.doi.org/10.1007/s00040-004-0763-z] Kraus, F B, Neumann, P & Moritz, R F A (2005) Genetic variance of mating frequency in the honey bee ( Apis mellifera L.). InsectSoc52:1–5. Kucharski, R, Maleszka, J, Foret, S & Maleszka, R (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science, 319, 1827-30. [http://dx.doi.org/10.1126/science.1153069] [PMID: 18339900] Laidlaw, H H, Jr (1984) Spermatophore size as determinant of paternity in an arctid moth (Utetheisa ornatrix). Proc Natl Acad Sci USA, 91, 7081-4. Conte, YL & Hefetz, A (2008) Primer pheromones in social hymenoptera. Annu Rev Entomol, 53, 523-42. [http://dx.doi.org/10.1146/annurev.ento.52.110405.091434] [PMID: 17877458] Lee, KV, Goblirsch, M, McDermott, E, Tarpy, DR & Spivak, M (2019) Is the Brood Pattern within a Honey Bee Colony a Reliable Indicator of Queen Quality? Insects, 10, 12. [http://dx.doi.org/10.3390/insects10010012] [PMID: 30626029] Lensky, Y & Demter, M (1985) Mating flights of the queen honeybee (Apis mellifera) in a subtropical climate. Comp Biochem Physiol A Comp Physiol, 81, 229-41. [http://dx.doi.org/10.1016/0300-9629(85)90127-6] Lodesani, M, Balduzzi, D & Galli, A (2004) A study on spermatozoa viability over time in honey bee ( Apis mellifera ligustica ) queen spermathecae. J Apic Res, 43, 27-8. [http://dx.doi.org/10.1080/00218839.2004.11101105] Maisonnasse, A, Alaux, C, Beslay, D, Crauser, D, Gines, C, Plettner, E & Le Conte, Y (2010) New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation? Front Zool, 7, 18. [http://dx.doi.org/10.1186/1742-9994-7-18] [PMID: 20565874] Mattila, HR & Seeley, TD (2014) Extreme polyandry improves a honey bee colony’s ability to track dynamic foraging opportunities via greater activity of inspecting bees. Apidologie (Celle), 45, 347-63. [http://dx.doi.org/10.1007/s13592-013-0252-3] Milne, CP, Jr, Phillips, JP & Krell, PJ (1988) A photomicrographic study of worker honeybee embryogenesis. J Apic Res, 27, 69-83. [http://dx.doi.org/10.1080/00218839.1988.11100784] Moritz, RF, Lattorff, HMG & Crewe, RM (2004) Honeybee workers (Apis mellifera capensis) compete for producing queen-like pheromone signals. Proc Biol Sci, 271 (Suppl. 3), S98-S100. [PMID: 15101431] Pflugfelder, J, Moritz, RFA & Crewe, RM (2003) Lethal fighting between honeybee queens and parasitic workers ( Apis mellifera ). Naturwissenschaften, 90, 378-81. [http://dx.doi.org/10.1007/s00114-003-0445-0] [PMID: 12955229] Nelson, DL & Gary, NE (1983) Honey productivity of honeybee colonies in relation to body weight, attractiveness and fecundity of the queen. J Apic Res, 22, 209-13.

Queen Honey Bee

Polyandrous Queen Honey Bee 31

[http://dx.doi.org/10.1080/00218839.1983.11100589] Nelson, DL & Smirl, C (1977) The effect of queen-related problems and swarming on brood and honey production of honey bee colonies in Manitoba. Manitoba Entomol, 11, 45-9. Neumann, P, Moritz, RFA & van Praagh, J (1999) Queen mating frequency in different types of honey bee mating apiaries. J Apic Res, 38, 11-8. [http://dx.doi.org/10.1080/00218839.1999.11100990] Niño, EL, Tarpy, DR & Grozinger, CM (2013) Differential effects of insemination volume and substance on reproductive changes in honey bee queens ( Apis mellifera L.). Insect Mol Biol, 22, 233-44. [http://dx.doi.org/10.1111/imb.12016] [PMID: 23414204] Oertel, E (1956) Observations on the flight of drone honey bees. Ann Entomol Soc Am, 49, 497-500. [http://dx.doi.org/10.1093/aesa/49.5.497] Pankiw, T, Winston, ML, Plettner, E, Slessor, KN, Pettis, JS & Taylor, OR (1996) Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.). J Chem Ecol, 22, 605-15. [http://dx.doi.org/10.1007/BF02033573] [PMID: 24227572] Patel, NG, Haydak, MH & Gochnauer, TA (1960) Electrophoretic components of the proteins in honeybee larval food. Nature, 186, 633-4. [http://dx.doi.org/10.1038/186633a0] [PMID: 14430832] Patrício, K & Cruz-Landim, C (2002) Mating influence in the ovary differentiation in adult queens of Apis mellifera L. (Hymenoptera, Apidae). Braz J Biol, 62, 641-9. [http://dx.doi.org/10.1590/S1519-69842002000400012] [PMID: 12659014] Pettis, JS, Collins, AM, Wilbanks, R & Feldlaufer, MF (2004) Effects of coumaphos on queen rearing in the honey bee, Apis mellifera. Apidologie (Celle), 35, 605-10. [http://dx.doi.org/10.1051/apido:2004056] Plettner, E, Otis, GW, Wimalaratne, PDC, Winston, ML, Slessor, KN, Pankiw, T & Punchihewa, PWK (1997) Species-and caste-determined mandibular gland signals in honeybees (Apis). J Chem Ecol, 23, 363-77. [http://dx.doi.org/10.1023/B:JOEC.0000006365.20996.a2] Plettner, E, Slessor, KN, Winston, ML, Robinson, GE & Page, RE (1993) Mandibular gland components and ovarian development as measures of caste differentiation in the honey bee (Apis mellifera L.). J Insect Physiol, 39, 235-40. [http://dx.doi.org/10.1016/0022-1910(93)90094-8] Plettner, E, Slessor, KN, Winston, ML & Oliver, JE (1996) Caste-selective pheromone biosynthesis in honeybees. Science, 271, 1851-3. [http://dx.doi.org/10.1126/science.271.5257.1851] Rangel, J & Fisher, A, II (2019) Factors affecting the reproductive health of honey bee (Apis mellifera) drones—a review. Apidologie (Celle), 50, 759-78. [http://dx.doi.org/10.1007/s13592-019-00684-x] Rangel, J, Keller, JJ & Tarpy, DR (2013) The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth. Insectes Soc, 60, 65-73. [http://dx.doi.org/10.1007/s00040-012-0267-1] Rangel, J & R Tarpy, D (2016) In-hive miticides and their effect on queen supersedure and colony growth in the honey bee (Apis mellifera). J Environ Anal Toxicol, 61000377 [http://dx.doi.org/10.4172/2161-0525.1000377] Ratnieks, FW (1993) Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies. Behav Ecol Sociobiol, 32, 191-8. [http://dx.doi.org/10.1007/BF00173777] Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2e980

32 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Rhodes, JW, Lacey, MJ & Harden, S (2007) Changes with age in queen honey bee (Apis mellifera) head chemical constituents (Hymenoptera: Apidae). Sociobiology, 50, 11-22. Roberts, WC (1944) Multiple mating of queen bees proved by progeny and flight tests. Gleanings in Bee Culture, 72, 225-59. Robinson, GE (1992) Regulation of division of labor in insect societies. Annu Rev Entomol, 37, 637-65. [http://dx.doi.org/10.1146/annurev.en.37.010192.003225] [PMID: 1539941] Rueppell, O, Johnson, N & Rychtář, J (2008) Variance-based selection may explain general mating patterns in social insects. Biol Lett, 4, 270-3. [http://dx.doi.org/10.1098/rsbl.2008.0065] [PMID: 18364307] Ruttner, F (1954) Mehrfache begattung der bienenkönigin. Zool Anz, 153, 99-105. Ruttner, F (1962) Drohnen-sammelplätze. Bienenvater, 83, 1-2. Ruttner, F (1988) Biogeography and taxonomy of honeybees Springer [http://dx.doi.org/10.1007/978-3-642-72649-1] Ruttner, H (1976) Investigations on the flight activity and the mating behaviour of the drones, 6. Flight on and over mountain ridges. Apidologie (Celle), 7, 331-41. [http://dx.doi.org/10.1051/apido:19760404] Sandrock, C, Tanadini, M, Tanadini, LG, Fauser-Misslin, A, Potts, SG & Neumann, P (2014) Impact of chronic neonicotinoid exposure on honeybee colony performance and queen supersedure. PLoS One, 9e103592 [http://dx.doi.org/10.1371/journal.pone.0103592] [PMID: 25084279] Schlüns, H, Moritz, RFA, Neumann, P, Kryger, P & Koeniger, G (2005) Multiple nuptial flights, sperm transfer and the evolution of extreme polyandry in honeybee queens. Anim Behav, 70, 125-31. [http://dx.doi.org/10.1016/j.anbehav.2004.11.005] Shehata, SM, Townsend, GF & Shuel, RW (1981) Seasonal physiological changes in queen and worker honeybees. J Apic Res, 20, 69-78. [http://dx.doi.org/10.1080/00218839.1981.11100475] Shi, YY, Huang, ZY, Zeng, ZJ, Wang, ZL, Wu, XB & Yan, WY (2011) Diet and cell size both affect queenworker differentiation through DNA methylation in honey bees (Apis mellifera, Apidae). PLoS One, 6e18808 [http://dx.doi.org/10.1371/journal.pone.0018808] [PMID: 21541319] Simon, UE, Moritz, RFA & Crewe, RM (2001) The ontogenetic pattern of mandibular gland components in queenless worker bees (Apis mellifera capensis Esch.). J Insect Physiol, 47, 735-8. [http://dx.doi.org/10.1016/S0022-1910(00)00167-0] [PMID: 11356420] Skirkevicius, A (2004) First symptoms of queen loss in a honeybee colony ( Apis mellifera ). Apidologie (Celle), 35, 565-73. [http://dx.doi.org/10.1051/apido:2004057] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Kaminski, LA, King, GGS & Winston, ML (1990) Semiochemicals of the honeybee queen mandibular glands. J Chem Ecol, 16, 851-60. [http://dx.doi.org/10.1007/BF01016495] [PMID: 24263600] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Snodgrass, RE (1956) Anatomy of the Honey Bee, Comstock PublAssoc., Ithaca, New York.

Queen Honey Bee

Polyandrous Queen Honey Bee 33

Spivak, M & Reuter, GS (1998) Performance of hygienic honey bee colonies in a commercial apiary. Apidologie (Celle), 29, 291-302. [http://dx.doi.org/10.1051/apido:19980308] Stabe, HA (1930) The Rate of Growth of Worker, Drone and Queen Larvae of the Honeybee, Apis mellifera Linn.1. J Econ Entomol, 23, 447-53. [http://dx.doi.org/10.1093/jee/23.2.447] Taber, S, III (1954) The frequency of multiple mating of queen honey bees. J Econ Entomol, 47, 995-8. [http://dx.doi.org/10.1093/jee/47.6.995] Taber, S, III & Wendel, J (1958) Concerning the number of times queen bees mate. J Econ Entomol, 51, 7869. [http://dx.doi.org/10.1093/jee/51.6.786] Strauss, K, Scharpenberg, H, Crewe, RM, Glahn, F, Foth, H & Moritz, RFA (2008) The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent? Behav Ecol Sociobiol, 62, 1523-31. [http://dx.doi.org/10.1007/s00265-008-0581-9] Szabo, TI (1993) Length of life of queens in honey bee colonies. Am Bee J, 133, 723-4. Tarpy, DR, Hatch, S & Fletcher, DJC (2000) The influence of queen age and quality during queen replacement in honeybee colonies. Anim Behav, 59, 97-101. [http://dx.doi.org/10.1006/anbe.1999.1311] [PMID: 10640371] Tarpy, DR, Keller, JJ, Caren, JR & Delaney, DA (2012) Assessing the mating ‘health’ of commercial honey bee queens. J Econ Entomol, 105, 20-5. [http://dx.doi.org/10.1603/EC11276] [PMID: 22420250] Tarpy, DR, Keller, JJ, Caren, JR & Delaney, DA (2011) Experimentally induced variation in the physical reproductive potential and mating success in honey bee queens. Insectes Soc, 58, 569-74. [http://dx.doi.org/10.1007/s00040-011-0180-z] Tarpy, DR & Mayer, MK (2009) The effects of size and reproductive quality on the outcomes of duels between honey bee queens ( Apis mellifera L.). Ethol Ecol Evol, 21, 147-53. [http://dx.doi.org/10.1080/08927014.2009.9522503] Tarpy, DR, Nielsen, R & Nielsen, DI (2004) A scientific note on the revised estimates of effective paternity frequency in Apis. Insectes Soc, 51, 203-4. [http://dx.doi.org/10.1007/s00040-004-0734-4] Tarpy, DR & Nielsen, DI (2002) Sampling error, effective paternity, and estimating the genetic structure of honey bee colonies (Hymenoptera: Apidae). Ann Entomol Soc Am, 95, 513-28. [http://dx.doi.org/10.1603/0013-8746(2002)095[0513:SEEPAE]2.0.CO;2] Tarpy, DR & Olivarez, R, Jr (2014) Measuring sperm viability over time in honey bee queens to determine patterns in stored-sperm and queen longevity. J Apic Res, 53, 493-5. [http://dx.doi.org/10.3896/IBRA.1.53.4.02] Traynor, KS, Pettis, JS, Tarpy, DR, Mullin, CA, Frazier, JL, Frazier, M & vanEngelsdorp, D (2016) In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States. Sci Rep, 6, 33207. [http://dx.doi.org/10.1038/srep33207] [PMID: 27628343] Thrasyvoulou, AT & Benton, AW (1982) Rates of growth of honeybee larvae. J Apic Res, 21, 189-92. [http://dx.doi.org/10.1080/00218839.1982.11100540] Tsvetkov, N, Samson-Robert, O, Sood, K, Patel, HS, Malena, DA, Gajiwala, PH, Maciukiewicz, P, Fournier, V & Zayed, A (2017) Chronic exposure to neonicotinoids reduces honey bee health near corn crops. Science, 356, 1395-7. [http://dx.doi.org/10.1126/science.aam7470] [PMID: 28663503]

34 Polyandrous Queen Honey Bee

Lovleen Marwaha

vanEngelsdorp, D, Tarpy, DR, Lengerich, EJ & Pettis, JS (2013) Idiopathic brood disease syndrome and queen events as precursors of colony mortality in migratory beekeeping operations in the eastern United States. Prev Vet Med, 108, 225-33. [http://dx.doi.org/10.1016/j.prevetmed.2012.08.004] [PMID: 22939774] Verbeek, B (1976) Investigation of the flight activity of young honey-bee queens under continental and insular conditions by means of photoelectronic control. Apidologie (Celle), 7, 151-68. [http://dx.doi.org/10.1051/apido:19760205] Wallner, K (1999) Varroacides and their residues in bee products. Apidologie (Celle), 30, 235-48. [http://dx.doi.org/10.1051/apido:19990212] Weaver, N (1957) Effects of larval age on dimorphic differentiation of the female honey bee. Ann Entomol Soc Am, 50, 283-94. [http://dx.doi.org/10.1093/aesa/50.3.283] Williams, JL (1987) Wind-directed pheromone trap for drone honey bees (Hymenoptera: Apidae). J Econ Entomol, 80, 532-6. [http://dx.doi.org/10.1093/jee/80.2.532] Wilson, EO (1976) The insect societiesThe Belknap Press of Havard University Press, Cambridge. Winston, ML (1987) The Biology of the Honey BeeHarvard University Press, London, UK. Woyke, J (1958) The process of mating in the honeybee. PszczelZeszNauk, 2, 1-42. Woyke, J (1960) Natural and artificial insemination of queen honeybees. PszczelZeszNauk, 4, 183-275. Woyke, J (1962) Natural and artificial insemination of queen honeybees. Bee World, 43, 21-5. [http://dx.doi.org/10.1080/0005772X.1962.11096922] Woyke, J (1964) Causes of repeated mating flights by queen honeybees. J Apic Res, 3, 17-23. [http://dx.doi.org/10.1080/00218839.1964.11100077] Woyke, J (1966) Wovonhängt die zahl der spermien in natürlichemwegebegattettenköniginnen ab? Z Bienenforsch, 8, 236-48.

der

samenblase

der

auf

Woyke, J (1966) Development of methods, results and perspectives of artificial insemination of queen bees. Manual and artificial insemination of queen bees. 1-18. Woyke, J (1996) Different reaction of Apis dorsata and Apis mellifera to brood infestation by parasitic mites. Proceedings of 3rd Asian Apicultural Association Conference on Bee Research and Beekeeping, Hanoi, Vietnam6 -10 October 1996172-5. Woyke, J (1971) Correlations between the age at which honeybee brood was grafted, characteristics of the resultant queens, and results of insemination. J Apic Res, 10, 45-55. [http://dx.doi.org/10.1080/00218839.1971.11099669] Wu, JY, Anelli, CM & Sheppard, WS (2011) Sub-lethal effects of pesticide residues in brood comb on worker honey bee (Apis mellifera) development and longevity. PLoS One, 6e14720 [http://dx.doi.org/10.1371/journal.pone.0014720] [PMID: 21373182] Zheng, HQ, Jin, SH, Hu, FL & Pirk, CWW (2009) Sustainable multiple queen colonies of honey bees, Apis mellifera ligustica. J Apic Res, 48, 284-9. [http://dx.doi.org/10.3896/IBRA.1.48.4.09] Zmarlicki, C & Morse, RA (1963) Drone congregation areas. J Apic Res, 2, 64-6. [http://dx.doi.org/10.1080/00218839.1963.11100059]

Polyandrous Queen Honey Bee, 2022, 35-56

35

CHAPTER 2

The Queen Honey Bee Duties in the Composite Colonies Abstract: In a honey bee colony, a polyandrous queen bee performs two important duties predominantly. The first is reproduction, for modulation of colonial strength; and the second is the secretion of queen pheromones for regulation of social organization, developmental specification, colonial productivity control, retinue behaviour induction, worker ovarian suppression, foraging control, swarming reduction, other queen rearing inhibition, etc. In the female caste of honey bees, reproduction is uni-righted by a polyandrous queen, which mates preferably with multiple drones of other colonies during a nuptial flight in a Drone Congregation Area(DCA) and thereafter lays fertilised or unfertilized eggs depending on in-situ and ex-situ hive ambience, whereas worker honey bees perform the remaining tasks, including hive construction, brood rearing, foraging for food and nectar, honey production, protection and general organisation of the colony, pollen grain storage, water collection for the colony, ventilation in the hive, and the removal of carcases. In other words, worker bees perform all tasks except for reproduction and colony dominance. The specific duties assigned reflect the rectitudinous behaviour of the honey bee colony. Additionally, the specific division of labour enhances the competence of all honey bee castes. The Queen's honey bee is considerably fertile due to differential genomic expression, proteomics, and developmental specification. Further, her reproducibility is influenced by different biotic and abiotic factors prevailing within and outside the hive. In this chapter, a brief description of two predominant duties of the queen, including reproduction and pheromonal secretion, is highlighted. Subsequent chapters provide elaborative views of reproduction and pheromones.

Keywords: Division of labour, Pheromonal secretion, Queen honey bee, Reproduction. 2.1. REPRODUCIBILITY OF QUEEN HONEY BEE 2.1.1. General Information on Queen Reproduction The distinguishing role of the queen honey bee is to modulate the strength of the colony with her peculiar characteristics of laying unfertilized (n = 16) or fertilised eggs (2n = 32), which eventually affects the colony size, behavior, and agricultural productivity (Fig. 2a). Further, a queen’s decision to lay fertilised and Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

36 Polyandrous Queen Honey Bee

Lovleen Marwaha

unfertilized eggs depends upon various factors, like the presence of capped or uncapped brood, food availability, temperature, parasites, the structure of the hive, etc. Fertilized eggs develop into a female caste, which could be either a queen or a worker, depending upon the larval diet, whereas unfertilized eggs develop into drones. Furthermore, the queen possesses a powerful mechanism to control fertilization, as she lays eggs in different hexagonal cells, separate from drones and workers.

Fig. (2a). It depicts a honey bee colony with a single fertile queen (female), facultative reproducible workers (female), and drones (male). The diploid genetic material directs the development of the female, whereas, in the case of males, only a single set of chromosomes is sufficient for development.

The honey bee queen is quite fertile as she possesses a properly developed and functional reproductive system, formed under the influence of a larval diet, hormones, and an epigenetically regulated instructional system. Each ovary of a single queen has 120–200 ovarian filaments which are responsible for egg production, whereas workers possess less than 20 ovarioles and, therefore, are less reproductively active. A polyandrous queen usually mates once during nuptial flight and stores ample sperm deposits for her entire life. Mating occurs during the early phase of queen life, and subsequently, she does not leave the hive except at the time of swarming (Wilson, 1971; Corbella and Goncalves, 1982; Winston, 1987; Seeley, 1985, 1995; Crozier and Pamilo, 1996;Tarpy et al., 2003, 2004, Tarpya and David, 2004; Tarpy and Seeley, 2006; Abdulaziz et al., 2013). During the nuptial flight,

Composite Colonies

Polyandrous Queen Honey Bee 37

the queen mates with an average of 12 drones. Furthermore, there is a variation in the number of drones that mate with the queen and the average amount of sperm received by her from each drone during mating (Tarpy et al., 2004). A postmating queen honey bee can lay approximately 1500 eggs daily. In a colony, usually a single queen is present during normal times, except for swarming preparation or during supersedure. Worker bees are sterile due to a degenerated reproductive system under the influence of low protein enriched larval food and queen pheromones. Therefore, worker honey bees perform other duties for the colony, which include collection of food, production of honey, protection, brood rearing, retinue behaviour, swarming and other kinds of social interaction (Fig. 2b, c).

Fig. (2b). Click to depict the natural way of nectar sucking by a forager. Honey bees suck sugary syrup and collect pollen from various flowers. Thereafter, workers transport nectar and pollen to the hive by filling in the honey stomach and pollen basket.

Worker bees perform various duties from the day of hatching; thereafter, duties are assigned according to their ages, initially cell cleaning (1-3 days), followed by brood rearing (3–11 days), queen attendant (7–12 days), wax production (12–17 days), foraging, protection, etc. Nurse bees workers control the diversified developmental pattern of the female caste. Workers' differential feeding patterns

38 Polyandrous Queen Honey Bee

Lovleen Marwaha

and larval developmental programming ultimately result in the development of queens and workers. In a hive, there are separate wax cells for the queen to lay fertilized eggs and unfertilized eggs. The queen rearing cell is the largest, followed by intermediatesized drone cells and small-sized worker cells (Fig. 2d). Drones reared in hexagonal wax cells of workers are small and less successful in mating (Seeley 1985; Winston 1989; Strand 1989; Berg et al. 1997; Francis et al., 1998).

Fig. (2c). Foragers taking sugary syrup outside the entrance of a bee hive. Workers prefer liquid sugary foods in comparison to granular food items. Liquid food is somehow correlated with their natural food preference.

Fig. (2d). A small section of a newly constructed hive with hexagonal wax worker cells. Worker honey bees attach eggs to the bottom of hexagonal wax cells.

Composite Colonies

Polyandrous Queen Honey Bee 39

2.1.2. Factors Influencing Queen Reproductive Characteristics In Apis mellifera, a honey bee colony is headed by a single queen, the only reproductive female in the colony. Therefore, the strength of the colony is dependent upon its reproductive potential and other factors (Fig. 2e). Furthermore, different factors which affect the quality of queens include genetic integrity, age of selected larvae for queen raising, social environment within a colony, royal jelly composition, and its quantity. Female caste development within colonies is highly plastic, as queens raised from younger larvae develop into superior quality queens with increased egg laying capacity (Barchuk et al., 2007; de Azevedo and Hartfelder, 2008; Toth et al., 2009). According to Woyke (1971), there is a negative correlation between queen grafting age and the sperm storage capacity of spermathecae. Similarly, Tarpy et al. (2011) demonstrated that queens raised from 0-day-old worker larvae can store more sperm than queens raised from 2-day-old worker larvae. Additionally, the quality of queen production is affected by several factors prevailing within the hive and outside conditions (Ruttner, 1976, 1980;Laidlaw, 1992; Alqarni, 1995; Kaftanoglu et al., 2000; Alqarni et al., 2011; Alghamdi et al., 2012).

Fig. (2e). Elucidation of various factors influencing the strength of a colony A Queen Honey Bee lays a fertilised or unfertilized egg depending upon available wax cells. As for different castes, there is variation in wax cell dimensions.

40 Polyandrous Queen Honey Bee

Lovleen Marwaha

The reproductive potential of a queen is measured by ovariole number and size, spermatheca size, sperm count, sperm viability, and other characteristics. (Woyke, 1971; Eid et al., 1980; Dedej et al., 1998; Hatch et al., 1999; Tarpy et al., 2000, 2004; Al-Abbadi, 2005; Akyol et al., 2008; Tarpy and Mayer, 2009; Delaney et al., 2011; Tarpy et al., 2011). Queen size, including body weight and thorax width, influences mating frequency, spermatheca influences sperm storage, and ovariole number and ovary size influence egg production (Eckert, 1934; Woyke, 1971; Nelson and Gary, 1983; CasagrandeJaloretto et al., 1984; Dedej et al., 1998; Hatch et al., 1999; Gilley et al., 2003; Delaney et al., 2011; Tarpy et al., 2011; Jackson et al., 2011; Alqarni et al., 2013). Queen thoracic width affects mating success and sperm storage capacity, as larger queens can mate with multiple drones, and larger spermathecae can store more sperm (Delaney et al., 2011). Colony health is dependent upon the queen's health, which can be influenced by numerous diseases and additionally by several biotic and abiotic factors. Further, several pests and pathogens can have negative effects on the queen's health. Pathogens and parasites are responsible for poor health and colony losses (Genersch et al., 2010; Cornman et al., 2012; Dainat et al., 2012; McMenamin and Genersch, 2015). Various disease causative agents for honey bees are Varroa Destructor, Deformed Wing Virus (DWV), Israeli Acute Bee Paralysis Virus (IAPV), and Acute Bee Paralysis Virus (ABPV) (Cox-Foster, et al., 2007; Highfield, et al., 2009; Generenz, et al., 2010; De Miranda, et al., 2010a,b; Francis, et al., 2013; McMenamin and Genersch, 2015). Nelson and Smirl (1977) reported that honey bee colonies headed by poor queens exhibited less colony strength and honey production. Similarly, Nelson and Gary (1983) measured the co-relation between honey production and retinue behavior with queen body weight. Furthermore, they concluded that colony strength and brood production are linked with queen weight. Rangel et al. (2013) reported that a queen raised from 0-day-old worker larvae is larger and can store more sperm in her spermathecae than a queen raised from 2-day-old worker larvae. They further reported that a colony headed by a superior-quality queen with more queen-like morphological features shows better growth than a low-quality queen with a worker-like appearance. Queens with worker-like morphology exhibit lower fecundity, fertility, and longevity (Nelson and Smirl, 1977; Nelson and Gary, 1983; Rangel et al., 2013). The above-mentioned studies indicate that a honey bee colony headed by a highquality queen builds stronger hives and stores more honey and pollen in comparison to colonies headed by a low-quality queen. It has been reported that a

Composite Colonies

Polyandrous Queen Honey Bee 41

queen developed from young larvae is larger in size than a queen developed from older worker larvae. Additionally, honey bee colony strength is dependent upon the genetic makeup of the queen, environmental conditions, and bee-keeping practices (Hoopingarner and Farrar, 1959; Merozov et al., 1971; Severson and Erickson, 1989). Matilla and Seeley (2007) reported that apicultural productivity, including swarm production, foraging rate, food storage, and population growth, is higher in genetically variant colonies than in genetically uniform colonies. Sammataro and Weiss (2013) reported that beeswax production is higher in sucrose syrup-fed colonies than in fructose corn syrup-fed colonies. In the honey bee queen, morphometric and reproductive organs are affected by bee race, rearing season, age of grafted larvae, and food availability (Komarov and Alpatov, 1936; Vagt, 1955; Cale, 1963; Avetisyan et al., 1967; Mirza et al., 1967; Pain et al., 1974; Moukayess, 1979; Shawer, 1980; Rawash et al., 1983; Casagrande-Jaloret al., 1984; Woyke, 1987). Worker honey bees exhibit adaptive responses. If the queen is not reproductively active, then workers start laying down eggs that are unfertilized and, therefore, develop into drones. In a specific method, workers contribute genomic contributions for the protection of the colony, before the complete failure of the colony. 2.1.3. Queen Making Decision in the Colony In a honey bee colony, there are group decisions about foraging, nest maintenance, and reproduction (Fig. 2f–2i). Queen rearing is a group decision in which workers collectively decide to raise an unmated queen. Tarpy et al. (2004) studied queen rearing, emergence, and the elimination stage of queen production. In honey, bee gynes produce piping sounds, which affect the emergence of other mature gynes (Simpson and Cherry, 1969; Bruinsma et al., 1981; Schneider and DeGrandi-Hoffman, 2003). A normal honey bee colony rears about 5–25 queens during swarming or queen loss or failure. Queen rearing occurs within larger queen cells, in which honey bee workers provide royal jelly to develop queen larvae and seal wax queen cells about 24 hours before pupae form. The total time duration required for the development of a queen is 15–16 days for honey bees (Roubik, 1989; DeGrandiHoffman and Watkins, 1998). Workers can consider the quality of selected larvae for queen rearing and further modulate the nutrition quality and quantity to direct the specific larval developmental phase (Fig. 2g - i). For queen rearing, usually selected workers, a larva is provided with huge quantities of royal food.

42 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (2f). Photograph of hive section, with uncapped and capped brood. Worker honey bees can be seen adding worker jelly to developing worker larvae. Larvae in different developmental phases can be easily visualized in the clicked hive portion.

Fig. (2g). Photo impression describing uncapped and capped worker cells. Further, worker honey bees can be visualized while adding worker jelly to developing larvae.

Composite Colonies

Polyandrous Queen Honey Bee 43

Fig. (2h). A picture of a strong colony headed by a good-quality queen. In the photo-captured section, larvae of variable instars can be detected. Additionally, nearby honey-filled wax cells can be traced, which worker bees use as larval food or as their food.

Fig. (2i). This image depicts a high-quality brood pattern from a strong colony with high productivity. The wax cells filled with unripe honey and pollen can be located. Both the aforementioned colonial products are provided by worker bees to develop larvae for growth. Additionally, worker bees can be viewed while performing various brood rearing tasks.

44 Polyandrous Queen Honey Bee

Lovleen Marwaha

A few explorations reported that for queen rearing, worker honey bees shake royal cells, with the movement of the body dorso-ventrally for 1-2 seconds at 16 HZ (Fletcher, 1978; Schneider et al., 2001; Schneider and Lewis, 2004). It has been reported that royal jelly cells initiated earlier are visited, incubated, and shaken comparatively more frequently than queen cells developed later (Schneider and De Grandi-Hoffman, 2002). 2.1.4. The Queen Elimination Procedure Multiple queens are typically produced during swarming, and the excess is removed using various methods (Michener, 1974). Therefore, multiple queens can coexist in a hive at a time of colony fission (swarming). The reared gynes are subjected to natural selection. It has been reported that worker honey bees randomly destroy some of the queen cells during the emergency queen rearing process (Hatch et al., 1999; Schneider and De Grandi-Hoffman, 2003). Queen reproductive potential is negatively correlated with the age of larvae when they enter into the queen development phase (Woyke, 1971; Gilley et al., 2003). Furthermore, during queen rearing, workers frequently destroy queen cells with low quality queens (Hatch et al., 1999). Queens can be killed during lethal fights between queens or queen cells can be killed by an early emergent queen (Gilley, 2001). A queen able to survive during early natural selection during rearing can replace her mother to dominate the colony. Workers can also eliminate certain specific reared queens by biasing the outcome of gyne-gyne interactions. According to Roubik (1989), excessive queen production is necessary for the selection of suitable genotypes (Visscher, 1993: Tarpy et al., 2003). Furthermore, regarding reproduction, a virgin queen generally does not lay eggs, but on treatment with CO2, they lay unfertilized eggs, which develop into drones due to haplodiploid sex determination (Mackensen, 1947; Kaftanoglu and Peng, 1982; Winston, 1987; Woyke and Jasinski, 1992). CO2 treatment influences mating behavior, ovary activation, and expression of genes in the brain (Nino et al., 2011). A post-mated queen can also lay unfertilized eggs on depletion of stored sperm (Mackensen, 1947). Additionally, a few reports suggest that workers show differential responses to older egg-laying queens inseminated with high and low volumes of semen (Nino et al., 2012).

Composite Colonies

Polyandrous Queen Honey Bee 45

2.2. QUEEN PHEROMONES 2.2.1. General Information about Queen Pheromones In a colony with a healthy and fecund queen, workers take proper care of the queen, as she acts as a predominant factor in influencing colony strength. Pheromones are categorized into two groups based on induced changes (Fig. 2j). Furthermore, for harmonious social interaction, the queen secretes certain volatile chemicals bouquets including 9-ODA(2E)-9-oxodecenoic acid), 9-HDA (9hydroxy-(E)-2-decenoic acid), 10-HDA (10-hrdroxy-2-decenoic acid), HVA (4hydroxy-3-methoxyphenylethanol), HOB (Methyl-p-hydroxybenzoate), 10HDAA (10-hydroxydecanoic acid), OLA (oligolactide), methyl oleate, decyl decanoate, linolenic acid, coniferyl alcohol, cetyl alcohol, etc. Honey bee queens produce pheromones through multiple glands, among which only two have been appreciably explored; one is the mandibular gland pheromone and the other is Dufourgland (Slessor, et al., 1990; Katzav-Gozansky, et al., 1997a,b; Slessor, et al., 2005).

Fig. (2j). Illustration of a releaser and primer pheromone released by a queen honey bee. Primer pheromones act at a physiological level to induce a long-term response in worker honey bees, whereas releaser pheromones induce weaker effects that induce short-term behavioral changes.

46 Polyandrous Queen Honey Bee

Lovleen Marwaha

2.2.2. Queen Mandibular Gland Pheromones The queen mandibular gland secretes volatile chemicals which are responsible for differential processes. The queen secretes about thirty different chemicals, among which major chemicals are known to induce retinue response in worker honey bees (Hoover et al., 2003; Richard et al., 2007; Kocher et al., 2009). The specific chemicals include (E)-9-oxodec-2-enoic acid (9-ODA), both enantiomers of 9hydroxy-2 (E)-decenoic acid (9-HDA), methyl p-hydroxybenzoate (HOB) and 4hydroxy-3-methoxyphenylethanol (HVA), 10-HDAA (10-hydroxydecanoic acid) (Slessor et al., 1988; Rangel et al., 2016). Queen mandibular pheromonal composition varies with the mating status of the queen (Slessor et al., 1990; Richard et al., 2007; Kocher et al., 2009; Kocher et al., 2010; Nino et al., 2012). Such queens vary in the composition of QMP, including 9-ODA, HOB, HVA, and 9-HDA. The virgin queen secretes more 9ODA than a single drone-inseminated queen, whereas the remaining pheromones of the QMP component remain the same. The physiological characteristics of 9ODA point to its role in drone attraction by virgin queens (Richard et al., 2007). Additionally, the chemical composition of the queen mandibular gland varies with her ovary activation and insemination volume (Slessor et al., 1990; Hoover et al., 2003; Richard, 2007; Kocher, 2009). Queen mandibular pheromones act as honest signals to suppress the development of worker honey bees and to prove her dominance in honey bee colonies (Keller and Nonacs, 1993; Strauss et al., 2008; Heinze and D’Ettorre, 2009; Wright, 2009; van Zweden, 2010; Kocher and Grozinger, 2011; Peso et al., 2012). Workers with a virgin queen have activated ovaries, whereas workers in a queenrighted colony have fewer activated ovaries (Peso et al., 2012). They studied the proportionality of the total 28 components and the activation of workers' ovaries. The chemical composition of the queen pheromone indicates the queen's mating status and, as a result, worker ovarian activation. Worker honey bees are more attracted to multi-drone inseminated queens than queens inseminated with a single drone (Richard et al., 2007; Strauss et al., 2008; Kocher et al., 2009; 2010; Nino et al., 2012). In a single drone inseminated (SDI) queen, colony workers show poorer activation of ovaries than a colony with a virgin queen, which suggests that 9-ODA is not primarily related to ovary activation in worker honey bees. A virgin queen secretes a higher concentration of 9-ODA than a mated queen. Further, worker honey bees are considerably more attracted to naturally mated than virgin or instrumentally inseminated queens (Richard et al., 2007; Kocher et al., 2009, 2010). In other words, QMP composition changes with the mating sta-

Composite Colonies

Polyandrous Queen Honey Bee 47

tus of the queen; therefore, workers elicit differential behavior for specific pheromones (elaborative description is specified in chapters 6 and 7). Below listed are the pheromonal functions of the specific gland (Slessor, et al., 1988; Winston, et al., 1989; Slessor, et al., 1990; Higo, et al 1992; Pankiw, et al., 1998; Grozinger, et al., 2003; Hoover, et al., 2003; Fischer and Grozinger, 2008). • Retinue Behaviour Induction In Workers: Under the influence of queen’s mandibular pheromone, colonial workers surround the queen, antennate, and lick her. • Delaying Workers' Maturity: QMPs delay maturity in the behaviour of worker honey bees to coordinate rearing of brood tasks. • Enhance Fat Content: Specific volatile chemicals enhance the fat content of worker honey bees to enable them to perform hive task accomplishments. • Genetic Expression Modulation: The volatiles change worker brain gene expression, which ultimately affects the development, physiology, and behaviour of workers. • Foraging Activity: QMPs increase foraging activity in QMPs in workers, which enhances colonial productivity. • Swarming: QMPs serve as attractants for workers, which co-ordinate the movement of swarms. • Inhibition of Worker Ovarian Development: Specific chemicals inhibit worker ovarian development. Queen honey bee presence reduces activation of worker honey bees' ovary. In queen-less colonies, there is activation of worker ovaries, and they can become egg-laying workers, which can lay unfertilized eggs. In the case of worker honey bees, pheromones can affect the activation of ovaries. Reproductively active queen and brood reduce the process of activation of worker honey bees' reproductive system (Verheijen-Voogd, 1959; Jay, 1968, 1972; Free, 1987; Ratnieks, 1993; Arnold et al., 1994; Visscher, 1996; Mohammedi et al., 1998; Hoover et al., 2003; Maisonnasse et al., 2009). 2.2.3. Dufour’s Gland In the queen honey bee queen, exudates of specific glands carry mainly tetradecyl hexadecanoate and hexadecyl tetradecanoate. The specific gland performs the

48 Polyandrous Queen Honey Bee

Lovleen Marwaha

following functions (Katzav-Gozansky, et al 2001: Martin, et al., 2002; Nino, et al., 2013): Marking of Eggs: chemical secretion of a specific gland is used to mark fertilized and unfertilized eggs laid by the queen in a colony of honey bees. This has been considered a disputed concept now. At present, a specific gland is now considered to secrete queen signal only ((Katzav-Gozansky, et al., 2001: Martin, et al., 2002; Nino, et al., 2013). Retinue Formation: The specific gland secretion helps in the induction of the retinual behavior. In a queen honey bee queen, the exudates of specific glands carry mainly tetradecyl hexadecanoate and hexadecyl tetradecanoate. The specific gland performs the following functions (Katzav-Gozansky, et al., 2001; Martin, et al., 2002; Nino, et al., 2013): • Marking of Eggs: Chemical secretion of a specific gland is used to mark fertilized and unfertilized eggs laid by the queen in a colony of honey bees. This is considered a disputed concept now. Currently, a specific gland is thought to secrete only a queen signal (Katzav-Gozansky et al., 2001; Martin et al., 2002; Nino et al., 2013). • Retinue Formation: The specific gland secretion helps in the induction of retinual behavior. In the honey bee queen, in addition to the mandibular gland, many other glands also produce various volatile chemicals required for the regulation of worker ovarian development, activation, and functionality. For instance, the tergal gland of the queen secretes certain pheromones required for the attraction of workers and the deactivation of worker honey bee ovaries (Wossler and Crewe, 1999; AlQarni et al., 2005). Queen-produced Dufour’s gland pheromones also facilitate attraction to workers (Wossler and Crewe, 1999b; Katzav-Gozansky et al., 2002; Keeling et al., 2003; Richard et al., 2007; Kocher et al., 2011; Richard et al., 2011), but it is presently unclear if it affects the worker's ovarian activation. The queen secretes chemicals in response to her mating condition, and these chemicals influence the ovarian maturation of workers. The specific topic needs to be fully characterized and will be an exciting future research area (Smith et al., 1993; Wossler and Crewe, 1999a; Katzav-Gozansky et al., 2001; Richard et al., 2011).

Composite Colonies

Polyandrous Queen Honey Bee 49

2.3. CONCLUSION The polyandrous queen honey bee proves her dominance in the colony by reproducing at a high rate and imposing pheromonal regulations on nest residents. Her volatile chemical bouquet further solidifies her monopoly. Worker honey bees exhibit submissive behavior by performing all other duties except for reproduction. Furthermore, workers facilitate the queen’s colony rightness by not rearing another queen, but with the condition that the queen might possess a high fertile potential and strong pheromonal emission. REFERENCES Alqarni, AS, Balhareth, HM & Owayss, AA (2013) Queen morphometric and reproductive characters of Apis mellifera jemenitica, a native honey bee to Saudi Arabia. Bull Insectol, 66, 239-44. Akyol, E, Yeninar, H & Kaftanoglu, O (2008) Live weight of queen honey bees (Apis mellifera L.) predicts reproductive characteristics. J Kans Entomol Soc, 81, 92-100. [http://dx.doi.org/10.2317/JKES-705.13.1] AL-Abbadi, A.A. (2005). Honeybee queen rearing methods and their relation to morphological and physiological characteristics and queen productivity. Ph.D. Thesis, Faculty of Agriculture, Alexandria University, Egypt. Alqarni, A.S. (1995). Morphometrical and biological studies on the native honeybee race, Apis mellifera L.; the Carniolan, A. m. carnica Pollmann and their F1 hybrid.- M.Sc. Thesis, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia. Al-Qarni, AS, Phelan, PL, Smith, BH & Cobey, S (2005) Tergal glandular secretions of naturally mated and istrumentally inseminated honeybee queens (Apis mellifera L.). Journal of King Saud University, 17, 125-37. Arnold, G, Le Conte, Y, Trouiller, J, Hervet, H & Chappe, B (1994) Inhibition of worker honeybee ovaries development by a mixture of fatty acid esters from larvae. Comptes rendus de l'Académie des sciences. Série 3, Sciences de la vie, 317, 511-5. Avetisyan, GA, Rakhmatov, KK & Ziedov, M (1967) Influence of rearing periods on the external and internal characteristics of queen bees 277-84.The 21st International Apiculture Congress of Apimondia, College Park, Maryland, USA Barchuk, AR, Cristino, AS, Kucharski, R, Costa, LF, Simões, ZLP & Maleszka, R (2007) Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera. BMC Dev Biol, 7, 70. [http://dx.doi.org/10.1186/1471-213X-7-70] [PMID: 17577409] Berg, S, Koeniger, N, Koeniger, G & Fuchs, S (1997) Body size and reproductive success of drones (Apis mellifera L). Apidologie (Celle), 28, 449-60. [http://dx.doi.org/10.1051/apido:19970611] Bruinsma, O, Kruijt, JP & van Dusseldorp, W (1981) Delay of emergence of honey bee queens in response to tooting sounds. Proc K Ned Akad Wet Ser C 84, 381-7. Cale, GH (1963) The production of queens, package bees, and royal jelly. The hive and the honeybee 437-62. Casagrande-Jaloretto, DC, Correabueno, O & Stort, AC (1984) Numero de ovariolos em rainhas de Apis mellifera. Naturalia (Sao Jose Rio Preto), 9, 73-9. Corbella, E & Gonçalves, LS (1982) Relationship between weight at emergence, number of ovarioles and spermathecal volume of Africanized honeybee queens (Apis mellifera L). Rev Bras Genet, 5, 835-40. Cornman, RS, Tarpy, DR, Chen, Y, Jeffreys, L, Lopez, D, Pettis, JS, vanEngelsdorp, D & Evans, JD (2012) Pathogen webs in collapsing honey bee colonies. PLoS One, 7e43562

50 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1371/journal.pone.0043562] [PMID: 22927991] Cox-Foster, DL, Conlan, S, Holmes, EC, Palacios, G, Evans, JD, Moran, NA, Quan, PL, Briese, T, Hornig, M, Geiser, DM, Martinson, V, vanEngelsdorp, D, Kalkstein, AL, Drysdale, A, Hui, J, Zhai, J, Cui, L, Hutchison, SK, Simons, JF, Egholm, M, Pettis, JS & Lipkin, WI (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318, 283-7. [http://dx.doi.org/10.1126/science.1146498] [PMID: 17823314] Crozier, RH & Pamilo, P (1996) Evolution of social insect coloniesOxford University Press, New York, NY. Dainat, B, Evans, JD, Chen, YP, Gauthier, L & Neumann, P (2012) Predictive markers of honey bee colony collapse. PLoS One, 7e32151 [http://dx.doi.org/10.1371/journal.pone.0032151] [PMID: 22384162] de Azevedo, SV & Hartfelder, K (2008) The insulin signaling pathway in honey bee (Apis mellifera) caste development — differential expression of insulin-like peptides and insulin receptors in queen and worker larvae. J Insect Physiol, 54, 1064-71. [http://dx.doi.org/10.1016/j.jinsphys.2008.04.009] [PMID: 18513739] DeGrandi-Hoffman, G & Watkins, JC (1998) Queen development time and the Africanization of European honey bees. Am Bee J, 138, 467-9. de Miranda, JR, Cordoni, G & Budge, G (2010) The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex. J Invertebr Pathol, 103 (Suppl. 1), S30-47. [http://dx.doi.org/10.1016/j.jip.2009.06.014] [PMID: 19909972] de Miranda, JR & Genersch, E (2010) Deformed wing virus. J Invertebr Pathol, 103 (Suppl. 1), S48-61. [http://dx.doi.org/10.1016/j.jip.2009.06.012] [PMID: 19909976] Dedej, S, Hartfelder, K, Aumeier, P, Rosenkranz, P & Engels, W (1998) Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee ( Apis mellifera carnica ). J Apic Res, 37, 183-90. [http://dx.doi.org/10.1080/00218839.1998.11100970] Delaney, DA, Keller, JJ, Caren, JR & Tarpy, DR (2011) The physical, insemination, and reproductive quality of honey bee queens (Apis mellifera L.). Apidologie (Celle), 42, 1-13. [http://dx.doi.org/10.1051/apido/2010027] Eckert, JE (1934) Studies in the number of ovarioles in queen honeybees in relation to body size. J Econ Entomol, 27, 629-35. [http://dx.doi.org/10.1093/jee/27.3.629] Fischer, P & Grozinger, CM (2008) Pheromonal regulation of starvation resistance in honey bee workers (Apis mellifera). Naturwissenschaften, 95, 723-9. [http://dx.doi.org/10.1007/s00114-008-0378-8] [PMID: 18414825] Ratnieks, FLW & Keller, L (1998) Queen control of egg fertilization in the honey bee. Behav Ecol Sociobiol, 44, 57-61. [http://dx.doi.org/10.1007/s002650050514] Francis, RM, Nielsen, SL & Kryger, P (2013) Varroa-virus interaction in collapsing honey bee colonies. PLoS One, 8e57540 [http://dx.doi.org/10.1371/journal.pone.0057540] [PMID: 23526946] Fletcher, DJC (1978) Vibration of queen cells by worker honeybees and its relation to the issue of swarms with virgin queens. J Apic Res, 17, 14-26. [http://dx.doi.org/10.1080/00218839.1978.11099894] Free, JB (1987) Pheromones of social beesCornell University Press, Ithaca, New York. Genersch, E, von der Ohe, W, Kaatz, H, Schroeder, A, Otten, C, Büchler, R, Berg, S, Ritter, W, Mühlen, W, Gisder, S, Meixner, M, Liebig, G & Rosenkranz, P (2010) The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Apidologie (Celle), 41, 332-52.

Composite Colonies

Polyandrous Queen Honey Bee 51

[http://dx.doi.org/10.1051/apido/2010014] Gilley, DC (2001) The behavior of honey bees (Apis mellifera ligustica) during queen duels. Ethology, 107, 601-22. [http://dx.doi.org/10.1046/j.1439-0310.2001.00692.x] Gilley, DC, Tarpy, DR & Land, BB (2003) Effect of queen quality on interactions between workers and dueling queens in honeybee ( Apis mellifera L.) colonies. Behav Ecol Sociobiol, 55, 190-6. [http://dx.doi.org/10.1007/s00265-003-0708-y] Grozinger, CM, Sharabash, NM, Whitfield, CW & Robinson, GE (2003) Pheromone-mediated gene expression in the honey bee brain. Proc Natl Acad Sci USA, 100 (Suppl. 2), 14519-25. [http://dx.doi.org/10.1073/pnas.2335884100] [PMID: 14573707] Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Heinze, J & d’Ettorre, P (2009) Honest and dishonest communication in social Hymenoptera. J Exp Biol, 212, 1775-9. [http://dx.doi.org/10.1242/jeb.015008] [PMID: 19482994] Highfield, AC, El Nagar, A, Mackinder, LCM, Noël, LMLJ, Hall, MJ, Martin, SJ & Schroeder, DC (2009) Deformed wing virus implicated in overwintering honeybee colony losses. Appl Environ Microbiol, 75, 7212-20. [http://dx.doi.org/10.1128/AEM.02227-09] [PMID: 19783750] Higo, HA, Colley, SJ, Winston, ML & Slessor, KN (1992) Effects of honey bee (Apis mellifera L.) queen mandibular gland pheromone on foraging and brood rearing. Can Entomol, 124, 409-18. [http://dx.doi.org/10.4039/Ent124409-2] Hoopingarner, R & Farrar, CL (1959) Genetic control of size in queen honey bees. J Econ Entomol, 52, 5478. [http://dx.doi.org/10.1093/jee/52.4.547] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Jackson, JT, Tarpy, DR & Fahrbach, SE (2011) Histological estimates of ovariole number in honey bee queens, Apis mellifera, reveal lack of correlation with other queen quality measures. J Insect Sci, 11, 1-11. [http://dx.doi.org/10.1673/031.011.8201] [PMID: 21870968] Jay, SC (1968) Factors influencing ovary development of worker honeybees under natural conditions. Can J Zool, 46, 345-7. [http://dx.doi.org/10.1139/z68-052] Jay, SC (1972) Ovary development of worker honeybees when separated from worker brood by various methods. Can J Zool, 50, 661-4. [http://dx.doi.org/10.1139/z72-090] Kaftanoglu, O, Akyol, E & Yeninar, H (2000) The effect of juvenile hormone analog on the development time and the quality of queen honeybees (Apis mellifera L. 351-7.Proceedings of the 2nd International Conference of Africanized honey bees and bee mites, Arizona, USA Kaftanoglu, O & Peng, YS (1982) Effects of insemination on the initiation of oviposition in the queen honeybee. J Apic Res, 21, 3-6. [http://dx.doi.org/10.1080/00218839.1982.11100508] Gozansky, TK, Soroker, V & Hefetz, A (1997) The biosynthesis of Dufour’s gland constituents in queens of the honeybee (Apis mellifera). Invert Neurosci, 3, 239-43. [http://dx.doi.org/10.1007/BF02480380]

52 Polyandrous Queen Honey Bee

Lovleen Marwaha

Katzav-Gozansky, T, Soroker, V, Hefetz, A, Cojocaru, M, Erdmann, DH & Francke, W (1997) Plasticity of caste-specific Dufour’s gland secretion in the honey bee (Apis mellifera L.). Naturwissenschaften, 84, 238-41. [http://dx.doi.org/10.1007/s001140050386] Katzav-Gozansky, T, Ibarra, F, Francke, W, Hefetz, A & Soroker, V (2001) Dufour’s gland secretion of the queen honeybee ( Apis mellifera ): an egg discriminator pheromone or a queen signal? Behav Ecol Sociobiol, 51, 76-86. [http://dx.doi.org/10.1007/s002650100406] Katzav-Gozansky, T, Soroker, V & Hefetz, A (2002) Honeybees Dufour’s gland - idiosyncrasy of a new queen signal. Apidologie (Celle), 33, 525-37. [http://dx.doi.org/10.1051/apido:2002035] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Keller, L & Nonacs, P (1993) The role of queen pheromones in social insects: queen control or queen signal? Anim Behav, 45, 787-94. [http://dx.doi.org/10.1006/anbe.1993.1092] Kocher, SD, Ayroles, JF, Stone, EA & Grozinger, CM (2010) Individual variation in pheromone response correlates with reproductive traits and brain gene expression in worker honey bees. PLoS One, 5e9116 [http://dx.doi.org/10.1371/journal.pone.0009116] [PMID: 20161742] Kocher, SD & Grozinger, CM (2011) Cooperation, conflict, and the evolution of queen pheromones. J Chem Ecol, 37, 1263-75. [http://dx.doi.org/10.1007/s10886-011-0036-z] [PMID: 22083225] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2009) Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees. Behav Ecol, 20, 1007-14. [http://dx.doi.org/10.1093/beheco/arp090] [PMID: 22476212] Komarov, PM & Alpatov, WW (1936) Variation of honeybees I. Variation of the body weight and of the reproductive system of the queen racial characteristics. Biological Abstracts, 10, 1234. Laidlaw, HH (1992) Production of queens and package bees. The hive and the honey bee, pp.989-1042. In: The hive and the honey bee (GRAHAM J. H., Ed.).- Dadant & Sons, Hamilton, Illinois, USA. Eid, MAA, Ewies, MA & Nasr, MS (1980) The weight of the newly emerged honeybee queen as an index of its potential productivity. Bulletin of Faculty of Agriculture. University of Cairo, 29, 91-111. Mackensen, O (1947) Effect of carbon dioxide on initial oviposition of artificially inseminated and virgin queen bees. J Econ Entomol, 40, 344-9. [http://dx.doi.org/10.1093/jee/40.3.344] [PMID: 20264497] Maisonnasse, A, Lenoir, JC, Costagliola, G, Beslay, D, Choteau, F, Crauser, D, Becard, JM, Plettner, E & Le Conte, Y (2009) A scientific note on E-β-ocimene, a new volatile primer pheromone that inhibits worker ovary development in honey bees. Apidologie (Celle), 40, 562-4. [http://dx.doi.org/10.1051/apido/2009024] Martin, SJ, Jones, GR, Châline, N, Middleton, H & Ratnieks, FL (2002) Reassessing the role of the honeybee (Apis mellifera) Dufour’s gland in egg marking. Naturwissenschaften, 89, 528-32. [http://dx.doi.org/10.1007/s00114-002-0367-2] [PMID: 12451458] Mattila, HR & Seeley, TD (2007) Genetic diversity in honey bee colonies enhances productivity and fitness. Science, 317, 362-4. [http://dx.doi.org/10.1126/science.1143046] [PMID: 17641199] McMenamin, AJ & Genersch, E (2015) Honey bee colony losses and associated viruses. Curr Opin Insect Sci, 8, 121-9. [http://dx.doi.org/10.1016/j.cois.2015.01.015] [PMID: 32846659]

Composite Colonies

Polyandrous Queen Honey Bee 53

Merozov, AV, Cherevko, YA & Gadelia, IV (1971) Winter rearing of queens and drones under glasshouse condition 317-22.Proceedings of 23rd International Apiculture Congress of Apimondia, Moscow, Russia Michener, CD (1974) The Social Behavior of the BeesHarvard University Press, Cambridge. Mirza, E, Dragan, M & Sherbanescus, S (1967) Seasonal variability in the weight of emerging queens 26973.The 21st International Apiculture Congress of Apimondia, College Park, Maryland, USA Mohammedi, A, Paris, A, Crauser, D & Le Conte, Y (1998) Effect of aliphatic esters on ovary development of queenless bees (Apis mellifera L.). Naturwissenschaften, 85, 455-8. [http://dx.doi.org/10.1007/s001140050531] Moukayess, KI (1979) Studies on the honeybee. Nelson, DL & Gary, NE (1983) Honey productivity of honeybee colonies in relation to body weight, attractiveness and fecundity of the queen. J Apic Res, 22, 209-13. [http://dx.doi.org/10.1080/00218839.1983.11100589] Nelson, DL & Smirl, C (1977) The effect of queen-related problems and swarming on brood and honey production of honey bee colonies in Manitoba. Manitoba Entomol, 11, 45-9. Niño, EL, Malka, O, Hefetz, A, Teal, P, Hayes, J & Grozinger, CM (2012) Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology. J Insect Physiol, 58, 1082-9. [http://dx.doi.org/10.1016/j.jinsphys.2012.04.015] [PMID: 22579504] Niño, EL, Malka, O, Hefetz, A, Tarpy, DR & Grozinger, CM (2013) Chemical profiles of two pheromone glands are differentially regulated by distinct mating factors in honey bee queens (Apis mellifera L.). PLoS One, 8e78637 [http://dx.doi.org/10.1371/journal.pone.0078637] [PMID: 24236028] Niño, EL, Tarpy, DR & Grozinger, C (2011) Genome-wide analysis of brain transcriptional changes in honey bee (Apis mellifera L.) queens exposed to carbon dioxide and physical manipulation. Insect Mol Biol, 20, 387-98. [http://dx.doi.org/10.1111/j.1365-2583.2011.2011.01072.x] [PMID: 21410804] Pain, J, Roger, B & Theurkauff, J (1974) Determination of a seasonal cycle for the content in 9-oxodec and 9-hydroxydec-2-enoic acids in the virgin queen’s heads of bees (Apis mellifera ligustica S.). Apidologie (Celle), 5, 319-55. [http://dx.doi.org/10.1051/apido:19740401] Pankiw, T, Huang, Z, Winston, ML & Robinson, GE (1998) Queen mandibular gland pheromone influences worker honey bee (Apis mellifera L.) foraging ontogeny and juvenile hormone titers. J Insect Physiol, 44, 685-92. [http://dx.doi.org/10.1016/S0022-1910(98)00040-7] [PMID: 12769952] Peso, M, Niño, EL, Grozinger, CM & Barron, AB (2013) Effect of honey bee queen mating condition on worker ovary activation. Insectes Soc, 60, 123-33. [http://dx.doi.org/10.1007/s00040-012-0275-1] Rangel, J, Keller, JJ, Tarpy, DR & Tarpy, DR (2013) The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth. Insectes Soc, 60, 65-73. [http://dx.doi.org/10.1007/s00040-012-0267-1] Ratnieks, FW (1993) Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies. Behav Ecol Sociobiol, 32, 191-8. [http://dx.doi.org/10.1007/BF00173777] Rawash, IA (1983) Effects of larva age and number of queen cell cups on the quality of the Carnio-Egyptian F1 hybrid of honeybee queens 320-6.Proceedings of the 2nd International Conference of Apiculture in Tropical Climates, New Delhi, India Richard, FJ, Schal, C, Tarpy, DR & Grozinger, CM (2011) Effects of instrumental insemination and insemination quantity on Dufour’s gland chemical profiles and vitellogenin expression in honey bee queens

54 Polyandrous Queen Honey Bee

Lovleen Marwaha

(Apis mellifera). J Chem Ecol, 37, 1027-36. [http://dx.doi.org/10.1007/s10886-011-9999-z] [PMID: 21786084] Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2e980 [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Roubik, DW (1992) Ecology and natural history of tropical beesCambridge University Press. Ruttner, F (1976) Les races d’abeilles de l’Afrique, pp. 347367. In: Le 25 Congrés International d’Apiculture Apimondia, Grenoble, France Ruttner, F (1980) Queen rearing, biological bases and technical recommendationApimondia Publishing House, Bucharest, Romania. Sammataro, D & Weiss, M (2013) Comparison of productivity of colonies of honey bees, Apis mellifera, supplemented with sucrose or high fructose corn syrup. J Insect Sci, 13, 1-13. [http://dx.doi.org/10.1673/031.013.1901] [PMID: 23886010] Schneider, SS & Degrandi-Hoffman, G (2003) The influence of paternity on virgin queen success in hybrid colonies of European and African honeybees. Anim Behav, 65, 883-92. [http://dx.doi.org/10.1006/anbe.2003.2133] Schneider, SS, Painter-Kurt, S & Degrandi-Hoffman, G (2001) The role of the vibration signal during queen competition in colonies of the honeybee, Apis mellifera. Anim Behav, 61, 1173-80. [http://dx.doi.org/10.1006/anbe.2000.1689] Seeley, TD (1985) Honeybee ecologyPrinceton University Press, Princeton, NJ. [http://dx.doi.org/10.1515/9781400857876] Seeley, TD (1995) The Wisdom of the Hive: The Social Physiology of Honey Bee ColoniesHarvard University Press, Cambridge, MA. [http://dx.doi.org/10.4159/9780674043404] Severson, DW & Erickson, EH, Jr (1989) Seasonal constraints on mating and insemination of queen honey bees in a continental climate. Apidologie (Celle), 20, 21-7. [http://dx.doi.org/10.1051/apido:19890103] Shawer, MB (1980) Simpson, J & Cherry, SM (1969) Queen confinement, queen piping and swarming in Apis mellifera colonies. Anim Behav, 17, 271-8. [http://dx.doi.org/10.1016/0003-3472(69)90012-8] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Kaminski, LA, King, GGS & Winston, ML (1990) Semiochemicals of the honeybee queen mandibular glands. J Chem Ecol, 16, 851-60. [http://dx.doi.org/10.1007/BF01016495] [PMID: 24263600] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Smith, RK, Spivak, M, Taylor, OR, Jr, Bennett, C & Smith, ML (1993) Maturation of tergal gland alkene profiles in European honey bee queens,Apis mellifera L. J Chem Ecol, 19, 133-42. [http://dx.doi.org/10.1007/BF00987478] [PMID: 24248518] Strand, MR (1989) Oviposition behavior and progeny allocation of the polyembryonic waspCopidosoma floridanum (Hymenoptera: Encyrtidae). J Insect Behav, 2, 355-69. [http://dx.doi.org/10.1007/BF01068061]

Composite Colonies

Polyandrous Queen Honey Bee 55

Strauss, K, Scharpenberg, H, Crewe, RM, Glahn, F, Foth, H & Moritz, RFA (2008) The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent? Behav Ecol Sociobiol, 62, 1523-31. [http://dx.doi.org/10.1007/s00265-008-0581-9] Tarpy, DR (2003) Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth. Proc Biol Sci, 270, 99-103. [http://dx.doi.org/10.1098/rspb.2002.2199] [PMID: 12596763] Tarpy, DR & Gilley, DC (2004) Group decision making during queen production in colonies of highly eusocial bees. Apidologie (Celle), 35, 207-16. [http://dx.doi.org/10.1051/apido:2004008] Tarpy, DR, Gilley, DC & Seeley, TD (2004) Levels of selection in a social insect: a review of conflict and cooperation during honey bee ( Apis mellifera ) queen replacement. Behav Ecol Sociobiol, 55, 513-23. [http://dx.doi.org/10.1007/s00265-003-0738-5] Tarpy, DR, Hatch, S & Fletcher, DJC (2000) The influence of queen age and quality during queen replacement in honeybee colonies. Anim Behav, 59, 97-101. [http://dx.doi.org/10.1006/anbe.1999.1311] [PMID: 10640371] Tarpy, DR, Keller, JJ, Caren, JR & Delaney, DA (2011) Experimentally induced variation in the physical reproductive potential and mating success in honey bee queens. Insectes Soc, 58, 569-74. [http://dx.doi.org/10.1007/s00040-011-0180-z] Tarpy, DR & Mayer, MK (2009) The effects of size and reproductive quality on the outcomes of duels between honey bee queens ( Apis mellifera L.). Ethol Ecol Evol, 21, 147-53. [http://dx.doi.org/10.1080/08927014.2009.9522503] Tarpy, DR, Nielsen, R & Nielsen, DI (2004) A scientific note on the revised estimates of effective paternity frequency in Apis. Insectes Soc, 51, 203-4. [http://dx.doi.org/10.1007/s00040-004-0734-4] Tarpy, DR & Seeley, TD (2006) Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens. Naturwissenschaften, 93, 195-9. [http://dx.doi.org/10.1007/s00114-006-0091-4] [PMID: 16518641] Toth, AL, Bilof, KBJ, Henshaw, MT, Hunt, JH & Robinson, GE (2009) Lipid stores, ovary development, and brain gene expression in Polistes metricus females. Insectes Soc, 56, 77-84. [http://dx.doi.org/10.1007/s00040-008-1041-2] Vagt, M (1955) Morphological investigations on emergency queens of bee reared from larvae of different ages. Bee World, 41, 23. van Zweden, JS (2010) The evolution of honest queen pheromones in insect societies. Commun Integr Biol, 3, 50-2. [http://dx.doi.org/10.4161/cib.3.1.9655] [PMID: 20539783] Verheijen-Voogd, C (1959) How worker bees perceive the presence of their queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 41, 527-82. [http://dx.doi.org/10.1007/BF00299267] Visscher, PK (1996) Reproductive conflict in honey bees: a stalemate of worker egg-laying and policing. Behav Ecol Sociobiol, 39, 237-44. [http://dx.doi.org/10.1007/s002650050286] Visscher, PK (1993) A theoretical analysis of individual interests and intracolony conflict during swarming of honey bee colonies. J Theor Biol, 165, 191-212. [http://dx.doi.org/10.1006/jtbi.1993.1185] [PMID: 8246516] Wilson, EO (1971) The Insect SocietiesHarvard University Press, Cambridge, MA. Winston, M (1987) The Biology of the Honey BeeHarvard University Press, Boston.

56 Polyandrous Queen Honey Bee

Lovleen Marwaha

Winston, ML, Slessor, KN, Willis, LG, Naumann, K, Higo, HA, Wyborn, MH & Kaminski, LA (1989) The influence of queen mandibular pheromones on worker attraction to swarm clusters and inhibition of queen rearing in the honey bee (Apis mellifera L.). Insectes Soc, 36, 15-27. [http://dx.doi.org/10.1007/BF02225877] Wossler, TC & Crewe, RM (1999) Honeybee queen tergal gland secretion affects ovarian development in caged workers. Apidologie (Celle), 30, 311-20. [http://dx.doi.org/10.1051/apido:19990407] Woyke, J (1987) Can the number of ovarioles in the ovaries been estimated by external characters of living queens 152-5.The 31st International Apicultural Congress, 19-25 August 1987Warsaw, Poland Woyke, J (1971) Correlations between the age at which honeybee brood was grafted, characteristics of the resultant queens, and results of insemination. J Apic Res, 10, 45-55. [http://dx.doi.org/10.1080/00218839.1971.11099669] Woyke, J & Jasinski, Z (1992) Natural mating of instrumentally inseminated queen bees [http://dx.doi.org/10.1051/apido:19920305] Wright, GA (2009) Bee pheromones: signal or agent of manipulation? Curr Biol, 19, R547-8. [http://dx.doi.org/10.1016/j.cub.2009.05.032] [PMID: 19640487]

Polyandrous Queen Honey Bee, 2022, 57-66

57

CHAPTER 3

The Queen Honey Bee Morphology, Development, and Reproductive System Abstract: The queen bee carries the same genetic information as worker bees. Still, the genomic expression is variable, eventually resulting in the development of an enormously sizeable female bee, with an enriched blend of pheromone possession, a comparatively long life span, better immunity, development, and physiology. Differential developmental patterns compared to the workers are due to the influence of royal jelly, ultimately inducing differential genomic expression. Furthermore, with profound pheromone secretion, the queen regulates the colony's development, differentiation, reproducibility, behaviour, communication, and task management. This chapter briefly describes honey bees' morphology, development, and reproductive system development.

Keywords: Anatomy, Physiology, Queen’s Morphology. 3.1. INTRODUCTION The queen honey bee differs from other castes of the colony by morphology, anatomy, physiology, development, reproduction, life span, and her role in the colony. The queen's predominant role is to reproduce and secrete queen substances. In contrast, workers perform various duties, including taking care of the brood, nest maintenance, defence, queen care, and foraging. Nurse honey bees regulate the developmental pattern of nest mates by controlling larval diet type, quality, and quantity. The size, ovariole number, spermathecae diameter, sperm count, and sperm viability of queens vary (Woyke, 1971; Dedej et al., 1998; Hatch et al., 1999; Tarpy et al., 2000; Tarpy and Mayer, 2009; Delaney et al., 2011; Tarpy et al., 2011). Queen body size is related to mating frequency, sperm storage, ovariole number, and ovary weight (Delaney et al., 2011; Tarpy et al., 2011; Woyke, 1971; Eckert, 1934; Jackson et al., 2011; Dedej et al., 1998; Hatch et al., 1999; Rangel et al., 2013; Richardson et al., 2018; De Souza et al., 2019; Richard et al., 2007). Queen honey bee morphology and reproductive organ development are affected by factors like bee race, rearing season, age of grafted larvae, and food availability Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

58 Polyandrous Queen Honey Bee

Lovleen Marwaha

(Moukayess, 1979; Woyke, 1987; Cale, 1963). Therefore, the quality of the queen is affected by various factors (Alqarni et al., 2011). 3.2. MORPHOLOGY OF THE QUEEN A detailed study by Sarhan et al., 2019 indicated that in Apis mellifera jemenitica(A. m. jemenitica), the average weights of virgin and mated queens are 0.139 ± 0.01 g and 0.143 ± 0.013 g, respectively. Further, they had speculated that the average fresh weight of A. m. jemenitica queen in queen-right and queen-less conditions was 0.136 ± 0.01 g and 0.141 ± 0.01 g, respectively. They further reported that the number of ovarioles in A. m. jemenitica virgin queens was 124 to 163, with a mean of 142.9 ± 9.47. In virgin and mated queens, they reported the size of spermathecae as 1.248 ± 0.103 mm and 1.25 ± 0.022 mm, respectively, whereas the mean weight of ovaries was 0.013 ± 0.003 g. The average number of stored sperm per spermathecae in mated queens was 4.202 ± 0.613 million, with the average number of sperm per drone recorded was 8,763,950 ± 1,633,203.15 with the viability of 79.54 ± 6.70%. The queen's head capsule's average dimension was 3.51 ± 0.23 mm. The queen’s right forewing length and width were 9.361 ± 0.41 mm and 3.217 ± 0.20 mm, respectively. Other morphological traits are considered indicators of queen quality, as the queen's weight correlates with the volume of spermatheca (Akyol et al., 2008). In contrast, other reports indicate that other factors like organ variation like ovariole number are correlated with other morphometric traits like queen size, body weight, and thorax width (Hatch et al., 1999). Queen characters indicate the quality of the queen. Akyol et al. (2008) correlated body size with spermathecal diameter. Further, they reported no correlation between ovariole number, thoracic width, wing length, wet weight, and ovariole number, which range from 100 to 180 in A. mellifera queens. Queen Ovariole's number is affected by the honey bee race (Corbella and Goncalves, 1982). The grafting stage of a bee further influences the spermathecal volume, as it is 1.18 mm3 when eggs are grafted, whereas, for 3-day-old larvae, it corresponds to 0.82 mm3 (Woyke, 1987). Almehmadi et al. (2011) reported that in the 3rd and 5th larval instar, in 1, 2, and 3-day-old pupae, along with newly emerged queens, several differences have been reported (Almehmadi et al., 2011). Furthermore, they said that queen ovaries grow continuously and become differentiated after the 5th instar larval and pupal stages. Queen honey bees possess well-developed 200–400 ovarioles, mandibles with a notch, no corbicula, and a round head, whereas workers possess 2–12 ovarioles, reduced spermathecae, a smooth mandible, and a triangular head. Queen honey bees weigh about 150–250 mg and have a large abdomen, ovaries with a

Development and Reproductive

Polyandrous Queen Honey Bee 59

comparatively large number of ovarian filaments, and large spermatheca (Snodgrass, 2018). In contrast, worker honey bees possess small ovarioles, hypopharyngeal glands, wax glands, notched mandibles, and pollen baskets. In addition, worker honey bees usually do not lay eggs in the presence of a reproductively active queen (Ruttner, 1983). Multiple traits have been used to discriminate between female castes, including ovariole number, mandible shape, and stinger shape. According to Ruttner (1983), using morphometry, different castes can be distinguished based on the size of their heads, mandibles, and basitarsus (DeSouza et al., 2015). The Queen honey bee’s morphometric and reproductive development is influenced by genomic integrity, rearing condition, grafted larvae age, and food availability (Alqarni et al., 2013). In other words, the quality of the queen bee’s honey is influenced by the above-described factors. According to some studies, there is no correlation between ovariole number and queen size, body weight, and thorax width (Casagrande-Jaloretto et al., 1984; Hatch et al., 1999). 3.3. DEVELOPMENT OF THE QUEEN HONEY BEE The female castes of Apis mellifera develop from a fertilised egg, either into queens or workers, depending on the larvae's diet. The development of a queen and a worker begins with diploid eggs, but the phenotype is determined by the diet of developing larvae (Hadak, 1970; Michener, 1974; Page and Peng, 2001). Only royal jelly is given to all larval castes for the queen, workers, and drones for the first three days of larvae development. Royal jelly is secreted by worker honey bees' hypopharyngeal glands and mandibular glands. After three days of larval development, differential diets given to queen and worker larvae induce final discrimination in further development. Queen larvae are fed on royal jelly throughout the developmental phases, whereas worker larvae are shifted to worker jelly after three days of larval developmental stages (Dietz and Haydak, 1971; Graham, 2006). A queen can be raised by workers grafting larvae developed from fertilised eggs before the first three days of larval development. If grafting of worker larvae is done after the 4th day of action, the formed queen will possess a small body with reduced ovarioles and a small spermatheca (Weaver, 1957; Woyke, 1971; Dedej et al., 1998; Tarpy et al., 2011). A larval diet somehow affects the development of phenotypes in the female caste of honey bees. The differential composition of worker jelly influences the development of ovaries in female castes (Wang et al., 2018). Therefore, it could be concluded that the queen and worker caste development begins after three days of action, as experimental data demonstrates the specific observation that developmental differentiation starts after three days of larval development.

60 Polyandrous Queen Honey Bee

Lovleen Marwaha

Queen development is correlated with her reproductive development, which indirectly influences the colony's strength, growth and productivity, disease resistance, and overwintering ability. Ovarian development measures the queen's reproductive capability, the size of spermathecae, and the queen's morphology (Hoopingarner and Farrar (1959; Woyke, 1971; Tarpy et al., 2011; De Souza et al., 2013; Rangel et al., 2013). The queen's morphology is considered an essential indicator of queen quality, which regulates the strength and productivity of the colony. Recently, many in vitro protocols have been available that control the development of queen-like and worker-like morphology (Patel et al., 2007; Mutti et al., 2011; Kaftanoglu et al., 2011; Crailsheim et al., 2013). However, in the absence of social control in an artificial environment, it affects the normal development of animals (Linksvayer et al., 2011; Leimar et al., 2012; De Souza et al., 2015). Honey bees reared under in vitro conditions exhibit differential developmental patterns, including queen worker-like or intercaste phenotypes. Intercaste has intermediate morphology regarding body size, mandible shape, hind leg characteristics, ovariole number, and spermatheca presence or absence (De Souza et al., 2015). De Souza et al. (2018) reported differences between in vitro and naturally occurring queens in terms of morphology, physiology, and development and concluded that the vitellogenin gene and MRJP1 are expressed differentially in both cases (Engels et al., 1990; Amdam and Omholt, 2003; Guidugli et al., 2005; Seehuus et al., 2006; CruzLandim, 2009). De Souza et al. (2018) concluded that in vitro developed queen-like or worker-like bees differ from naturally occurring queens and workers concerning body weight, ovariole number, and spermatheca size. 3.4. OVARIAN STRUCTURAL INTEGRITY (3A-B) Insect ovaries are composed of several ovarioles, representing a serially organised functional unit in which the oocyte forms from stem cells to fully chlorinated eggs. The queen honey bee's reproductive system includes ovaries, oviducts, spermathecae, and vaginas. A pair of ovaries in a queen honey bee forms large gourd-shaped masses with an enlarged posterior or basal end. In contrast, the anterior ovarian end is narrow, curved, and attached. As a queen lays eggs continuously throughout her life, her ovaries contain eggs of all stages. The ovarioles of adult honey bee workers exhibit successive developmental stages of oogenesis from tip to base. Ovarioles can be divided into regions depending on cell type and location of germ cell development. Brief description: the presence of

Development and Reproductive

Polyandrous Queen Honey Bee 61

various areas of an ovariole includes the following: • Terminal Filament • Germarium • Vitellarium • Pedicel Terminal Filament: Terminal filament contains a stack of aligned disc-shaped cells oriented transversely, and the filament portion contains cells of somatic and germinal origin (Tanaka and Hartfelder, 2004). Germarium: In the germarium region, there are undifferentiated germ cells known as oogonia, whereas in the mid-germarium area, early oocytes become surrounded by approximately 39–59 trophoblast nurse cells. Vitellarium: At the vitellarium stage, developing oocytes become enlarged and encapsulated by a monolayer of columnar follicle cells, which are somatic. Nurse cells possess massive nuclei compared to the cytoplasm, which indicates substantial transcriptional activity (Gutzeit et al., 1993). Pedicel: The terminal portion of each ovariole is called a pedicel, having a somatic origin. Pedicels act as plugs to the opening of the oviduct and undergo degeneration during egg laying (Buning, 1994).

Fig. (3a). Reproductive system of a queen bee with different associated organs.

62 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (3b). Reproductive System of a Queen Honey bee from the lateral side.

Conclusion: The previous description shows that due to differential larval nutrition, a variant genomic expression occurs in the queen honey bee, which results in specific anatomy, morphology, physiology, reproducibility, immunity, pheromone profile, life span, and overall role in the colony. The queen bee’s enormously developed reproductive system empowers her to modulate colony strength and dominate the honey bee colony. REFERENCES Akyol, E, Yeninar, H, Korkmaz, A & Çakmak, İ (2008) An observation study on the effects of queen age on some characteristics of honey bee colonies. Ital J Anim Sci, 7, 19-25. [http://dx.doi.org/10.4081/ijas.2008.19] Almehmadi, RM, Alghamdi, AA, Wongsiri, S, Chanchao, C & Aljedani, DM (2011) Histological studies on ovary differentiation in Yemini queen honeybees, Apis mellifera jemenitica (Hymenoptera: Apidae), during post-embryonic development. Pan-Pac Entomol, 87, 177-87. [http://dx.doi.org/10.3956/2011-08.1] Alqarni, AS, Balhareth, HM & Owayss, AA (2013) Queen morphometric and reproductive characters of Apis mellifera jemenitica, a native honey bee to Saudi Arabia. Bull Insectol, 66, 239-44. Alqarni, AS, Hannan, MA, Owayss, AA & Engel, MS (2011) The indigenous honey bees of Saudi Arabia (Hymenoptera, Apidae, Apis mellifera jemenitica Ruttner): Their natural history and role in beekeeping. ZooKeys, 83-98. [PMID: 22140343] Al-Sarhan, R, Adgaba, N, Tadesse, Y, Alattal, Y, Al-Abbadi, A, Single, A & Al-Ghamdi, A (2019) Reproductive biology and morphology of Apis mellifera jemenitica (Apidae) queens and drones. Saudi J Biol Sci, 26, 1581-6. [http://dx.doi.org/10.1016/j.sjbs.2018.10.012] [PMID: 31762630] Amdam, GV & Omholt, SW (2003) The hive bee to forager transition in honeybee colonies: the double repressor hypothesis. J Theor Biol, 223, 451-64.

Development and Reproductive

Polyandrous Queen Honey Bee 63

[http://dx.doi.org/10.1016/S0022-5193(03)00121-8] [PMID: 12875823] Cale, GH, Gowen, JW & Carlile, WR (1963) Pink an eye color and viability gene in honey bees. J Hered, 54, 163-6. [http://dx.doi.org/10.1093/jhered/54.4.163] Casagrande-Jaloretto, DC, Correa, BO & Stort, AC (1984) Ovariole number in queens of Apis mellifera. Naturalia (Sao Jose Rio Preto), 9, 73-9. Corbella, E & Gonçalves, LS (1982) Relationship between weight at emergence, number of ovarioles and spermathecal volume of Africanized honey bee queens Apis-mellifera L). Rev Bras Genet, 5, 835-40. Crailsheim, K, Brodschneider, R, Aupinel, P, Behrens, D, Genersch, E, Vollmann, J & Riessberger-Gallé, U (2013) Standard methods for artificial rearing of Apis mellifera larvae. J Apic Res, 52, 1-16. [http://dx.doi.org/10.3896/IBRA.1.52.1.05] da Cruz-Landim, C & Poiani, SB (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae). Anim Biol Leiden Neth, 59, 299-311. [http://dx.doi.org/10.1163/157075609X454935] De Souza, DA, Kaftanoglu, O, De Jong, D, Page, RE, Amdam, GV & Wang, Y (2018) Differences in the morphology, physiology and gene expression of honey bee queens and workers reared in vitro versus in situ. Biol Open, 7, bio.036616. [http://dx.doi.org/10.1242/bio.036616] De Souza, DA, Kaftanoglu, O, De Jong, D, Page, RE, Jr, Amdam, GV & Wang, Y (2018) Differences in the morphology, physiology and gene expression of honey bee queens and workers reared in vitro versus in situ. Biol Open, 7, bio.036616. [http://dx.doi.org/10.1242/bio.036616] [PMID: 30341101] De Souza, DA, Wang, Y, Kaftanoglu, O, De Jong, D, Amdam, GV, Gonçalves, LS & Francoy, TM (2015) Morphometric identification of queens, workers and intermediates in in vitro reared honey bees (Apis mellifera). PLoS One, 10, e0123663. [http://dx.doi.org/10.1371/journal.pone.0123663] [PMID: 25894528] de Souza, FS, Kevill, JL, Correia-Oliveira, ME, de Carvalho, CAL & Martin, SJ (2019) Occurrence of deformed wing virus variants in the stingless bee Melipona subnitida and honey bee Apis mellifera populations in Brazil. J Gen Virol, 100, 289-94. [http://dx.doi.org/10.1099/jgv.0.001206] [PMID: 30628883] Dedej, S, Hartfelder, K, Aumeier, P, Rosenkranz, P & Engels, W (1998) Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee ( Apis mellifera carnica ). J Apic Res, 37, 183-90. [http://dx.doi.org/10.1080/00218839.1998.11100970] Delaney, DA, Keller, JJ, Caren, JR & Tarpy, DR (2011) The physical, insemination, and reproductive quality of honey bee queens (Apis mellifera L.). Apidologie (Celle), 42, 1-13. [http://dx.doi.org/10.1051/apido/2010027] Dietz, A & Haydak, MH (1971) Caste determination in honey bees. I. The significance of moisture in larval food. J Exp Zool, 177, 353-7. [http://dx.doi.org/10.1002/jez.1401770309] [PMID: 5096132] Eckert, JE (1934) Studies in the number of ovarioles in queen honeybees in relation to body size. J Econ Entomol, 27, 629-35. [http://dx.doi.org/10.1093/jee/27.3.629] Engels, W & Imperatriz-Fonseca, VL (1990) Caste development, reproductive strategies, and control of fertility in honey bees and stingless bees. Social insects, Springer, Berlin, Heidelberg 167-230. [http://dx.doi.org/10.1007/978-3-642-74490-7_9] Gilley, DC, Tarpy, DR & Land, BB (2003) Effect of queen quality on interactions between workers and dueling queens in honeybee ( Apis mellifera L.) colonies. Behav Ecol Sociobiol, 55, 190-6.

64 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1007/s00265-003-0708-y] Graham, JM (1992) The Hive and the Honey BeeDadant & Sons, Inc., Hamilton, IL. Graham, S, Myerscough, MR, Jones, JC & Oldroyd, BP (2006) Modelling the role of intracolonial genetic diversity on regulation of brood temperature in honey bee (Apis mellifera L.) colonies. Insectes Soc, 53, 22632. [http://dx.doi.org/10.1007/s00040-005-0862-5] Guidugli, KR, Nascimento, AM, Amdam, GV, Barchuk, AR, Omholt, S, Simões, ZLP & Hartfelder, K (2005) Vitellogenin regulates hormonal dynamics in the worker caste of a eusocial insect. FEBS Lett, 579, 4961-5. [http://dx.doi.org/10.1016/j.febslet.2005.07.085] [PMID: 16122739] Gutzeit, HO, Zissler, D & Fleig, R (1993) Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles. Rouxs Arch Dev Biol, 202, 181-91. [http://dx.doi.org/10.1007/BF00365309] [PMID: 28305996] Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Haydak, MH (1970) Honey bee nutrition. Annu Rev Entomol, 15, 143-56. [http://dx.doi.org/10.1146/annurev.en.15.010170.001043] Hoopingarner, R & Farrar, CL (1959) Genetic control of size in queen honey bees. J Econ Entomol, 52, 5478. [http://dx.doi.org/10.1093/jee/52.4.547] Jackson, JT, Tarpy, DR & Fahrbach, SE (2011) Histological estimates of ovariole number in honey bee queens, Apis mellifera, reveal lack of correlation with other queen quality measures. J Insect Sci, 11, 1-11. [http://dx.doi.org/10.1673/031.011.8201] [PMID: 21870968] Kaftanoglu, O, Linksvayer, TA & Page, RE, Jr (2011) Rearing honey bees, Apis mellifera, in vitro 1: effects of sugar concentrations on survival and development. J Insect Sci, 11, 96. [PMID: 22208776] Leimar, O, Hartfelder, K, Laubichler, MD & Page, RE, Jr (2012) Development and evolution of caste dimorphism in honeybees - a modeling approach. Ecol Evol, 2, 3098-109. [http://dx.doi.org/10.1002/ece3.414] [PMID: 23301175] Lindauer, M (1952) Ein Beitrag zur Frage der Arbeitsteilung im Bienenstaat. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 34, 299-345. [http://dx.doi.org/10.1007/BF00298048] Linksvayer, TA, Kaftanoglu, O, Akyol, E, Blatch, S, Amdam, GV & Page, RE, Jr (2011) Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism. J Evol Biol, 24, 1939-48. [http://dx.doi.org/10.1111/j.1420-9101.2011.02331.x] [PMID: 21696476] Michener, CD (1974) The Social Behaviour of Bees: A Comparative StudyBelknap Press of Harvard University Press, Cambridge. Moukayess, KI (1979) Studies on the honeybee [anatomical, histological. Mutti, N.S., Dolezal, A.G., Wolschin, F., Mutti, J.S., Gill, K.S. and Amdam, G.V., 2011. IRS and TOR nutrient-signalling pathways act via juvenile hormones to influence honey bee caste fate. J Exp Biol, 214, 3977-84. Page, RE, Jr & Peng, CYS (2001) Aging and development in social insects with emphasis on the honey bee, Apis mellifera L. Exp Gerontol, 36, 695-711. [http://dx.doi.org/10.1016/S0531-5565(00)00236-9] [PMID: 11295509] Patel, A, Fondrk, MK, Kaftanoglu, O, Emore, C, Hunt, G, Frederick, K & Amdam, GV (2007) The making of a queen: TOR pathway is a key player in diphenic caste development. PLoS One, 2, e509.

Development and Reproductive

Polyandrous Queen Honey Bee 65

[http://dx.doi.org/10.1371/journal.pone.0000509] [PMID: 17551589] R. E. SNODGRASS.THE ANATOMY OF THE HONEY BEE.134-140. RamziAl-Sarhan NuruAdgaba YilmaTadesse YehyaAlattal AmalAl Abbadi ArifSingle AhmadAl-Ghamdi Reproductive biology and morphology of Apis mellifera jemenitica (Apidae) queens and drones Rangel, J, Keller, JJ & Tarpy, DR (2013) The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth. Insectes Soc, 60, 65-73. [http://dx.doi.org/10.1007/s00040-012-0267-1] Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2, e980. [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Richardson, RT, Ballinger, MN, Qian, F, Christman, JW & Johnson, RM (2018) Morphological and functional characterization of honey bee, Apis mellifera, hemocyte cell communities. Apidologie (Celle), 49, 397-410. [http://dx.doi.org/10.1007/s13592-018-0566-2] Ruttner, F (1983) Queen Rearing: Biological Basis and Technical InstructionApimondia Publishing House, Bucharest, Romania. Ruttner, F & Maul, V (1983) Experimental analysis of reproductive interspecies isolation of Apis mellifera L. and Apis cerana Fabr. Apidologie (Celle), 14, 309-27. [http://dx.doi.org/10.1051/apido:19830405] Seehuus, SC, Krekling, T & Amdam, GV (2006) Cellular senescence in honey bee brain is largely independent of chronological age. Exp Gerontol, 41, 1117-25. [http://dx.doi.org/10.1016/j.exger.2006.08.004] [PMID: 17052880] Snodgrass, RE (2018) Anatomy of the honey beeCornell University Press. De Souza, DA, Bezzera-Laure, MAF, Francoy, TM & Gonçalves, LS (2013) Experimental evaluation of the reproductive quality of Africanized queen bees (Apis mellifera) on the basis of body weight at emergence. Genet Mol Res, 12, 5382-91. [http://dx.doi.org/10.4238/2013.November.7.13] [PMID: 24301910] Tanaka, ED & Hartfelder, K (2004) The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropod Struct Dev, 33, 431-42. [http://dx.doi.org/10.1016/j.asd.2004.06.006] [PMID: 18089049] Tarpy, DR & Mayer, MK (2009) The effects of size and reproductive quality on the outcomes of duels between honey bee queens ( Apis mellifera L.). Ethol Ecol Evol, 21, 147-53. [http://dx.doi.org/10.1080/08927014.2009.9522503] Tarpy, DR, Hatch, S & Fletcher, DJC (2000) The influence of queen age and quality during queen replacement in honeybee colonies. Anim Behav, 59, 97-101. [http://dx.doi.org/10.1006/anbe.1999.1311] [PMID: 10640371] Tarpy, DR, Keller, JJ, Caren, JR & Delaney, DA (2011) Experimentally induced variation in the physical reproductive potential and mating success in honey bee queens. Insectes Soc, 58, 569-74. [http://dx.doi.org/10.1007/s00040-011-0180-z] Wang, H & Yi, JH (2018) An improved optimization method based on krill herd and artificial bee colony with information exchange. Memet Comput, 10, 177-98. [http://dx.doi.org/10.1007/s12293-017-0241-6] Weaver, N (1957) Effects of larval age on dimorphic differentiation of the female honey bee. Ann Entomol Soc Am, 50, 283-94. [http://dx.doi.org/10.1093/aesa/50.3.283] Woyke, J (1971) Correlations between the age at which honeybee brood was grafted, characteristics of the resultant queens, and results of insemination. J Apic Res, 10, 45-55.

66 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1080/00218839.1971.11099669] Woyke, J (1987) Length of stay of the parasitic mite Tropilaelaps clareae outside sealed honeybee brood cells as a basis for its effective control. J Apic Res, 26, 104-9. [http://dx.doi.org/10.1080/00218839.1987.11100745]

Polyandrous Queen Honey Bee, 2022, 67-82

67

CHAPTER 4

Royal Jelly as Larval Food for Honey Bees Abstract: Larval feeds for different castes of honey bees include exclusively royal jelly from 4–9 days of development for the queen, and for worker larvae, royal jelly and worker jelly for 4-6 and 6–9 days respectively, whereas for drone larvae, royal jelly and a blended composite mixture of honey and pollen grain for 4-6 and 6–9 days respectively. For the queen, worker, and drone larvae, larval feeds include royal jelly and worker jelly for 4-6 and 6–9 days respectively. Royal jelly is a thick, creamy substance that is produced by the hypopharyngeal and mandibular glands of worker honey bees. Its primary components include water, hydrocarbons, proteins, lipids, minerals, vitamins, and a small amount of various types of polyphenols. Because the queen eats different larvae than the worker bees, this triggers a chain reaction of biochemical reactions, which ultimately leads to a high concentration of juvenile and ecdysone hormones being released. These hormones, in turn, regulate the expression of different genes in a sequential manner. Queen larvae have a variant proteomic that promotes the healthy development of the female reproductive system, which in turn leads to profound fertility and immune protection, as well as a longer life span for the queen.

Keywords: Composition, Honey bees, Royal jelly, Worker jelly. 4.1. INTRODUCTION 4.1.1. Information That Is General Regarding Royal Jelly Royal Jelly, also known as RJ, is a thick fluid that is packed with protein and is secreted by the hypopharyngeal and mandibular glands of worker Apis mellifera honey bees. It is a protein-rich product that is produced by worker honey bees, and it is given to larvae of all different castes for the first three days. This product is responsible for the beginning stages of larval development, and it is given to larvae of all different castes. After that, the worker and drone larvae are fed a mixture of worker jelly, honey, and pollen, while the queen larvae are only given royal jelly to consume while they are in the larval phase (Arct and Pytkowska, 2008; Rzepecka-Stojko, et al., 2015; Panche, et al., 2016). Because it improves the queen honey bee's general Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

68 Polyandrous Queen Honey Bee

Lovleen Marwaha

health and fertility, royal jelly helps honey bee queens live longer. This is accomplished by delaying the ageing process. Because RJ makes the queen quite fertile, she has the ability to lay up to 3000 eggs per day and has a life span of about 3-5 years. This is in contrast to the life span of worker honey bees, which ranges from 2–6 months (Bonamigo et al., 2017; Paula et al., 2017). Royal jelly is a valuable bee product because it possesses anti-bacterial and antioxidative properties in addition to immunomodulation and anti-tumorin characteristics. These characteristics make royal jelly an excellent anti-tumorin agent. Royal jelly contains major proteins like MRJPs, including MRJP1MRJP9, which constitute about 80–90% of total proteins present in the concerned secretion (Sver, et al., 1996; Albert, and Klaudiny, 2004; Albert, et al., 2004; Sver, et al., 1996; Albert, and Klaudiny, 2004; Albert, et al., 2004; 80–90% of total proteins present in the (Silici, et al., 2009; Ramadan, and Al-Ghamdi, 2012; Fujita, et al., 2013; Lin, et al., 2018). In addition, MRJPs and glycoproteins are linked to oligosaccharides via a covalent bond at the N-terminal residue (Schmitzová et al., 1998). Posttranslational modifications to proteins can be accomplished through the glycosylation pathway, which is a complex modification pathway (Apweiler et al., 1999). Protein glycosylation is responsible for regulating a number of processes, including immune activity, cell growth, and cell proliferation (Dzik, 2001). The type of glycan chain monomer present on a glycoprotein can have an effect on how well that glycoprotein performs its function (Lin et al., 2019). 4.2. COMPOSITION RJ is an emulsion that is made up of a combination of protein, sugar, and lipid that is suspended in water. The watery component of royal jelly ranges in concentration from 60–70%, and its pH can range anywhere from 3.6–4.2. Proteins, carbohydrates, lipids, vitamins, flavonoids, minerals, polyphenols, and other biologically active substances are all found in royal jelly. Other components include flavonoids (De et al., 2013). In addition, the mineral content accounts for approximately 1.5% of the total weight and contains copper, iron, zinc, calcium, potassium, manganese, biotin, sodium salts, folic acid, niacin, inositol, riboflavin, pantothenic acid, thiamine, and vitamin E. (Silici et al., 2009; Kamakura, et al., 2014; Kanelis, et al., 2015;

Royal Jelly

Polyandrous Queen Honey Bee 69

Melliou and Chinou, 2017). RJ is made up of flavanones like hesperetin, naringenin, and isosakuranetin; flavones like apigenin, acacetin, and its glucoside; flavonols like isorhamnetin and luteolin glucoside; and isoflavonoids like kaempferol glucosides, coumestrol, genistein, and formononetin. The water content of royal jelly ranges from about 50 to 70 percent, with possible ranges of 7 to 21.2 percent (Kanelis, et al., 2015; Kolayli, et al., 2016; Melliou and Chinou, 2017). Royal jelly contains trehalose, gentiobiose, maltose, isomaltose, raffinose, melezitose, and erlose (Amoedo, et al., 2007; Nabas, et al., 2014). The percentage of protein found in RJ can range anywhere from 8% to 9% (Oroli et al., 2013; Nabas et al., 2014). The Major Royal Jelly Proteins, also known as MRJPs, account for approximately 90 percent of the total protein content (Kamakura, 2011). RJ is made up of a number of different amino acids, some of which are lysine, cystine, proline, valine, aspartic acid, serine, glutamic acid, glycine, cysteine, threonine, tyrosine, alanine, phenylalanine, leucine-isoleucine, hydroxyproline, and glutamine (Silici, et al., 2009). Roughly eighty to ninety percent of the fatty acids in RJ are unique fatty acids that have different structures. 10-hydroxydecanoic acid, also known as 10-HDA, is the principal fatty acid found in royal jelly (Kolayli, et al., 2016). In addition to these, RJ also contains 10-hydroxy-2-decenoic acid (10H2DA) and sebacic acid (SA) (Makino, et al., 2016). RJ is made up of a variety of other components, some of which are phenolic compounds and flavonoids (Nabas, et al., 2014). 4.2.1. Sugar The sugar content of royal jelly ranges from 7.5–15%, with 90% of the sugar being made up of fructose and glucose while only 0.8–3.6% of the sugar being made up of sucrose. A small amount of various other types of sugars, such as trehalose, maltose, ribose, melibiose, and erlose, can be found in royal jelly (Wei et al., 2017). Royal jelly composition varies with season, botanical origin, geographical location, bee species, and method of extraction. In addition to having a higher sugar content than worker jelly, royal jelly is also known to induce epigenetic modification.

70 Polyandrous Queen Honey Bee

Lovleen Marwaha

The incorporation of fructose and glucose into worker jelly stimulates developmental processes more closely aligned with those of the queen type. Royal jelly serves as a phagostimulant by kicking off insulin/insulin-like signalling cascades and mTOR pathways. These pathways have an effect on the bees' ability to consume food and the beginning stages of queen development (Zuluaga et al., 2016). 4.2.2. Lipid About 7–8% of royal jelly is composed of lipids, with the majority of these lipids having a chain length ranging from 8–12 carbon atoms. There are three different types of acids that can be found in royal jelly: 10hydroxy-2-decenoic acid (10H2DA), 10-hydroxydecanoic acid (10-HDA), and sebacic acid (SA) (Paula et al., 2017). RJ inhibits histone deacetylases, which are responsible for the hydrolysis of acetyl-lysine residues of histones. 10-hydroxydecanoic acid causes epigenetic effects on caste specification in Apis mellifera (Zuluaga, et al., 2016; Pasupuleti et al., 2017). Because 10-HDA possesses potent bactericidal properties, it can protect against virulent bacterial infections caused by Paenibacillus larvae (Boisard et al., 2014) as well as toxins produced by Staphylococcus aureus (Graikou et al., 2016). In addition, the particular component is effective against cells that cause colon cancer as well as neurodegenerative diseases (Bankova, 2005; Salatino, 2018). 10-HDA is a chemical that is used in the production of anticancer drugs as well as cosmetics (Daugsch et al., 2008; Bittencourt, et al., 2015). The anti-inflammatory effects of 10-HDA, SA, and 10H2DA were aided by the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B signalling pathways (Bankova, 2005; Andrade, et al., 2017). It has been shown that oestrogen receptors, including ER and ER, are more active after consuming royal jelly (Zhang, et al., 2017). The neuroblastoma SH-SY5Y cell line underwent apoptosis as a result of 6hydroxydopamine treatment (Betances-Salcedo, et al., 2017).

Royal Jelly

Polyandrous Queen Honey Bee 71

4.2.3. Protein The protein found in royal jelly makes up the bulk of this substance, accounting for approximately 80% of the total volume of RJ. Cell adhesion, cell growth, cell differentiation, and immune function are all dependent on the glycosylation and phosphorylation of MRJPs. Proteins in royal jelly stimulate growth and increase the amount of juvenile hormones in female larvae, which allows the proteins to modulate the developmental synchronicity of the larvae. The use of royal jelly has been shown to promote cell proliferation, as well as antimicrobial and cytokine suppressive activities (Xin et al., 2016). Jiang et al., 2018, reported that MRJPs have an anti-senescence activity in human cultured cells. This activity was observed in human cultured cells. MRJP1's secondary structure is made up of 9.6 percent alpha helices, 38.3 percent beta sheets, and 20 percent beta turns (Xue, et al., 2017). Monomer and oligomer are the two different manifestations of MRJP1 that can be found in nature. Oligomer components have a high resistance to heat and are growth stimulants that are significantly more potent than MRJP2 and MRJP3, respectively (Moriyama, et al., 2015). As a result of its ability to inhibit the activity of the citric acid enzyme in C. elegans, MRJP1 possesses nematicidal properties (Bilal, et al., 2018). Induction of apoptosis, reduction of free radical production, and inhibition of tumour necrosis factor (TNF)- production are the mechanisms by which MRJP2 and its isoform X1 have the ability to fight cancer and lessen the toxicity caused by CCl4 exposure (Abu-Serie, and Habashy, 2019). Jelleines, royalisin, and aspimin are some of the additional proteins that can be found in royal jelly. Because this particular chemical possesses antimicrobial properties, it helps bee larvae better defend themselves against a wide variety of infections. Royalisin and jelleine both have antimicrobial properties, which impair the ability of bacterial membranes to perform their normal functions. Royal jelly contains a protein with properties similar to those of apolipoprotein III, which is known to inhibit the growth of microorganisms. RJ has an effect on the functioning of the glucose oxidase enzyme, which is responsible for the catalyzation of the conversion of glucose to hydrogen peroxide (Fratini et al., 2016). RJ analysis revealed the presence of glucuronic acid termini, sulfation of mannose residues, and core -mannosylation of the N-glycans, as shown by LCMALDI-TOF MS glycomic analyses, Western blotting, and arraying data (Hykollari, et al., 2018).

72 Polyandrous Queen Honey Bee

Lovleen Marwaha

4.2.4. Phenols, Flavonoids And Free Amino Acids Pinobanksin, octanoic acids, dodecanoic acid, and 1,2-benzene dicarboxylic acid, in addition to their esters, are all components of royal jelly. The flavonoid component can be broken down into four distinct groups. • flavanones including, but not limited to, hesperetin, isosakuranetin, and naringenin. • flavones including, but not limited to, acacetin, apigenin and its glucoside, chrysin, and luteolin glucoside. • flavonols including, but not limited to, isorhamnetin and kaempferol glucoside. • Isoflavonoids, such as genistein, coumestrol, and formononetin RJ has properties that are protective against cell death and inflammation (Kocot, et al., 2018). Royal jelly obtained from larvae less than 24 hours old has lower levels of protein and phenolic compounds than royal jelly obtained from younger larvae with longer development times. As a result, former RJ exhibited a more powerful free radicle scavenge ring than larvae that have been alive for longer than 24 hours (Liu, et al., 2008). In addition to this, essential oils can be found in RJ (Xue et al., 2017). Amino acids such as valine, serine, glutamic acid, glycine, cysteine, alanine, threonine, tyrosine, hydroxyproline, phenylalanine, leucineisoleucine, and glutamine are found in royal jelly. Royal jelly also contains phenylalanine, hydroxyproline, and tyrosine (Kocot, et al., 2018; Pina, et al., 2018). The use of royal jelly has been shown to lengthen the lifespan of C. elegans (Honda, et al., 2011). In addition, due to the extraordinary properties it possesses, RJ makes mammals live for a significantly longer period of time. 4.2.5. Vitamins, Minerals And Other Bioactive Sustances A, B1, B2, B5, B6, B8, B9, B12, C, and E are among the vitamins that can be found in RJ (Xue et al., 2017; Kocot et al., 2018). Pantothenic acid, which is found in RJ, is known to make people live longer (Gardner, 1948). Minerals make up about 1.5% of the total content of royal jelly (Kocot et al., 2018). A significant number of minerals, including K, Mg, Na, P, Ca, Cu, S, Fe, Al, Zn, Sr, Ba, Cd, Bi, Pb, Hg, Sn, Te, W, Tl, Cr, Sb, Ni, Mn, V, Ti, Co, and Mo are found in royal jelly. These minerals play an important role in the structure and function of the substance. In addition, the various components that make up RJ are shaped by the presence of minerals and trace elements. RJ also has acetylcholine in it, which is a neurotransmitter (Wessler et al., 2016), and neurotransmitters play a role in the formation of memories as well as how well cognitive processes work. RJ is composed of free bases such as adenosine, guanosine, uridine, cytidine, and iridin in addition to adenosine monophosphate

Royal Jelly

Polyandrous Queen Honey Bee 73

(AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) (ATP). Phosphate, ATP, and AMP are all found in relatively high concentrations in RJ (Wu et al., 2015; Xue et al., 2017; Kunugi and Ali, 2019). RJ has a higher concentration of sugar, protein, and minerals and a lower proportion of water (Wang, et al., 2016). Ovarian development is dependent on royal jelly's ability to stimulate gene expression through epigenetic modification (Chittka and Chittka, 2010). Because of royal jelly, a queen can have a life span of about three to five years and lay between two thousand and three thousand eggs every single day (Fratini, et al., 2016). Because royal jelly is produced in significantly higher quantities for queen larvae compared to that of worker larvae, it is possible to harvest it from queen cells (Isidorov et al., 2012). 4.2.6. The Importance of Royal Jelly to the Developmental Process The primary component of royal jelly is called royalactin, and it is responsible for stimulating queen development by acting on the epidermal growth factor receptor. Because queen larvae are only fed royal jelly, they are able to reproduce and have a longer lifespan than other stages of the insect. Changes in metabolism, hormonal level, and epigenetic modification caused by DNA methylation, histone modification, and non-protein coding RNAs are all responsible for the development of the mature queen from the queen larvae, which develop over the course of six larval days and consume a significant amount of royal jelly (Barchuk, et al., 2007; Foret, et al., 2012; Maleszka, 2014; Ashby, et al., 2016). Additionally, the relevant point has been investigated by Maleszka (2019), and their findings highlighted major developmental phases as well as the impact of royal jelly. After the first three days, only the queen larva is fed royal jelly (Weaver, 1970; Ashby, et al., 2016; Wang, et al., 2016). Initially, all female larvae are fed royal jelly for the first three days (Weaver, 1970; Ashby, et al., 2016; Wang, et al., 2016). (Shuel and Dixon, 1960; Weaver, et al., 1966). It has been demonstrated through testing that female larvae that were fed a lower concentration of RJ did not develop all of the queen characteristics, but instead possessed intermediate characteristics (Maleszka, 2014; De Souza, 2015; Buttstedt et al. 2016). Even larvae that are fed royalactin that does not contain RJ can mature into queens (Buttstedt, et al., 2016). RJ administered during the first three days of the colony's life is insufficient to effectively stimulate queen development (Maleszka, et al., 2014; Foret, et al., 2012). It has been demonstrated that the methylation machinery present in female larvae can stimulate the development of the queen (Kucharski, et al., 2008). Methionine, a methyl group, and other essential amino acids are found in relatively high concentrations in royal jelly. Methylation cannot occur without these amino acids (Drapeau, et al.,

74 Polyandrous Queen Honey Bee

Lovleen Marwaha

2006). In addition, RJ contains a few peculiar fatty acids that are unaffected by the temperature of 40 degrees Celsius. According to the findings of one investigation, RJ is composed of an ethanol-soluble, protease-resistant RJ fraction that is capable of inducing queen development (Rembold, et al., 1974). A glycoprotein known as royalactin is a monomeric component of the protein known as MRJP1 (Maleszka, 2019). 4.3. SECRETION OF ROYAL JELLY A few studies suggested that RJ proteins are produced by three glands, specifically the hypopharyngeal, post-cerebral, and thoracic glands. These glands are located in the torso (Fujita et al.2013). The hypopharyngeal glands are located on the bee's head, and each gland is composed of hundreds of acini connected to the axial duct. The glands each have an opening located close to the sub-oral portion of the hypopharynx. The size of acini grows and shrinks throughout the development of honey bees, reaching its maximum size approximately six days after hatching and beginning a gradual decline after fifteen days (Albert et al. 2014, Ji et al.2014). Special glands in the head of the worker bee are responsible for the production of Major Royal Jelly Proteins, also known as MRJPs. These glands facilitate protein secretion. MRJP1 and 2 possess antioxidant activity, MRJP3 possesses nutritional function (Schmitzová, et al., 1998; Bliková, et al., 2009;Buttstedt, et al., 2013; Vezeteu, et al., 2017; Buttstedt, et al., 2018). In the case of Apis mellifera, the MRJP family is composed of approximately nine proteins that act as the primary components. These proteins are denoted by the designations MRJP1–9 (Drapeau et al., 2006; Helbing, et al., 2017). Ecdysone and juvenile hormone are two of the hormones that are responsible for regulating the synthesis of MRJP precursor and vitellogenin in the hemolymph (Barchuk et al., 2002). After administration of the juvenile hormone analogue methoprene or 20-hydroxyecdysone (20E) to nurse honey bees, there is a reduction in the concentration of MRJP2 in the honey bees' bodies (Ueno et al., 2015). RJ can be evaluated with the help of 10-HDA, which is widely regarded as the most significant component. The enzyme glucose oxidase is used to determine how stable RJ is, and its activity can be influenced by both temperature and storage conditions. Even when kept at a temperature of 4 degrees Celsius, the concentration of certain enzymes decreases after being stored. Furosine content was utilised by Marconi et al. (2002) as a marker for determining the freshness of RJ, which is the result of Maillard's reaction (Messia et al., 2003; Sabatini, et al., 2009).

Royal Jelly

Polyandrous Queen Honey Bee 75

Additionally, RJ is a yellowish, creamy, acidic, and viscous fluid that is secreted by the hypopharyngeal and mandibular glands of worker honey bees. It has a wet weight of 60–70% water, 6–18% hydrocarbons, 9–18% proteins, 3–8% lipids, 0.8–3.0% minerals, and a small number of polyphenols and vitamins (Karaali, and Eke, 1988; Sabatini, and De Almeida, 2009). Antibacterial properties, antioxidant effects, anti-inflammatory properties, vasodilatory and hypotensive activities, disinfectant action, antitumor properties, and anti-hypercholesterolaemic activity are all present in RJ. In addition, RJ has antitumor properties (Hu et al., 2017). The research conducted by Wang et al. (2015) has the objective of determining the chemical make-up of RJ by taking into account factors such as moisture, total protein, 10-HDA, carbohydrates, and minerals. The calibre and quantity of the food provided to the young birds plays a role in the establishment of caste (Shuel and Dixon 1960). Wang et al. (2015, further reported that there is a difference in chemical constituents between RJ and WJ, as RJ possesses lower water content. Royalactin and 10-HDA content in royal jelly induce the development of the queen and control different developmental phases by influencing the secretion of different hormones, the major regulator of the moulting process. Wang et al. (2015, further reported that there is a difference in chemical constituents between RJ and WJ (Kamakura, 2011; Wang, et al., 2015). 4.4. DIET OF OTHER CASTES An investigation that was carried out by Martin et al., 2019, revealed the results of a study that showed workers and drones are fed on pollen with a high pollen content and pollen grain, both of which have a higher concentration of polyunsaturated fatty acids (PUFA) than royal jelly. The composition of the membrane fatty acids has been evaluated using a variety of castes, such as larvae, pupa, emergents, and various adult stages. It has been observed that the PUFA compositions of all stages are comparable; however, as the life cycle progresses, there is an increase in the concentration of PUFA in the drones and workers, whereas the queen possesses the lowest level of PUFA. There is a fivefold increase in the concentration of PUFA in worker bees after they emerge from the hive; however, this increase is followed by a decrease in the concentration of PUFA and an increase in oxidative damage over the course of a worker honey bee's entire life. These two factors are responsible for the worker honey bee's shorter life span. During the larval stage, workers are fed worker jelly to help them develop properly. In other words, the larval diet plays a role in the development of specific castes. Worker larvae are fed two distinct types of food, including royal jelly (RJ) and worker jelly (WJ) (Fig. 4a) (Haydak 1970). When compared to WJ, the

76 Polyandrous Queen Honey Bee

Lovleen Marwaha

moisture to day-old content of RJ is significantly lower. The amount of sugar found in royal jelly has been shown to have an effect on the determination of caste. According to Asencot and Lensky (1988), the amount of sugar in RJ that is fed to queen larvae that are 1-3 days old contains 12.4 percent, which is approximately four times that of RJ than in WJ. It was demonstrated through testing that the addition of sugar to WJ can steer development in the direction of queen type (Asencot and Lensky 1985). The 57-kDa protein found in royal jelly has been shown to play a role in the determination of caste in honey bees through the epidermal growth factor receptor (EGFR)-mediated signalling pathway (Kamakura 2011).

Fig. (4a). Developmental phases of different castes within the honey bee colony. The honey bee is a holometabolous insect, with developmental phases including eggs, larvae, pupae, and adults. Carefully studying the above diagram, it can be concluded that for all three castes, during 3-6 days, royal jelly is the major larval food. In the case of the female caste, larvae remain totipotent for the first 3 days. During the first 7-9 days, differentiating larval food is provided to different castes. The larval phase is completed within uncapped wax cells, whereas pupation is completed within capped wax cells. The influence of royal jelly can be analyzed, which accelerates pupation development and increases the life span and reproductive potential of the queen.

CONCLUSION Royal jelly is a protein-rich secretion that is necessary for the early larval growth of all instars. This secretion also has an effect on the various developmental processes. In the case of queen honey bee larvae, consuming only royal jelly as their sole source of nutrition increases genomic expression, which in turn regulates the formation of the female reproductive system and overall development. The reproductive potential of the queen honey bee is influenced by the royal jelly product that is produced by the worker bees, and as a result, her dominance in the colony.

Royal Jelly

Polyandrous Queen Honey Bee 77

REFERENCES Abu-Serie, MM & Habashy, NH (2019) Two purified proteins from royal jelly with in vitro dual anti-hepatic damage potency: Major royal jelly protein 2 and its novel isoform X1. Int J Biol Macromol, 128, 782-95. [http://dx.doi.org/10.1016/j.ijbiomac.2019.01.210] [PMID: 30711561] Albert, Š & Klaudiny, J (2004) The MRJP/YELLOW protein family of Apis mellifera: Identification of new members in the EST library. J Insect Physiol, 50, 51-9. [http://dx.doi.org/10.1016/j.jinsphys.2003.09.008] [PMID: 15037093] Andrade, JKS, Denadai, M, de Oliveira, CS, Nunes, ML & Narain, N (2017) Evaluation of bioactive compounds potential and antioxidant activity of brown, green and red propolis from Brazilian northeast region. Food Res Int, 101, 129-38. [http://dx.doi.org/10.1016/j.foodres.2017.08.066] [PMID: 28941675] Apweiler, R, Hermjakob, H & Sharon, N (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta, Gen Subj, 1473, 4-8. [http://dx.doi.org/10.1016/S0304-4165(99)00165-8] [PMID: 10580125] Arct, J & Pytkowska, K (2008) Flavonoids as components of biologically active cosmeceuticals. Clin Dermatol, 26, 347-57. [http://dx.doi.org/10.1016/j.clindermatol.2008.01.004] [PMID: 18691514] Asencot, M & Lensky, Y (1988) The effect of soluble sugars in stored royal jelly on the differentiation of female honeybee (Apis mellifera L.) larvae to queens. Insect Biochem, 18, 127-33. [http://dx.doi.org/10.1016/0020-1790(88)90016-9] Ashby, R, Forêt, S, Searle, I & Maleszka, R (2016) MicroRNAs in honey bee caste determination. Sci Rep, 6, 18794. [http://dx.doi.org/10.1038/srep18794] [PMID: 26739502] Bankova, V (2005) Chemical diversity of propolis and the problem of standardization. J Ethnopharmacol, 100, 114-7. [http://dx.doi.org/10.1016/j.jep.2005.05.004] [PMID: 15993016] Barchuk, AR, Bitondi, MMG & Simões, ZLP (2002) Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera. J Insect Sci (Ludhiana), 2, 1-8. [http://dx.doi.org/10.1673/031.002.0101] [PMID: 15455035] Barchuk, AR, Cristino, AS, Kucharski, R, Costa, LF, Simões, ZLP & Maleszka, R (2007) Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera. BMC Dev Biol, 7, 70. [http://dx.doi.org/10.1186/1471-213X-7-70] [PMID: 17577409] Betances-Salcedo, E, Revilla, I, Vivar-Quintana, A & González-Martín, M (2017) Flavonoid and antioxidant capacity of propolis prediction using near infrared spectroscopy. Sensors (Basel), 17, 1647. [http://dx.doi.org/10.3390/s17071647] [PMID: 28718789] Bilal, B & Azim, MK (2018) Nematicidal activity of ‘major royal jelly protein’-containing glycoproteins from Acacia honey. Exp Parasitol, 192, 52-9. [http://dx.doi.org/10.1016/j.exppara.2018.07.011] [PMID: 30040959] Bíliková, K, Mirgorodskaya, E, Bukovská, G, Gobom, J, Lehrach, H & Šimúth, J (2009) Towards functional proteomics of minority component of honeybee royal jelly: The effect of post-translational modifications on the antimicrobial activity of apalbumin2. Proteomics, 9, 2131-8. [http://dx.doi.org/10.1002/pmic.200800705] [PMID: 19322786] Boisard, S, Le Ray, AM, Gatto, J, Aumond, MC, Blanchard, P, Derbré, S, Flurin, C & Richomme, P (2014) Chemical composition, antioxidant and anti-AGEs activities of a French poplar type propolis. J Agric Food Chem, 62, 1344-51. [http://dx.doi.org/10.1021/jf4053397] [PMID: 24443994] Bonamigo, T, Campos, JF, Oliveira, AS, Torquato, HFV, Balestieri, JBP, Cardoso, CAL, Paredes-Gamero,

78 Polyandrous Queen Honey Bee

Lovleen Marwaha

EJ, de Picoli Souza, K & dos Santos, EL (2017) Antioxidant and cytotoxic activity of propolis of Plebeia droryana and Apis mellifera (Hymenoptera, Apidae) from the Brazilian Cerrado biome. PLoS One, 12e0183983 [http://dx.doi.org/10.1371/journal.pone.0183983] [PMID: 28898258] Buttstedt, A, Ihling, CH, Pietzsch, M & Moritz, RFA (2016) Royalactin is not a royal making of a queen. Nature, 537, E10-2. [http://dx.doi.org/10.1038/nature19349] [PMID: 27652566] Buttstedt, A, Moritz, RFA & Erler, S (2013) More than royal food - Major royal jelly protein genes in sexuals and workers of the honeybee Apis mellifera. Front Zool, 10, 72. [http://dx.doi.org/10.1186/1742-9994-10-72] [PMID: 23356547] Buttstedt, A, Moritz, RFA & Erler, S (2014) Origin and function of the major royal jelly proteins of the honeybee ( Apis mellifera ) as members of the yellow gene family. Biol Rev Camb Philos Soc, 89, 255-69. [http://dx.doi.org/10.1111/brv.12052] [PMID: 23855350] Buttstedt, A, Mureşan, CI, Lilie, H, Hause, G, Ihling, CH, Schulze, SH, Pietzsch, M & Moritz, RFA (2018) How honeybees defy gravity with royal jelly to raise queens. Curr Biol, 28, 1095-1100.e3. [http://dx.doi.org/10.1016/j.cub.2018.02.022] [PMID: 29551410] Chittka, A & Chittka, L (2010) Epigenetics of Royalty. PLoS Biol, 8e1000532 [http://dx.doi.org/10.1371/journal.pbio.1000532] [PMID: 21072243] Daugsch, A, Moraes, CS, Fort, P & Park, YK (2008) Brazilian red propolis--chemical composition and botanical origin. Evid Based Complement Alternat Med, 5, 435-41. [http://dx.doi.org/10.1093/ecam/nem057] [PMID: 18955226] De Paula, R, Rabalski, I, Messia, MC, Abdel-Aal, ESM & Marconi, E (2017) Effect of processing on phenolic acids composition and radical scavenging capacity of barley pasta. Food Res Int, 102, 136-43. [http://dx.doi.org/10.1016/j.foodres.2017.09.088] [PMID: 29195932] Moreira, FL, de Souza, GHB, Rodrigues, IV, Lopes, NP & de Oliveira, ARM (2013) A non-michaelian behavior of the in vitro metabolism of the pentacyclic triterpene alfa and beta amyrins by employing rat liver microsomes. J Pharm Biomed Anal, 84, 14-9. [http://dx.doi.org/10.1016/j.jpba.2013.05.038] [PMID: 23777643] De Souza, DA, Wang, Y, Kaftanoglu, O, De Jong, D, Amdam, GV, Gonçalves, LS & Francoy, TM (2015) Morphometric identification of queens, workers and intermediates in in vitro reared honey bees (Apis mellifera). PLoS One, 10e0123663 [http://dx.doi.org/10.1371/journal.pone.0123663] [PMID: 25894528] Drapeau, MD, Albert, S, Kucharski, R, Prusko, C & Maleszka, R (2006) Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees. Genome Res, 16, 1385-94. [http://dx.doi.org/10.1101/gr.5012006] [PMID: 17065613] Dzik, S (2001) Science 291:2370-2376. Transfus Med Rev, 15, 323. Foret, S, Kucharski, R, Pellegrini, M, Feng, S, Jacobsen, SE, Robinson, GE & Maleszka, R (2012) DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees. Proc Natl Acad Sci USA, 109, 4968-73. [http://dx.doi.org/10.1073/pnas.1202392109] [PMID: 22416128] Fratini, F, Cilia, G, Mancini, S & Felicioli, A (2016) Royal Jelly: An ancient remedy with remarkable antibacterial properties. Microbiol Res, 192, 130-41. [http://dx.doi.org/10.1016/j.micres.2016.06.007] [PMID: 27664731] Fujita, T, Kozuka-Hata, H, Ao-Kondo, H, Kunieda, T, Oyama, M & Kubo, T (2013) Proteomic analysis of the royal jelly and characterization of the functions of its derivation glands in the honeybee. J Proteome Res, 12, 404-11. [http://dx.doi.org/10.1021/pr300700e] [PMID: 23157659] Gardner, TS (1948) The use of Drosophila melanogaster as a screening agent for longevity factors; the effects

Royal Jelly

Polyandrous Queen Honey Bee 79

of biotin, pyridoxine, sodium yeast nucleate, and pantothenic acid on the life span of the fruit fly. J Gerontol, 3, 9-13. [http://dx.doi.org/10.1093/geronj/3.1.9] [PMID: 18856648] Graikou, K, Popova, M, Gortzi, O, Bankova, V & Chinou, I (2016) Characterization and biological evaluation of selected Mediterranean propolis samples. Is it a new type? Lebensm Wiss Technol, 65, 261-7. [http://dx.doi.org/10.1016/j.lwt.2015.08.025] Haydak, MH (1970) Honey bee nutrition. Annu Rev Entomol, 15, 143-56. [http://dx.doi.org/10.1146/annurev.en.15.010170.001043] Helbing, S, Lattorff, HMG, Moritz, RFA & Buttstedt, A (2017) Comparative analyses of the major royal jelly protein gene cluster in three Apis species with long amplicon sequencing. DNA Res, 24, 279-87. [http://dx.doi.org/10.1093/dnares/dsw064] [PMID: 28170034] Hidaka, S, Okamoto, Y, Uchiyama, S, Nakatsuma, A, Hashimoto, K, Ohnishi, T & Yamaguchi, M (2006) Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model. Evid Based Complement Alternat Med, 3, 339-48. [http://dx.doi.org/10.1093/ecam/nel019] [PMID: 16951718] Kunugi, H & Mohammed Ali, A (2019) Royal jelly and its components promote healthy aging and longevity: from animal models to humans. Int J Mol Sci, 20, 4662. [http://dx.doi.org/10.3390/ijms20194662] [PMID: 31547049] Honda, Y, Fujita, Y, Maruyama, H, Araki, Y, Ichihara, K, Sato, A, Kojima, T, Tanaka, M, Nozawa, Y, Ito, M & Honda, S (2011) Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans. PLoS One, 6e23527 [http://dx.doi.org/10.1371/journal.pone.0023527] [PMID: 21858156] Hua, FL, Likova, KB & Casabiancac, H (2017) Standard methods for Apis mellifera royal jelly research. J Apic Res, 1-69. Hykollari, A, Malzl, D, Eckmair, B, Vanbeselaere, J, Scheidl, P, Jin, C, Karlsson, NG, Wilson, IBH & Paschinger, K (2018) Isomeric separation and recognition of anionic and zwitterionic N-glycans from royal jelly glycoproteins. Mol Cell Proteomics, 17, 2177-96. [http://dx.doi.org/10.1074/mcp.RA117.000462] [PMID: 30104209] Isidorov, VA, Bakier, S & Grzech, I (2012) Gas chromatographic–mass spectrometric investigation of volatile and extractable compounds of crude royal jelly. J Chromatogr B Analyt Technol Biomed Life Sci, 885-886, 109-16. [http://dx.doi.org/10.1016/j.jchromb.2011.12.025] [PMID: 22245368] Jiang, C, Liu, X, Li, C, Qian, H, Chen, D, Lai, C & Shen, L (2018) Anti-senescence effect and molecular mechanism of the major royal jelly proteins on human embryonic lung fibroblast (HFL-I) cell line. J Zhejiang Univ Sci B, 19, 960-72. [http://dx.doi.org/10.1631/jzus.B1800257] [PMID: 30507079] Kamakura, M (2011) Royalactin induces queen differentiation in honeybees. Nature, 473, 478-83. [http://dx.doi.org/10.1038/nature10093] [PMID: 21516106] Kamakura, M, Fukuda, T, Fukushima, M & Yonekura, M (2001) Storage-dependent degradation of 57-kDa protein in royal jelly: a possible marker for freshness. Biosci Biotechnol Biochem, 65, 277-84. [http://dx.doi.org/10.1271/bbb.65.277] [PMID: 11302159] Kanelis, D, Tananaki, C, Liolios, V, Dimou, M, Goras, G, Rodopoulou, MA, Karazafiris, E & Thrasyvoulou, A (2015) A suggestion for royal jelly specifications / Prijedlog globalnih standarda za matičnu mliječ. Arh Hig Rada Toksikol, 66, 275-84. [http://dx.doi.org/10.1515/aiht-2015-66-2651] [PMID: 26751859] Karaali, A, Meydanoǧlu, F & Eke, D (1988) Studies on composition, freeze-drying and storage of Turkish royal jelly. J Apic Res, 27, 182-5. [http://dx.doi.org/10.1080/00218839.1988.11100799]

80 Polyandrous Queen Honey Bee

Lovleen Marwaha

Kolayli, S, Sahin, H, Can, Z, Yildiz, O, Malkoc, M & Asadov, A (2016) A member of complementary medicinal food: anatolian royal jellies, their chemical compositions, and antioxidant properties. J Evid Based Complementary Altern Med, 21, NP43-8. [http://dx.doi.org/10.1177/2156587215618832] [PMID: 26620573] Kocot, J, Kiełczykowska, M, Luchowska-Kocot, D, Kurzepa, J & Musik, I (2018) Antioxidant potential of propolis, bee pollen, and royal jelly: possible medical application. Oxid Med Cell Longev, 2018, 1-29. [http://dx.doi.org/10.1155/2018/7074209] [PMID: 29854089] Kucharski, R, Maleszka, J, Foret, S & Maleszka, R (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science, 319, 1827-30. [http://dx.doi.org/10.1126/science.1153069] [PMID: 18339900] Lin, N, Chen, S, Zhang, H, Li, J & Fu, L (2018) Quantification of major royal jelly protein 1 in fresh royal jelly by ultraperformance liquid chromatography–tandem mass spectrometry. J Agric Food Chem, 66, 12708. [http://dx.doi.org/10.1021/acs.jafc.7b05698] [PMID: 29381065] Lin, N, Li, J, Shao, R & Zhang, H (2019) Site-Specific Analysis of N-Linked Glycosylation Heterogeneity from Royal Jelly Glycoproteins. J Agric Food Chem, 67, 9411-22. [http://dx.doi.org/10.1021/acs.jafc.9b03080] [PMID: 31393126] Liu, JR, Yang, YC, Shi, LS & Peng, CC (2008) Antioxidant properties of royal jelly associated with larval age and time of harvest. J Agric Food Chem, 56, 11447-52. [http://dx.doi.org/10.1021/jf802494e] [PMID: 19007163] López-Gutiérrez, N, Aguilera-Luiz, MM, Romero-González, R, Vidal, JLM & Garrido Frenich, A (2014) Fast analysis of polyphenols in royal jelly products using automated TurboFlow™-liquid chromatography–Orbitrap high resolution mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 973, 17-28. [http://dx.doi.org/10.1016/j.jchromb.2014.09.038] [PMID: 25464090] Makino, J, Ogasawara, R, Kamiya, T, Hara, H, Mitsugi, Y, Yamaguchi, E, Itoh, A & Adachi, T (2016) Royal jelly constituents increase the expression of extracellular superoxide dismutase through histone acetylation in monocytic THP-1 cells. J Nat Prod, 79, 1137-43. [http://dx.doi.org/10.1021/acs.jnatprod.6b00037] [PMID: 27049436] Maleszka, R (2014) The social honey bee in biomedical research: realities and expectations. Drug Discov Today Dis Models, 12, 7-13. [http://dx.doi.org/10.1016/j.ddmod.2014.06.001] Marconi, E, Caboni, MF, Messia, MC & Panfili, G (2002) Furosine: a suitable marker for assessing the freshness of royal jelly. J Agric Food Chem, 50, 2825-9. [http://dx.doi.org/10.1021/jf0114987] [PMID: 11982406] Martin, N, Hulbert, AJ, Brenner, GC, Brown, SHJ, Mitchell, TW & Else, PL (2019) Honeybee caste lipidomics in relation to life-history stages and the long life of the queen. J Exp Biol, 222jeb.207043 [http://dx.doi.org/10.1242/jeb.207043] [PMID: 31672733] Melliou, E & Chinou, I (2014) Chemistry and bioactivities of royal jelly. Studies in Natural Products Chemistry, 43, 261-90. [). Elsevier.]. [http://dx.doi.org/10.1016/B978-0-444-63430-6.00008-4] Messia, MC, Caboni, MF & Marconi, E (2003) Valutazione della freschezza della gelatina reale. Moriyama, T, Ito, A, Omote, S, Miura, Y & Tsumoto, H (2015) Heat resistant characteristics of major royal jelly protein 1 (MRJP1) oligomer. PLoS One, 10e0119169 [http://dx.doi.org/10.1371/journal.pone.0119169] [PMID: 26020775] Nabas, Z, Haddadin, M, Haddadin, J & Nazer, I (2014) Chemical composition of royal jelly and effects of synbiotic with two different locally isolated probiotic strains on antioxidant activities. Pol J Food Nutr Sci, 64, 171-80.

Royal Jelly

Polyandrous Queen Honey Bee 81

[http://dx.doi.org/10.2478/pjfns-2013-0015] Oršolić, N (2013) [Royal jelly: component efficiency, analysis, and standardisation]. Arh Hig Rada Toksikol, 64, 445-61. [PMID: 24084354] Panche, AN, Diwan, AD & Chandra, SR (2016) Flavonoids: an overview. J Nutr Sci, 5e47 [http://dx.doi.org/10.1017/jns.2016.41] [PMID: 28620474] Pasupuleti, VR, Sammugam, L, Ramesh, N & Gan, SH (2017) Honey, propolis, and royal jelly: a comprehensive review of their biological actions and health benefits. Oxid Med Cell Longev, 2017, 1-21. [http://dx.doi.org/10.1155/2017/1259510] [PMID: 28814983] Pina, A, Begou, O, Kanelis, D, Gika, H, Kalogiannis, S, Tananaki, C, Theodoridis, G & Zotou, A (2018) Targeted profiling of hydrophilic constituents of royal jelly by hydrophilic interaction liquid chromatography–tandem mass spectrometry. J Chromatogr A, 1531, 53-63. [http://dx.doi.org/10.1016/j.chroma.2017.11.019] [PMID: 29198446] Ramadan, MF & Al-Ghamdi, A (2012) Bioactive compounds and health-promoting properties of royal jelly: A review. J Funct Foods, 4, 39-52. [http://dx.doi.org/10.1016/j.jff.2011.12.007] Rembold, H, Lackner, B & Geistbeck, I (1974) The chemical basis of honeybee, Apis mellifera, caste formation. Partial purification of queen bee determinator from royal jelly. J Insect Physiol, 20, 307-14. [http://dx.doi.org/10.1016/0022-1910(74)90063-8] Rzepecka-Stojko, A, Stojko, J, Kurek-Górecka, A, Górecki, M, Kabała-Dzik, A, Kubina, R, Moździerz, A & Buszman, E (2015) Polyphenols from bee pollen: structure, absorption, metabolism and biological activity. Molecules, 20, 21732-49. [http://dx.doi.org/10.3390/molecules201219800] [PMID: 26690100] Sabatini, AG, Marcazzan, GL, Caboni, MF, Bogdanov, S & Almeida-Muradian, LBD (2009) Quality and standardisation of royal jelly. Journal of ApiProduct and ApiMedical Science, 1, 16-21. [http://dx.doi.org/10.3896/IBRA.4.01.1.04] Salatino, A & Salatino, MLF (2018) Brazilian red propolis: legitimate name of the plant resin source. MOJ Food Processing & Technology, 6, 21-2. [http://dx.doi.org/10.15406/mojfpt.2018.06.00139] Schmitzová, J, Klaudiny, J, Albert, Š, Schröder, W, Schreckengost, W, Hanes, J, Júdová, J & Šimúth, J (1998) A family of major royal jelly proteins of the honeybee Apis mellifera L. Cell Mol Life Sci, 54, 102030. [http://dx.doi.org/10.1007/s000180050229] [PMID: 9791542] Shuel, RW & Dixon, SE (1960) The early establishment of dimorphism in the female honeybee,Apis mellifera L. Insectes Soc, 7, 265-82. [http://dx.doi.org/10.1007/BF02224497] Silici, S, Ekmekcioglu, O, Eraslan, G & Demirtas, A (2009) Antioxidative effect of royal jelly in cisplatininduced testes damage. Urology, 74, 545-51. [http://dx.doi.org/10.1016/j.urology.2009.05.024] [PMID: 19616287] Šver, L, Oršolić, N, Tadić, Z, Njari, B, Valpotić, I & Bašic, I (1996) A royal jelly as a new potential immunomodulator in rats and mice. Comp Immunol Microbiol Infect Dis, 19, 31-8. [http://dx.doi.org/10.1016/0147-9571(95)00020-8] [PMID: 8654043] Vezeteu, TV, Bobiş, O, Moritz, RFA & Buttstedt, A (2017) Food to some, poison to others - honeybee royal jelly and its growth inhibiting effect on European Foulbrood bacteria. MicrobiologyOpen, 6e00397 [http://dx.doi.org/10.1002/mbo3.397] [PMID: 27743422] Ueno, T, Takeuchi, H, Kawasaki, K & Kubo, T (2015) Changes in the gene expression profiles of the hypopharyngeal gland of worker honeybees in association with worker behavior and hormonal factors. PLoS One, 10e0130206

82 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1371/journal.pone.0130206] [PMID: 26083737] Wang, Y, Ma, L, Zhang, W, Cui, X, Wang, H & Xu, B (2016) Comparison of the nutrient composition of royal jelly and worker jelly of honey bees (Apis mellifera). Apidologie (Celle), 47, 48-56. [http://dx.doi.org/10.1007/s13592-015-0374-x] Weaver, N (1966) Physiology of caste determination. Annu Rev Entomol, 11, 79-102. [http://dx.doi.org/10.1146/annurev.en.11.010166.000455] [PMID: 5321586] Wei, CC, Chang, CH & Liao, VHC (2017) Anti-Parkinsonian effects of β-amyrin are regulated via LGG-1 involved autophagy pathway in Caenorhabditis elegans. Phytomedicine, 36, 118-25. [http://dx.doi.org/10.1016/j.phymed.2017.09.002] [PMID: 29157804] Wessler, I, Gärtner, HA, Michel-Schmidt, R, Brochhausen, C, Schmitz, L, Anspach, L, Grünewald, B & Kirkpatrick, CJ (2016) Honeybees produce millimolar concentrations of non-neuronal acetylcholine for breeding: possible adverse effects of neonicotinoids. PLoS One, 11e0156886 [http://dx.doi.org/10.1371/journal.pone.0156886] [PMID: 27285384] Wu, L, Chen, L, Selvaraj, JN, Wei, Y, Wang, Y, Li, Y, Zhao, J & Xue, X (2015) Identification of the distribution of adenosine phosphates, nucleosides and nucleobases in royal jelly. Food Chem, 173, 1111-8. [http://dx.doi.org/10.1016/j.foodchem.2014.10.137] [PMID: 25466132] Xin, X, Chen, Y, Chen, D, Xiao, F, Parnell, LD, Zhao, J, Liu, L, Ordovas, JM, Lai, CQ & Shen, L (2016) Supplementation with major royal-jelly proteins increases lifespan, feeding, and fecundity in Drosophila. J Agric Food Chem, 64, 5803-12. [http://dx.doi.org/10.1021/acs.jafc.6b00514] [PMID: 27388939] Xue, X, Wu, L & Wang, K (2017) Chemical Composition of Royal Jelly. [http://dx.doi.org/10.1007/978-3-319-59689-1_8] Zhang, C, Shen, X, Chen, J, Jiang, X & Hu, F (2017) Identification of free radical scavengers from Brazilian green propolis using off‐line HPLC‐DPPH assay and LC‐MS. J Food Sci, 82, 1602-7. [http://dx.doi.org/10.1111/1750-3841.13730] [PMID: 28561958] Zuluaga, C, Martínez, A, Fernández, J, López-Baldó, J, Quiles, A & Rodrigo, D (2016) Effect of high pressure processing on carotenoid and phenolic compounds, antioxidant capacity, and microbial counts of bee-pollen paste and bee-pollen-based beverage. Innov Food Sci Emerg Technol, 37, 10-7. [http://dx.doi.org/10.1016/j.ifset.2016.07.023]

Polyandrous Queen Honey Bee, 2022, 83-110

83

CHAPTER 5

Quality Influencing Factors and Disease Resistance in Queen of Apis mellifera (Hymenoptera: Apidae) Abstract: Before the 4th instar larval phase, worker larvae exhibit totipotency to develop into either female caste. In subsequent larval stages, differential expression of various genetic elements occurs under the prominent induction of royal jelly, developmental hormones, and volatile queen emission. In the honey bee female caste, anatomical reproductive disproportionality establishes due to this diversification of genomic expression. Exponential fertility and pheromonal profiling of the queen regulate colonial strength, colonial productivity, submissive behaviour, and the development of workers. Different factors prevailing within the hive or outside of the colony premises influence the queen's quality. For example, the queen's fecundity is negatively proportional to the age of the worker larva before entering the queen differentiation pathway. Further, numerous additional factors like genomic content, physiology, quality and quantity of royal jelly, colonial food storage, social environment, queen pheromones, etc. influence queen reproductive potential. Further, queens have differential immune protective characteristics for different pathogens and parasites. This chapter highlights influencing factors for nonsynchronous ovarian development and variant immune-protective measures in female honey bees. The subsequent chapters elucidate the details of workers' ovarian programmed cell death under the regulation of multiple factors.

Keywords: Female Caste Development, Influencing Factors, Queen Quality. 5.1. INTRODUCTION In more primitive social insects such as polistes wasps, the suppression of worker ovarian development involves overly aggressive dominance interactions; whereas, in more advanced eusocial insects such as honey bees, reproductive dominance occurs by imposing workers' ovarian development inhibition via primer pheromones produced by the queen, larval nutritional discrimination, and by differential expression of various genetic elements. The specific factors aggregately result in the formation of two variant female castes within a honey bee colony, represented by a fertile queen and facultative reproducing worker honey bees with the same genome (Velthuis, 1976; Ratnieks and Visscher, 1989, Willis et al., 1990; Winston and Slessor, 1992; Keller and Nonacs, 1993,Ratnieks, Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

84 Polyandrous Queen Honey Bee

Lovleen Marwaha

1993; Rangel et al., 2013; McMenamin and Genersch, 2015; Rangel et al., 2015; Ashby et al., 2016; Wojceichowski et al., 2018; Yin et al., 2018; Rangel and Fisher, 2019). Further, honey bees' female castes exhibit diametric behavioural phenotypes but plastic developmental fates (Barchuk et al., 2007; de Azevedo and Hartfelder, 2008; Toth et al., 2009). The differential ovarian phenotype development facilitates the colony's physiological reproductive division of labour. Therefore, in queen and worker honey bees, there is variation in the size of ovaries and physiological activity within ovaries. In the worker honey bee ovary, there is the induction of massive programmed cell death (PCD), which destroys most of the ovariole primordial cells in the ovary during larval development. Analysis of transcriptional activity in workers' ovaries depicts differential gene expression associated with PCD. Eventually, the PCD process regulates the ovarian development in worker honey bees. Available reports indicate that a queen, which develops from younger larvae, possesses large body measurements and large spermathecae, therefore can mate with many males and can store comparatively more sperm. Further evidence is available that queens raised from younger worker larvae produce significantly more workers, enhancing colonial productivity than queens developed from older worker larvae (Tarpy et al., 2002, 2004; Rangel et al., 2013; Wojceichowski et al., 2018). Conclusively, the queen's quality is influenced collectively by numerous factors, including the stage of larvae, when it enters into the queen's developmental path, larval diet, various genetic elements, and various other environmental cues. Numerous studies have witnessed that the reproductive potential of honey bee queens varied with a change in ovariole number, spermathecae diameter, sperm counts, and sperm viability. Furthermore, the queen's body size is related to mating frequency, sperm storage, ovariole number, and ovarian weight (Eckert, 1934; Woyke, 1971; Dedej et al., 1998; Hatch et al., 1999; Tarpy et al., 2000; Gilley et al., 2003; Tarpy and Mayer, 2009; Delaney et al., 2011; Jackson et al., 2011; Tarpy et al., 2011). Additionally, polyandrous characteristics, i.e., mating with multiple drones, further influence the strength and productivity of a colony. After 1-2 weeks of post-emergence, the queen honey bee takes one or several mating flights to mate with drones of other honey bee colonies (Kraus et al., 2005). Mating usually occurs in drone congregation areas, away from natal colonies, to avoid inbreeding (Tarpy and Nielsen, 2002; Tarpy et al., 2004; Schlüns et al., 2005; Koeniger et al., 2014). Usually, 6–26 drones mate with the queen honey bee, with an average number of 12–14. Therefore, the honey bee queen is polyandrous (Snodgrass, 1956; Tarpy et al., 2002; Tarpy and Nielsen, 2002; Tarpy et al., 2004; Kraus et

Factors and Disease

Polyandrous Queen Honey Bee 85

al., 2005; Delaney et al., 2011). In addition, genetic marker analysis of worker honey bees revealed that the queen's offspring reflects the patriline subfamilies with which she had mated (Delaney et al., 2011; Koeniger et al., 2014). Additionally, multiple mating increases the genetic diversity of species, which increases the likelihood that a particular species can withstand new biotic and abiotic threats (Rueppell et al., 2008). Furthermore, multiple mating increases the number of stored sperm, enhances queen attractiveness, regulates the division of labour in the colony, stabilizes brood nest temperature, improves communication between workers, reduces the incidence of disease, and enhances colonial flourishment and multiplication (Tarpy, 2003; Palmer et al., 2003; Richard et al., 2007; Delaney et al., 2011; Tarpy et al., 2013; Williams et al., 2015). All these therefore enhance colony growth and survival. Overall, multiple mating affected the mating number and resulted in enhanced intra-colony genetic diversity of nest mates, which collectively improved the quality of the queen (Tarpy, 2003). Further, the queen honey bee secretes different pheromones to monopolize reproductive prevalence within the colony and suppress worker honey bees' normal development (Fig. 5a). In contrast, worker honey bees are facultatively reproducible, take reproductive command of the colony when they find an opportunity and also mimic the pheromonal efficacy of the queen to some extent (Velthuis and Van, 1964; Velthuis, 1967; Velthuis, 1976; Velthuis, 1985; Villa et al., 2005; Vanengelsdorp, 2008; Wheeler et al., 2014).

Fig. (5a). The diagram comprehensively summarises the functional integrity of different queen pheromones that facilitate her colonial dominance.

86 Polyandrous Queen Honey Bee

Lovleen Marwaha

The superior quality queen can modulate the overall colonial strength, caste ratio, productivity, swarming, aggressively docile nature of the colony, and disease resistance with her reproducibility and volatile chemical emissions from various pheromonal glands. Queen mandibular glands of Apis mellifera secrete prime pheromones, which induce programmed cell death during the development of ovarioles in worker honey-bees ovaries (Fig. 5a) et al., 1962; Furgala, 1962; Butler and Fairey, 1963; Fig, 1964; David, 1970; Bouletreau-Merle, 1978; ChaudNetto and Bueno, 1979; Crewe et al., 2000; Gençer et al., 2002; Dodologlu and Gene, 2003; Gilley et al., 2003; Amdam et al., 2006, Cox-Foster et al., 2007 Breeze, et al., 2011; Gauthier et al., 2011; Chairman et al., 2016; Rangel and Trapy, 2016; De et al., 2008; 2010; Di et al., 2016; Duncan, 2016; FernandezNicolas, and Belles, 2016; Gajger et al., 2017; De Souza, et al., 2018; Brutscher, et al., 2019)

Fig. (5b). Section of comb showing worker honey bees performing different duties. The hive section contains capped brood wax cells and pollen grain-filled cells. Developing worker larvae are fed on a composite mixture of workers' glandular secretion, pollen, and honey. Therefore, unripe honey and pollen cells are close to workers' larval cells.

Further, the queen honey bee possesses different immune protection measures than worker honey bees, against pathogens and pests, including mites, bacteria, and viruses. Various pathogens and parasites are responsible for poor health and increased colony losses (McMenamin and Genersch, 2015; Cornman et al., 2012; Dainat et al., 2012; Genersch et al., 2010; Genersch, 2010). Viruses including Deformed Wing Virus (DWV), Israeli Acute Bee Paralysis Virus (IAPV), and Acute Bee Paralysis Virus (ABPV) act as a significant threat to honey bee colonies (Boecking et al., 2008; Genersch et al., 2010; Le Conte et al., 2010;

Factors and Disease

Polyandrous Queen Honey Bee 87

Guzmán-Novoa et al., 2010; Nazzi et al., 2012, Di Prisco, et al., 2016). Additionally, Acarapis woodi, Nosema apis, and Nosema ceranae also act as challenging pests of honey bee colonies (Cox-Foster et al., 2007; Highfield et al., 2009; De Miranda and Genersch, 2010; De Miranda et al., 2010; Genersch et al., 2010; Nazzi et al., 2012; Francis et al., 2013; McMenamin and Genersch, 2015). Under extreme pathogen/parasite/pest attack, colony collapse can occur due to critical damage to the colony's comb, brood, adults, and stored food resources. Honey bee colony collapse is a complex phenomenon that can occur due to multiple biotic and abiotic factors (Fig. 5c) (Vanengelsdorp, et al., 2010; Potts, et al., 2010; Smith, et al., 2013; Meixner, et al., 2014, Goulson, et al., 2015).

Fig. (5c). Click of a highly damaged hive, heavily infested by wax moths. Observation says here that honey bee colonies abandoned the hive under heavy infection. At high temperatures, wax moth infestation becomes a critical challenge. Under such conditions, the honey bee colony usually prefers to swarm.

5.2. STRONG QUEEN AND COLONY Usually, queens with higher reproductive potential establish stronger colonies with exponential growth and sustainable survivability (Fig., 5b, 5d - 5h) (Hoopingarner, and Farrar, 1959; Renner and Baumann, 1964; Kerr et al., 1974; Nelson and Gary, 1983; Leonardo, 1985; Oldroyd et al., 1990; Laidlaw, 1992; Hartfelder, 1993; Hartfelder and Steinbrück, 1997; Kaftanoglu, 2000; Hepperle and Hartfelder, 2001; Haarmann et al., 2002; Harano et al., 2007; Jay, 1970; Jay, 1972; Jay and Nelson, 1973; Liu, 1992; Morse and Calderone, 2000; Reginato and Cruz-Landim, 2000; Reginato and Cruz-Landim, 2003; Koç and Karacaoglu, 2004;

88 Polyandrous Queen Honey Bee

Lovleen Marwaha

Martin and Serrao, 2004; Koeniger, et al., 2005; Richard, et al., 2007; Patel, et al., 2007; Highfield, et al., 2009; Hayworth, et al., 2009; Nazzi, et al.,2009; Ring, 2009; Le Conte, 2010; Vanengelsdorp and Meixner, 2010; Lyke, et al., 2010; Richard, et al., 2011; Hatjina, et al., 2014; Masry, et al., 2015; Lago, et al., 2016; Milchreit, et al., 2016; Polsinelli and Yu, 2018; Klein, et al., 2019; Odemer and Odemer, 2019) . A superior quality queen can modulate the strength of workers, which can construct a comparatively more substantial wax comb and considerably enhance the honey as well as pollen storage resources of the colony in comparison to a low-quality queen-headed colony. Additionally, a strong colony exhibits more remarkable winter survival due to plentiful stored food before winter (Winston, 1987). On the other hand, many reports indicate that a queen is superseded if she is diseased, injured, or has lower insemination success (Cook, 1968; Richard et al., 2007). Usually, apiarists prefer to do re-queening for desired colony growth and productivity. However, according to Rhodes et al., 2004, during re-queening, the survival of the introduced queen in the colony increases with the queen's age, reproductive potential and pheromonal quality. Their study further indicated that the survival rate of bees increased strongly when the newly introduced queen honey bee was between 7 and 24 days of age. As a result, there can be a decrease in colony population rapidly during queen replacement initially or after several weeks afterward (Tarpy et al., 2000). An array of glandular pheromones the queen produces in a honey bee colony regulates task organization and productivity (Kocher and Grozinger, 2011). Therefore, a superior quality queen from first instar worker larvae can enhance colony productivity by modulating worker attractiveness toward her, brood rearing, honey production through mandibular gland components, etc. On the other hand, the queen honey bee, which possesses lower self-fitness, usually exhibits a head colony with weak growth and productivity (Rangel et al., 2013). Various studies explain the variation in reproductive fitness of honey bee queens raised from different workers' larva instar in terms of variation in body size, ovariole number, diameter and number, and viability of drone spermatozoa stored in queens spermathecae. (Dedej et al., 1998; Hatch et al., 1999; Tarpy et al., 2000; Tarpy and Mayer, 2009; Delaney et al., 2011; Tarpy et al., 2011; Linksvayer et al., 2011; Rangel et al., 2013). Some studies indicate a positive correlation between queen weight and thoracic width with queen fecundity, egg-laying potential, ovary weight and ovariole number (Nelson and Smirl, 1977; Nelson and Gary, 1983; Dedej et al., 1998; Hatch et al., 1999; Gilley et al., 2003). In addition, Delaney et al., 2011 observed

Factors and Disease

Polyandrous Queen Honey Bee 89

that queen thoracic width and spermathecae size are directly related to sperm storage count, as a large queen can mate with many drones and store vast quantities of sperm in her spermathecae. Koeniger et al. (2014) reported different drones had produced variations in sperm numbers. Further, according to Schluins et al. (2003), the variation in sperm number follows the size of a drone, and Kraus et al. (2003) considered that sperm production depends upon the rearing condition of drones. Finally, Rangel et al., 2013 conclusively remarked that the reproductive potential of a queen affects a honey bee colony's phenotype. 5.3. QUEEN QUALITY AND ASSOCIATED FACTORS 5.3.1. Larval Selection Influence The developmental fate of the queen is highly plastic, which is influenced by queen rearing environment (Barchuk et al., 2007; de Azevedo and Hartfelder, 2008; Linksvayer et al., 2011; Rangel et al., 2013). Concerned plasticity of queen formation influences her morphology, anatomy, physiology, reproduction, disease resistance, life span, retinue behaviour induction tendency, colonial regulation, and productivity. The queens raised from younger worker larvae exhibited higher body measurements compared to queens who developed from older worker larvae. In other words, the adult queen phenotype varies as per the age at which larvae enter the queen developmental pathway (Hatch et al., 1999; Tarpy et al., 2011; Range et al., 2013). Many reports witness that workers in the queen-less colony select worker larvae from 1-3 instars for queen raising (Hatch et al., 1999). Tarpy et al. (2011) observed that queens developed from zero-day-old worker larvae exhibited higher stored sperm count in spermathecae than those raised from 2-day old worker larvae. Rangel et al., 2013, had compared the development of a queen from a 0day-old worker larva with a 2-day-old worker honey bee larva. Further, they have concluded that the queen developed from a younger larva has a large body, high ovariole number, and comparatively larger sperm storage in spermathecae.

90 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (5d). Photograph of the queen with poor retinue induction potential. Pheromonal profiling of the queen determine workers' attraction toward the queen . Above clicked colony queen possess low pheromonal induced retinue in workers.

Fig. (5e). Click from the strong colony with the queen at the lower portion of the hive. Queen secretes reasonable proportionality of pheromones and induces a more robust retinue response. Pheromonal secretion of the queen is dependent upon her age, food availability, genotype, colonial environment, abiotic factors, etc

Factors and Disease

Polyandrous Queen Honey Bee 91

Fig. (5f). Pictorial presentation of hive section from a colony. Worker honey bees are engaged in brood rearing activities.

Fig. (5g). Few worker bees at the entrance of the brood chamber. Usually, workers deposit footprint pheromones at the entrance, facilitating the rest of the nestmates to locate the colony.

92 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (5h). A brood chamber with frames having bee comb on either side of each comb.

Sagili et al., 2018) experimented by manipulating the nutritional status of oneday-old larvae by depriving them of brood food for four hours. Subsequently, worker honey bees were allowed to choose larvae for rearing queens from nutritional-deprived and non-deprived larvae. They concluded that more worker honey bees get attracted to the non-deprived larval queen than the deprived larvae queen. Further, De Souza et al.,2018 considered the conjugative influence of larval age with variable developmental hormones concentration on queen rearing event. Their drawn conclusion indicated that younger larvae eventually develop into imagoes with comparatively more queen-like morphology, and juvenile hormone application during the larval stage positively influences the structural integrity of ovaries concomitantly with the number of ovarioles. Furthermore, it has been concluded that younger age and high juvenile hormone concentration collectively affect ovarian system functionality. Wojciechowski et al., 2018 concluded that metabolic flux acting via epigenetics regulation, particularly DNA methylation and micro RNA, establishes the gene expression pattern for caste-specific development. Further, they produced the first genome-wide map of the chromatin structure of honey bees during larval development. They conclusively remarked that developmental canalization into queen or worker is virtually irreversible. There had been a difference in

Factors and Disease

Polyandrous Queen Honey Bee 93

H3Kume3, H3K27ac, and H3K36m3 gene expression correlated with castespecific transcription. Further, they demonstrated the critical role of chromatin modification in the establishment and maintenance of caste-specific transcriptional program in honey bees (detail about responsible genetic element is highlighted in the next chapter). 5.3.2. Other Factors The quality of a queen bee depends on genotype, nutrition, rearing methods, rearing season, age of larvae at grafting time, number of larvae grafted per cell, and environmental factors (Kaftanoglu et al., 1992). Therefore, geneticists generally implement selective breeding programs to improve the quality of bees for quantitative honey production, gentleness, and disease resistance (Rotenbuhler, 1964; Spivak, 1996; Spivak and Gilliam, 1998; Harbo and Harris, 1999; Spivak and Reuter, 2001; Wilkes and Oldroyd, 2002). Furthermore, the rearing conditions of larvae, including colonial volatiles, temperature, nutrition, etc., influence the queen's quality. In addition, the live weight of the queen at the time of emergence is a good criterion for determining queen quality. Finally, there is a substantial paternal genetic influence on the quality of the queen. Eventually, the quality of the queen is influenced by the genetic variance of the drone, the age of larvae, the strength of the colony, and the number of nurse bees specifically caring for queen cells. Sometimes colony rejects the newly introduced queen, the reason for the presence of the old queen in the colony. However, the good queen starts producing pheromones, becoming accepted after the introduction. Furthermore, the genetics of the colony, climatic conditions, nectar, pollen flow, and queen introduction methods further affect queen acceptance within the colony. 5.4. OVARIAN WEIGHT AND FUNCTIONALITY AS QUEEN QUALITY INDEXES Woyke (1971) observed negative proportionality of the queen's body weight, and ovariole number of the virgin queen as per age selected larval before stepping into divergent caste-specific differentiation. Available reports suggest that queen weight is correlated with the weight of the ovary, size and number of ovarioles, the diameter of spermathecae, and the number of stored spermatozoa (Woyke, 1971; Dedej et al., 1998; Kaftanoglu et al., 2000; Kahya et al., 2008; Delaney et al., 2011; Tarpy et al., 2011; 2012). Therefore body weight is a good indicator of queen quality. Further, as ovaries occupy a sizeable abdominal area of the queen,

94 Polyandrous Queen Honey Bee

Lovleen Marwaha

the ovarian weight acts as the physical criterion to assess the reproductive potential of the queen honey bee (Winston, 1987; Kahya et al., 2008; Gilley et al., 2003). Delaney et al., 2011 concluded that, similarly, the queen's thorax width is proportionately associated with her stored sperm number and mating frequency (Tarpy et al., 2002: Delaney et al., 2011: Tarpy et al., 2012; Jackson et al., 2011). The virgin queen's ovaries are smaller than the egg-laying queen's, which indicates a positive link between ovarian size and functionality (Shehata et al., 1981; Patricio and Cruz-Landim, 2002). According to Shehata et al., 1981 ovaries of the egg-laying queen are eight times larger than those of virgins (Shehata et al., 1981). Ovarian development initiates actively after mating and a specific developmental phase is associated with distinct gene expression patterns in the brain and ovaries, which further impact in physiology and behaviour of the queen (Richard et al., 2007; Kocher et al., 2008; Nino et al., 2013). During winter, egg laying capacity and development of the queen decreases or stops, highlighting the influence of temperature on the structural and functional organization of ovaries (Shehata et al., 1981). Ovary size and fertility of the queen are interconnected (Tarpy et al., 2000), under various environmental challenges (Kahya et al., 2008). Several ovarioles can be counted during any specific time in queen but are generally more reliable after post-mating a few months (Carreck et al., 2013). In addition to the ovariole number, the length of the ovariole is also linked to the size of the ovaries, which reflects the physiological status of the ovary. Further, in queen honey bees, left and right ovaries can be symmetrical present (Carreck et al., 2013) or may not (Jackson et al., 2011). After mating, spermatozoa are stored in spermathecae for the remaining queen's life (Winston, 1987). Therefore, stored sperm number and viability of same are some factors that affect the reproductive capacity of the queen and her lifespan (Akyol et al., 2008, Al-Lawati, et al., 2009). 5.5. QUALITY OF QUEEN ON THE BASICS OF SPERMATHECAE Further, the size of spermathecae and the number of spermatozoa are good indicator of queen quality (Kaftanoglu et al., 1992) as the size of spermathecae influence the storage of more sperms, hence fertility. Queen with greater sperm storage can lay more fertilized eggs, therefore better chances of survival in the colony. Queen with comparative smaller spermathecae possesses more probability of being superseded. Experimental data suggest that queens raised from younger larvae have larger spermathecae (Hatch et al., 1999; Tarpy et al., 2000; Gilley et al., 2003). Queen weight significantly influences the onset of oviposition, the acceptance ratio of queens, the diameter of spermathecae, and the number of

Factors and Disease

Polyandrous Queen Honey Bee 95

spermatozoa in spermathecae (Akyol et al., 2008). The spermathecal wall is composed of a single layer of columnar epithelium lined internally with thick mucinous cuticles (Carreck et al., 2013; Hatjina et al., 2014). Spermathecae are rarely filled with spermatozoa in post-mated queen under experimental conditions, as procurable explorations witnesses about 47% filling of spermathecae (Jackson et al., 2011; Tarpy, et al., 2011). Queen longevity is related to an adequate number of sperm in the spermatheca because with depletion of stored sperm queen starts laying unfertilized eggs due to depletion of stored sperm (Winston, 1987). Post-mating queen possesses storage of about 100 million spermatozoa (Woyke, 1962). About 3-5% of semen discharged by each drone gets stored (Woyke, 1962). Several stored spermatozoa reflect the quality of the queen (Delaney et al., 2011; Tarpy et al., 2012). According to Woyke, 1962, a queen with less than 3 million sperm in her spermathecae is an inadequate mated. Various reports indicate that 13.6-19.0% of commercial produced queens possess sperm stored below the threshold (Camazine et al., 1998; Skowronek et al., 2002, 2004; Delaney et al., 2011; Tarpy et al., 2012). Furthermore, spermatozoa's viability is crucial for mating and reproductive success (Den Boer et al., 2009). Secretion from the spermathecal gland of the queen helps in the viability of sperm for several years (King et al., 2011; Zareie et al., 2013). Sperm motility had been reported in the spermathecae of the older queen (Lodesani et al.,2004). However, the movement pattern is slower in older queens than in younger ones (Al-Lawati et al., 2009). Furthermore, sperm viability is affected by temperature spikes, which result in colony failure (Pettis et al., 2016). 5.6. DISEASES AND REPRODUCTIVITY OF QUEEN HONEY BEE Apis mellifera live in eusocial colonies headed by a single queen with specific duties of exponential reproduction and pheromonal emission with multiple functions. Therefore, the queen's health directly influences the wealth of workers, colony strength, caste ratio, productivity, and behaviour like aggressiveness/ calmness of the colony. Like the worker, the Queen honey bee is susceptible to various infections (Rueppell et al., 2016). Queen honey bees can suffer from Acarapis woodi, an obligate endoparasite of honey bees. According to Pettis et al., 1989, a 1-day-old queen carries an average of 6.5 mites, whereas a 10-day-old queen can carry ten mites. Furthermore, there is a further increase in the chances of infection to the queen in case of severe

96 Polyandrous Queen Honey Bee

Lovleen Marwaha

infection to workers (Pettis et al., 1989; Villa et al., 2005). However, the impact of Acarapis woodi on the queen's performance is unknown (Amiri et al., 2017). Queen is considerably less susceptible to infection of tracheal mites because of the less post-capping stage of the queen brood and additionally the presence of octanoic acid in royal jelly (Calderone et al., 2002; Nazzi et al., 2009). Varroa mites prefer parasite drones over queens and workers, as drones have a longer development time (Fuchs, 1990; Calderone et al., 2002). Nosema apis (N. apis)and Nosema ceranae(N. ceranae) can infect the queen honey bee by attacking the midgut of the epithelial Cell (De Graaf et al., 1994; Gauthier et al., 2011; Fries et al., 2013). Nosema ceranae attach to queen ovaries, and it has been detected that infection can occur to queen larvae also (Traver and Fell, 2012; Roberts et al., 2015). Transmission of Nosema spp. can occur presumably in the adult stage, even during mating. However, the semen of drones can kill N. apis spores, which reduces the risk of disease transmission during mating. Nosemosis can badly affect queen physiology (Higes et al., 2009; Alaux et al., 2011). Available reports indicate that queens infected with N. apis oviposit late in comparison to healthy ones (Hassanein, 1951; Loskotova et al., 1980) exhibit changed pheromone production (Alaux et al., 2011), and further oocyte degeneration can cause infertility to her (Liu, 1992). N. apis infection can reduce the queen's lifespan to an average of 50 days, resulting in queen supersedure (Loskotova et al., 1980). There is an increase in the level of vitellogenin and another antioxidant enzyme in infected queens to counter imposed physiological challenges (Alaux et al., 2011). Counter nutritive change, as a protection measure, is too costly for the queen to survive in the long term. Deformed Wing Virus (DWV) is more common in colonies with high mite infection (Fievet et al., 2006). Infected drones can fly and move in congregation areas to mate with younger queens (Yañez et al., 2012; Amiri et al.,2016). Deformed wing virus had been detected from semen collected from queen spermatheca (Yue et al., 2006; Yañez et al., 2012; Amiri et al., 2016). It has been reported that the virus could be transmitted from an infected queen to her offspring (Yue et al., 2005; Chen et al., 2006; De Miranda et al., 2008; Ravoet et al., 2015). DWV has been observed to affect queens' heads, fat bodies, gut, and ovaries (Fievet et al., 2006; Chen et al., 2006; Gauthier et al., 2011; Amiri et al., 2016). Severe infection can cause ovarian tissue degeneration, affecting sperm viability (Gauthier et al., 2011). Similarly, Chronic Bee Paralysis Virus (CBPV) had been detected in relatively low concentration in queen (Chen et al., 2005; Chen et al., 2006; Gregorc and

Factors and Disease

Polyandrous Queen Honey Bee 97

Bakonyi, 2012). Infected queen honey bees show symptoms including trembling legs, spread, disjunct wings, and a bloated abdomen filled with hemolymph. Honey bee queens possess similar susceptibility to C BPV-like workers (Amiri et al., 2014). Sacbrood Virus (SBV) had been detected in the queen, mostly in ovaries, which affects the functionality of the concerned organ (Chen et al., 2005; Fievet et al., 2006; Ravoet et al., 2015). Black Queen Cell Virus (BQCV) has been reported to infect queen larvae and pupae. The specific virus had been detected in honey bee queens, mainly in the gut, faecal matter, and ovaries (Chen et al., 2005; Chen et al., 2006). Conclusively, diverse viruses can be problematic to the queen, but most viruses remain asymptomatic in the queen with potential transmission efficacy to the next generations(Gregorc, and Bakonyi, 2012; Ravoet et al., 2015). Overall remarks for the above description is, somewhere queen's quality expressed in the form of her fecundity and volatile emission aggregately is dependent upon her genomic content, larval age before entering into caste diversification, larval diet composition, and other environmental factors. A variant larval diet and comparatively shorter developmental phase additionally empower her with more contra susceptibility to various infections/infestation from different pathogens, parasites, and pests. CONCLUSION Special diet and superior developmental process impart unique characteristics to the queen honey bee, which improve her fecundity, fertility, physiology, immune system, and life span. Due to differential genomic expression, her immunological response becomes better than other castes within the colony. Queen honey bee becomes resistant to various pathogens and parasites. REFERENCES Akyol, E, Yeninar, H & Kaftanoglu, O (2008) Live weight of queen honey bees (Apis mellifera L.) predicts reproductive characteristics. J Kans Entomol Soc, 81, 92-100. [http://dx.doi.org/10.2317/JKES-705.13.1] Al-Lawati, H, Kamp, G & Bienefeld, K (2009) Characteristics of the spermathecal contents of old and young honeybee queens. J Insect Physiol, 55, 117-22. [http://dx.doi.org/10.1016/j.jinsphys.2008.10.010] [PMID: 19027748] Alaux, C, Folschweiller, M, McDonnell, C, Beslay, D, Cousin, M, Dussaubat, C, Brunet, JL & Conte, YL (2011) Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera). J Invertebr Pathol, 106, 380-5. [http://dx.doi.org/10.1016/j.jip.2010.12.005] [PMID: 21156180] Amdam, GV, Csondes, A, Fondrk, MK & Page, RE, Jr (2006) Complex social behaviour derived from

98 Polyandrous Queen Honey Bee

Lovleen Marwaha

maternal reproductive traits. Nature, 439, 76-8. [http://dx.doi.org/10.1038/nature04340] [PMID: 16397498] Amiri, E, Meixner, MD & Kryger, P (2016) Deformed wing virus can be transmitted during natural mating in honey bees and infect the queens. Sci Rep, 6, 33065. [http://dx.doi.org/10.1038/srep33065] [PMID: 27608961] Amiri, E, Strand, M, Rueppell, O & Tarpy, D (2017) Queen quality and the impact of honey bee diseases on queen health: potential for interactions between two major threats to colony health. Insects, 8, 48. [http://dx.doi.org/10.3390/insects8020048] [PMID: 28481294] Arnold, G, Le Conte, Y, Trouiller, J, Hervet, H & Chappe, B (1994) Inhibition of worker honeybee ovaries development by a mixture of fatty acid esters from larvae. Comptes rendus de l'Académie des sciences Série 3, Sciences de la vie,, 317, 511-5. Ashby, R, Forêt, S, Searle, I & Maleszka, R (2016) MicroRNAs in honey bee caste determination. Sci Rep, 6, 18794. [http://dx.doi.org/10.1038/srep18794] [PMID: 26739502] Barchuk, AR, Cristino, AS, Kucharski, R, Costa, LF, Simões, ZLP & Maleszka, R (2007) Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera. BMC Dev Biol, 7, 70. [http://dx.doi.org/10.1186/1471-213X-7-70] [PMID: 17577409] Boecking, O & Genersch, E (2008) Varroosis–the ongoing crisis in bee keeping. J Verbraucherschutz Lebensmsicherh, 3, 221-8. [http://dx.doi.org/10.1007/s00003-008-0331-y] Boulétreau, J (1978) Ovarian activity and reproductive potential in a natural population of Drosophila melanogaster. Oecologia, 35, 319-42. [http://dx.doi.org/10.1007/BF00345140] [PMID: 28310277] Breeze, TD, Bailey, AP, Balcombe, KG & Potts, SG (2011) Pollination services in the UK: How important are honeybees? Agric Ecosyst Environ, 142, 137-43. [http://dx.doi.org/10.1016/j.agee.2011.03.020] Brutscher, LM, Baer, B & Niño, EL (2019) Putative drone copulation factors regulating honey bee (Apis mellifera) queen reproduction and health: A review. Insects, 10, 8. [http://dx.doi.org/10.3390/insects10010008] [PMID: 30626022] Burgett, M & Kitprasert, C (1992) Tracheal mite infestation of queen honey bees. J Apic Res, 31, 110-1. [http://dx.doi.org/10.1080/00218839.1992.11101269] Butler, CG, Callow, RK & Johnston, NC (1962) The isolation and synthesis of queen substance, 9-oxodectrans -2-enoic acid, a honeybee pheromone. Proc R Soc Lond B Biol Sci, 155, 417-32. [http://dx.doi.org/10.1098/rspb.1962.0009] Butler, CG & Fairey, EM (1963) The role of the queen in preventing oogenesis in worker honey-bees. J Apic Res, 2, 14-8. [http://dx.doi.org/10.1080/00218839.1963.11100051] Calderone, NW & Kuenen, LPS (2003) Differential tending of worker and drone larvae of the honey bee, Apis mellifera, during the 60 hours prior to cell capping. Apidologie (Celle), 34, 543-52. [http://dx.doi.org/10.1051/apido:2003054] Camazine, S, Cakmak, I, Cramp, K, Finley, J, Fisher, J, Frazier, M & Rozo, A (1998) How healthy are commercially-produced US honey bee queens?. American bee journal (USA) Carreck, NL, Andree, M, Brent, CS, Cox-Foster, D, Dade, HA, Ellis, JD, Hatjina, F & van Englesdorp, D (2013) Standard methods for Apis mellifera anatomy and dissection. J Apic Res, 52, 1-40. [http://dx.doi.org/10.3896/IBRA.1.52.4.03] Chaimanee, V, Evans, JD, Chen, Y, Jackson, C & Pettis, JS (2016) Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the

Factors and Disease

Polyandrous Queen Honey Bee 99

organophosphate acaricide coumaphos. J Insect Physiol, 89, 1-8. [http://dx.doi.org/10.1016/j.jinsphys.2016.03.004] [PMID: 26979384] Chaud-Netto, J & Bueno, OC (1979) Number of ovarioles in workers of Apis mellifera adansonii and Apis mellifera ligustica: a comparative study. J Apic Res, 18, 260-3. [http://dx.doi.org/10.1080/00218839.1979.11099980] Chen, YP, Pettis, JS, Collins, A & Feldlaufer, MF (2006) Prevalence and transmission of honeybee viruses. Appl Environ Microbiol, 72, 606-11. [http://dx.doi.org/10.1128/AEM.72.1.606-611.2006] [PMID: 16391097] Chen, YP, Pettis, JS, Corona, M, Chen, WP, Li, CJ, Spivak, M, Visscher, PK, DeGrandi-Hoffman, G, Boncristiani, H, Zhao, Y, vanEngelsdorp, D, Delaplane, K, Solter, L, Drummond, F, Kramer, M, Lipkin, WI, Palacios, G, Hamilton, MC, Smith, B, Huang, SK, Zheng, HQ, Li, JL, Zhang, X, Zhou, AF, Wu, LY, Zhou, JZ, Lee, ML, Teixeira, EW, Li, ZG & Evans, JD (2014) Israeli acute paralysis virus: epidemiology, pathogenesis and implications for honey bee health. PLoS Pathog, 10e1004261 [http://dx.doi.org/10.1371/journal.ppat.1004261] [PMID: 25079600] Chen, Y, Pettis, JS & Feldlaufer, MF (2005) Detection of multiple viruses in queens of the honey bee Apis mellifera L. J Invertebr Pathol, 90, 118-21. [http://dx.doi.org/10.1016/j.jip.2005.08.005] [PMID: 16214161] Collins, AM & Pettis, JS (2013) Correlation of queen size and spermathecal contents and effects of miticide exposure during development. Apidologie (Celle), 44, 351-6. [http://dx.doi.org/10.1007/s13592-012-0186-1] Cook, VA (1968) Severe clipping of queens’ wings increases supersedure rate. NZ Beekpr, 30, 32. Cornman, RS, Tarpy, DR, Chen, Y, Jeffreys, L, Lopez, D, Pettis, JS, vanEngelsdorp, D & Evans, JD (2012) Pathogen webs in collapsing honey bee colonies. PLoS One, 7e43562 [http://dx.doi.org/10.1371/journal.pone.0043562] [PMID: 22927991] Cox-Foster, DL, Conlan, S, Holmes, EC, Palacios, G, Evans, JD, Moran, NA, Quan, PL, Briese, T, Hornig, M, Geiser, DM, Martinson, V, vanEngelsdorp, D, Kalkstein, AL, Drysdale, A, Hui, J, Zhai, J, Cui, L, Hutchison, SK, Simons, JF, Egholm, M, Pettis, JS & Lipkin, WI (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318, 283-7. [http://dx.doi.org/10.1126/science.1146498] [PMID: 17823314] Czekońska, K (2000) The influence of Nosema apis on young honey-bee queens and transmission of the disease from queens to workers. Apidologie (Celle), 31, 701-6. [http://dx.doi.org/10.1051/apido:2000154] Crewe, RM & Velthuis, HHW (1980) False queens: A consequence of mandibular gland signals in worker honeybees. Naturwissenschaften, 67, 467-9. [http://dx.doi.org/10.1007/BF00405650] Crozier, RH & Pamilo, P (1996) Evolution of social insect coloniesOxford University. De Souza, DA, Kaftanoglu, O, De Jong, D, Page, RE, Jr, Amdam, GV & Wang, Y (2018) Differences in the morphology, physiology and gene expression of honey bee queens and workers reared in vitro versus in situ. Biol Open, 7bio.036616 [http://dx.doi.org/10.1242/bio.036616] [PMID: 30341101] Dainat, B, Evans, JD, Chen, YP, Gauthier, L & Neumann, P (2012) Predictive markers of honey bee colony collapse. PLoS One, 7e32151 [http://dx.doi.org/10.1371/journal.pone.0032151] [PMID: 22384162] Danty, E, Arnold, G, Burmester, T, Huet, JC, Huet, D, Pernollet, JC & Masson, C (1998) Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera. Insect Biochem Mol Biol, 28, 387-97. [http://dx.doi.org/10.1016/S0965-1748(98)00011-3] [PMID: 9692239] David, JR (1970) Le nombre d’ovarioles chez la Drosophila: relation avec la fecondite et valeur adaptive.

100 Polyandrous Queen Honey Bee

Lovleen Marwaha

Arch Zool Exp Gén, 111, 357-70. de Azevedo, SV & Hartfelder, K (2008) The insulin signaling pathway in honey bee (Apis mellifera) caste development — differential expression of insulin-like peptides and insulin receptors in queen and worker larvae. J Insect Physiol, 54, 1064-71. [http://dx.doi.org/10.1016/j.jinsphys.2008.04.009] [PMID: 18513739] De Graaf, DC, Raes, H, Sabbe, G, De Rycke, PH & Jacobs, FJ (1994) Early development of Nosema apis (Microspora: Nosematidae) in the midgut epithelium of the honey-bee (Apis mellifera). J Invertebr Pathol, 63, 74-81. [http://dx.doi.org/10.1006/jipa.1994.1012] de Groot, AP & Voogd, S (1954) On the ovary development in queenless worker bees (Apis mellifica L.). Experientia, 10, 384-5. [http://dx.doi.org/10.1007/BF02160554] [PMID: 13210347] de Miranda, JR, Cordoni, G & Budge, G (2010) The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex. J Invertebr Pathol, 103 (Suppl. 1), S30-47. [http://dx.doi.org/10.1016/j.jip.2009.06.014] [PMID: 19909972] de Miranda, JR & Fries, I (2008) Venereal and vertical transmission of deformed wing virus in honeybees (Apis mellifera L.). J Invertebr Pathol, 98, 184-9. [http://dx.doi.org/10.1016/j.jip.2008.02.004] [PMID: 18358488] de Miranda, JR & Genersch, E (2010) Deformed wing virus. J Invertebr Pathol, 103 (Suppl. 1), S48-61. [http://dx.doi.org/10.1016/j.jip.2009.06.012] [PMID: 19909976] De Souza, DA, Kaftanoglu, O, De Jong, D, Page, RE, Jr, Amdam, GV & Wang, Y (2018) Differences in the morphology, physiology and gene expression of honey bee queens and workers reared in vitro versus in situ. Biol Open, 7bio.036616 [http://dx.doi.org/10.1242/bio.036616] [PMID: 30341101] Dedej, S, Hartfelder, K, Aumeier, P, Rosenkranz, P & Engels, W (1998) Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee ( Apis mellifera carnica ). J Apic Res, 37, 183-90. [http://dx.doi.org/10.1080/00218839.1998.11100970] Delaney, DA, Keller, JJ, Caren, JR & Tarpy, DR (2011) The physical, insemination, and reproductive quality of honey bee queens (Apis mellifera L.). Apidologie (Celle), 42, 1-13. [http://dx.doi.org/10.1051/apido/2010027] Delaplane, KS & Harbo, JR (1987) Drone production by young versus old worker honey-bees in queenless colonies. Apidologie (Celle), 18, 115-20. [http://dx.doi.org/10.1051/apido:19870201] den Boer, SPA, Boomsma, JJ & Baer, B (2009) Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage. J Insect Physiol, 55, 538-43. [http://dx.doi.org/10.1016/j.jinsphys.2009.01.012] [PMID: 19232404] Di Prisco, G, Annoscia, D, Margiotta, M, Ferrara, R, Varricchio, P, Zanni, V, Caprio, E, Nazzi, F & Pennacchio, F (2016) A mutualistic symbiosis between a parasitic mite and a pathogenic virus undermines honey bee immunity and health. Proc Natl Acad Sci USA, 113, 3203-8. [http://dx.doi.org/10.1073/pnas.1523515113] [PMID: 26951652] Dodologlu, A & Gene, F (2003) Comparison of some features of queens reared from different honey-bee (Apis mellifera L.) genotypes. J Appl Anim Res, 24, 105-9. [http://dx.doi.org/10.1080/09712119.2003.9706442] Duncan, EJ, Hyink, O & Dearden, PK (2016) Notch signalling mediates reproductive constraint in the adult worker honeybee. Nat Commun, 7, 12427. [http://dx.doi.org/10.1038/ncomms12427] [PMID: 27485026] Eckert, JE (1934) Studies in the number of ovarioles in queen honey-bees in relation to body size. J Econ

Factors and Disease

Polyandrous Queen Honey Bee 101

Entomol, 27, 629-35. [http://dx.doi.org/10.1093/jee/27.3.629] Evans, JD & Wheeler, DE (1999) Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proc Natl Acad Sci USA, 96, 5575-80. [http://dx.doi.org/10.1073/pnas.96.10.5575] [PMID: 10318926] Farrar, CL (1947) Nosema losses in package bees as related to queen supersedure and honey yields. J Econ Entomol, 40, 333-8. [http://dx.doi.org/10.1093/jee/40.3.333] [PMID: 20264495] Fernandez-Nicolas, A & Belles, X (2016) CREB-binding protein contributes to the regulation of endocrine and developmental pathways in insect hemimetabolan pre-metamorphosis. Biochim Biophys Acta, Gen Subj, 1860, 508-15. [http://dx.doi.org/10.1016/j.bbagen.2015.12.008] Fievet, J, Tentcheva, D, Gauthier, L, de Miranda, J, Cousserans, F, Colin, ME & Bergoin, M (2006) Localization of deformed wing virus infection in queen and drone Apis mellifera L. Virol J, 3, 16. [http://dx.doi.org/10.1186/1743-422X-3-16] [PMID: 16569216] Francis, RM, Nielsen, SL & Kryger, P (2013) Patterns of viral infection in honey bee queens. J Gen Virol, 94, 668-76. [http://dx.doi.org/10.1099/vir.0.047019-0] [PMID: 23223622] Francis, RM, Nielsen, SL & Kryger, P (2013) Varroa-virus interaction in collapsing honey bee colonies. PLoS One, 8e57540 [http://dx.doi.org/10.1371/journal.pone.0057540] [PMID: 23526946] Fries, I, Chauzat, MP, Chen, YP, Doublet, V, Genersch, E, Gisder, S, Higes, M, McMahon, DP, MartínHernández, R, Natsopoulou, M, Paxton, RJ, Tanner, G, Webster, TC & Williams, GR (2013) Standard methods for Nosema research. J Apic Res, 52, 1-28. [http://dx.doi.org/10.3896/IBRA.1.52.1.14] Fuchs, S (1990) Preference for drone brood cells by Varroa jacobsoni Oud in colonies of Apis mellifera carnica. Apidologie (Celle), 21, 193-9. [http://dx.doi.org/10.1051/apido:19900304] Furgala, B (1962) The effect of the intensity of Nosema inoculum on queen supersedure in the honey bee, Apis mellifera Linnaeus. J Insect Pathol, 4, 429-32. Fyg, W (1964) Anomalies and diseases of the queen honey bee. Annu Rev Entomol, 9, 207-24. [http://dx.doi.org/10.1146/annurev.en.09.010164.001231] Gajger, IT, Sakač, M & Gregorc, A (2017) Impact of thiamethoxam on honey bee queen (Apis mellifera carnica) reproductive morphology and physiology. Bull Environ Contam Toxicol, 99, 297-302. [http://dx.doi.org/10.1007/s00128-017-2144-0] [PMID: 28748329] Gauthier, L, Ravallec, M, Tournaire, M, Cousserans, F, Bergoin, M, Dainat, B & de Miranda, JR (2011) Viruses associated with ovarian degeneration in Apis mellifera L. queens. PLoS One, 6e16217 [http://dx.doi.org/10.1371/journal.pone.0016217] [PMID: 21283547] Gençer, HV, Firatli, C & Firatli, Ç (2000) Effects of supplemental feeding of queen rearing colonies and larval age on the acceptance of grafted larvae and queen traits. Pak J Biol Sci, 3, 1319-22. [http://dx.doi.org/10.3923/pjbs.2000.1319.1322] Genersch, E, von der Ohe, W, Kaatz, H, Schroeder, A, Otten, C, Büchler, R, Berg, S, Ritter, W, Mühlen, W, Gisder, S, Meixner, M, Liebig, G & Rosenkranz, P (2010) The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Apidologie (Celle), 41, 332-52. [http://dx.doi.org/10.1051/apido/2010014] Genersch, E (2010) Honey bee pathology: current threats to honey bees and beekeeping. Appl Microbiol Biotechnol, 87, 87-97.

102 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1007/s00253-010-2573-8] [PMID: 20401479] Gerhardt, J & Kirschner, M (1997) Cells, Embryos and EvolutionBlackwell, Malden, MA. Gilley, DC, Tarpy, DR & Land, BB (2003) Effect of queen quality on interactions between workers and dueling queens in honeybee ( Apis mellifera L.) colonies. Behav Ecol Sociobiol, 55, 190-6. [http://dx.doi.org/10.1007/s00265-003-0708-y] Goulson, D, Nicholls, E, Botías, C & Rotheray, EL (2015) Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science, 3471255957 [http://dx.doi.org/10.1126/science.1255957] [PMID: 25721506] Grandperrin, D & Cassier, P (1983) Anatomy and ultrastructure of the Koschewnikow’s gland of the honey bee, Apis mellifera L. (Hymenoptera : Apidae). Int J Insect Morphol Embryol, 12, 25-42. [http://dx.doi.org/10.1016/0020-7322(83)90033-8] Gregorc, A & Bakonyi, T (2012) Viral infections in queen bees (Apis mellifera carnica) from rearing apiaries. Acta Vet Brno, 81, 15-9. [http://dx.doi.org/10.2754/avb201281010015] Guzmán-Novoa, E, Eccles, L, Calvete, Y, Mcgowan, J, Kelly, PG & Correa-Benítez, A (2010) Varroa destructor is the main culprit for the death and reduced populations of overwintered honey bee ( Apis mellifera ) colonies in Ontario, Canada. Apidologie (Celle), 41, 443-50. [http://dx.doi.org/10.1051/apido/2009076] Haarmann, T, Spivak, M, Weaver, D, Weaver, B & Glenn, T (2002) Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations. J Econ Entomol, 95, 28-35. [http://dx.doi.org/10.1603/0022-0493-95.1.28] [PMID: 11942761] Harano, KI, Sasaki, M & Sasaki, K (2007) Effects of reproductive state on rhythmicity, locomotor activity and body weight in European honey-bee, Apis mellifera (Hymenoptera, Apini) queens. Sociobiology, 50, 189-200. Harbo, JR & Harris, JW (1999) Selecting honey bees for resistance to Varroa jacobsoni. Apidologie (Celle), 30, 183-96. [http://dx.doi.org/10.1051/apido:19990208] Harmon, MA, Boehm, MF, Heyman, RA & Mangelsdorf, DJ (1995) Activation of mammalian retinoid X receptors by the insect growth regulator methoprene. Proc Natl Acad Sci USA, 92, 6157-60. [http://dx.doi.org/10.1073/pnas.92.13.6157] [PMID: 7597096] Hartfelder, K (1993) Structure and function of the prothoracic gland in honey bee ( Apis mellifera L.) development. Invertebr Reprod Dev, 23, 59-74. [http://dx.doi.org/10.1080/07924259.1993.9672294] Hartfelder, K & Steinbrück, G (1997) Germ cell cluster formation and cell death are alternatives in castespecific differentiation of the larval honey bee ovary. Invertebr Reprod Dev, 31, 237-50. [http://dx.doi.org/10.1080/07924259.1997.9672582] Hassanein, MH (1951) Studies on the effect of infection with Nosema apis on the physiology of the queen honey-bee. J Cell Sci, s3-92, 225-31. [http://dx.doi.org/10.1242/jcs.s3-92.18.225] Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Hatjina, F, Bieńkowska, M, Charistos, L, Chlebo, R, Costa, C, Dražić, MM, Filipi, J, Gregorc, A, Ivanova, EN, Kezić, N, Kopernicky, J, Kryger, P, Lodesani, M, Lokar, V, Mladenovic, M, Panasiuk, B, Petrov, PP, Rašic, S, Skerl, MIS, Vejsnæs, F & Wilde, J (2014) A review of methods used in some European countries for assessing the quality of honey bee queens through their physical characters and the performance of their colonies. J Apic Res, 53, 337-63.

Factors and Disease

Polyandrous Queen Honey Bee 103

[http://dx.doi.org/10.3896/IBRA.1.53.3.02] Hayworth, MK, Johnson, NG, Wilhelm, ME, Gove, RP, Metheny, JD & Rueppell, O (2009) Added weights lead to reduced flight behavior and mating success in polyandrous honey bee queens (Apis mellifera). Ethology, 115, 698-706. [http://dx.doi.org/10.1111/j.1439-0310.2009.01655.x] Hepburn, HR (1994) Reproductive cycling and hierarchical competition in Cape honeybees, Apis mellifera capensis Esch. Apidologie (Celle), 25, 38-48. [http://dx.doi.org/10.1051/apido:19940105] Hepburn, HR, Magnuson, P, Herbert, L & Whiffler, LA (1991) The development of laying workers in field colonies of the Cape honey bee. J Apic Res, 30, 107-12. [http://dx.doi.org/10.1080/00218839.1991.11101242] Hepperle, C & Hartfelder, K (2001) Differentially expressed regulatory genes in honey bee caste development. Naturwissenschaften, 88, 113-6. [http://dx.doi.org/10.1007/s001140000196] [PMID: 11402838] Higes, M, Martín, R & Meana, A (2006) Nosema ceranae, a new microsporidian parasite in honeybees in Europe. J Invertebr Pathol, 92, 93-5. [http://dx.doi.org/10.1016/j.jip.2006.02.005] [PMID: 16574143] Highfield, AC, El Nagar, A, Mackinder, LCM, Noël, LMLJ, Hall, MJ, Martin, SJ & Schroeder, DC (2009) Deformed wing virus implicated in overwintering honeybee colony losses. Appl Environ Microbiol, 75, 7212-20. [http://dx.doi.org/10.1128/AEM.02227-09] [PMID: 19783750] Hoopingarner, R & Farrar, CL (1959) Genetic control of size in queen honey bees. J Econ Entomol, 52, 5478. [http://dx.doi.org/10.1093/jee/52.4.547] Jackson, JT, Tarpy, DR & Fahrbach, SE (2011) Histological estimates of ovariole number in honey bee queens, Apis mellifera, reveal lack of correlation with other queen quality measures. J Insect Sci, 11, 1-11. [http://dx.doi.org/10.1673/031.011.8201] [PMID: 21870968] Jay, SC & Nelson, EV (1973) The effects of laying worker honeybees ( Apis mellifera L.) and their brood on the ovary development of other worker honeybees. Can J Zool, 51, 629-32. [http://dx.doi.org/10.1139/z73-091] Jay, SC (1972) Ovary development of worker honeybees when separated from worker brood by various methods. Can J Zool, 50, 661-4. [http://dx.doi.org/10.1139/z72-090] Jay, SC (1970) The effect of various combinations of immature queen and worker bees on the ovary development of worker honeybees in colonies with and without queens. Can J Zool, 48, 169-73. [http://dx.doi.org/10.1139/z70-019] Kaftanoglu, O, Kumova, U & Yeninar, H (1992) Ana Arı Yetistiriciliginin Onemi ve Ana Arı Kalitesini Etkileyen Faktörler. Dogu Anadolu Bölgesi, 1, 48-60. Kaftanoglu, O, Akyol, E & Yeninar, H (2000) Kahya, Y, Gençer, HV & Woyke, J (2008) Weight at emergence of honey bee ( Apis mellifera caucasica ) queens and its effect on live weights at the pre and post mating periods. J Apic Res, 47, 118-25. [http://dx.doi.org/10.1080/00218839.2008.11101437] Keller, L & Nonacs, P (1993) The role of queen pheromones in social insects: queen control or queen signal? Anim Behav, 45, 787-94. [http://dx.doi.org/10.1006/anbe.1993.1092] Kerr, WE, Blum, MS, Pisani, JF & Stort, AC (1974) Correlation between amounts of 2-heptanone and isoamyl acetate in honey-bees and their aggressive behaviour. J Apic Res, 13, 173-6.

104 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1080/00218839.1974.11099775] .

King, M, Eubel, H, Millar, AH & Baer, B (2011) Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera. J Insect Physiol, 57, 409-14. [http://dx.doi.org/10.1016/j.jinsphys.2010.12.011] [PMID: 21192944] Klein, S, Pasquaretta, C, He, XJ, Perry, C, Søvik, E, Devaud, JM, Barron, AB & Lihoreau, M (2019) Honey bees increase their foraging performance and frequency of pollen trips through experience. Sci Rep, 9, 6778. [http://dx.doi.org/10.1038/s41598-019-42677-x] [PMID: 31043647] Koç, AU & Karacaoglu, M (2004) Effects of rearing season on the quality of queen honey-bees (Apis mellifera L.) raised under the conditions of aegean region. Mellifera, 4, 34-7. Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2008) Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera). BMC Genomics, 9, 232. [http://dx.doi.org/10.1186/1471-2164-9-232] [PMID: 18489784] Koeniger, G, Koeniger, N, Tingek, S & Phiancharoen, M (2005) Variance in spermatozoa number among Apis dorsata drones and among Apis mellifera drones. Apidologie (Celle), 36, 279-84. [http://dx.doi.org/10.1051/apido:2005009] Koeniger, G, Koeniger, N, Ellis, J & Connor, LJ (2014) Mating biology of honey bees (Apis mellifera)Wicwas Press LLC. Kraus, FB, Neumann, P, Scharpenberg, H, Van Praagh, J & Moritz, RFA (2003) Male fitness of honeybee colonies (Apis mellifera L.). J Evol Biol, 16, 914-20. [http://dx.doi.org/10.1046/j.1420-9101.2003.00593.x] [PMID: 14635906] Kraus, FB, Neumann, P & Moritz, RFA (2005) Genetic variance of mating frequency in the honeybee (Apis mellifera L.). Insectes Soc, 52, 1-5. [http://dx.doi.org/10.1007/s00040-004-0766-9] Lago, DC, Humann, FC, Barchuk, AR, Abraham, KJ & Hartfelder, K (2016) Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development. Insect Biochem Mol Biol, 79, 1-12. [http://dx.doi.org/10.1016/j.ibmb.2016.10.001] [PMID: 27720811] Graham, JM (1992) Le Conte, Y, Arnold, G, Trouiller, J, Masson, C & Chappe, B (1990) Identification of a brood pheromone in honeybees. Naturwissenschaften, 77, 334-6. [http://dx.doi.org/10.1007/BF01138390] Le Conte, Y, Ellis, M & Ritter, W (2010) Varroa mites and honey bee health: can Varroa explain part of the colony losses? Apidologie (Celle), 41, 353-63. [http://dx.doi.org/10.1051/apido/2010017] Leonardo, AMC (1985) Developmental Cycle of the Mandibular Glands of Apis mellifera Workers 2. Effect of Queenlessness. J Apic Res, 24, 76-9. [http://dx.doi.org/10.1080/00218839.1985.11100652] Linksvayer, TA, Rueppell, O, Siegel, A, Kaftanoglu, O, Page, RE, Jr & Amdam, GV (2009) The genetic basis of transgressive ovary size in honeybee workers. Genetics, 183, 693-707. [http://dx.doi.org/10.1534/genetics.109.105452] [PMID: 19620393] Liu, TP (1992) Oöcytes degeneration in the queen honey bee after infection by Nosema apis. Tissue Cell, 24, 131-8. [http://dx.doi.org/10.1016/0040-8166(92)90087-N] [PMID: 18621203] Lodesani, M, Balduzzi, D & Galli, A (2004) A study on spermatozoa viability over time in honey bee ( Apis mellifera ligustica ) queen spermathecae. J Apic Res, 43, 27-8. [http://dx.doi.org/10.1080/00218839.2004.11101105] Loskotova, J, Peroutka, M & Vesely, V (1980) Nosema disease of honey-bee queens (Apis mellifica L.).

Factors and Disease

Polyandrous Queen Honey Bee 105

Apidologie (Celle), 11, 153-61. [http://dx.doi.org/10.1051/apido:19800205] Lyko, F, Foret, S, Kucharski, R, Wolf, S, Falckenhayn, C & Maleszka, R (2010) The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol, 8e1000506 [http://dx.doi.org/10.1371/journal.pbio.1000506] [PMID: 21072239] Martins, GF & Serrão, JE (2004) A comparative study of the ovaries in some Brazilian bees (Hymenoptera; Apoidea). Pap Avulsos Zool, 44, 45-53. [http://dx.doi.org/10.1590/S0031-10492004000300001] Masry, SHD, Abd El-Wahab, TE & Hassona, NM (2015) Origin, weight at emergence of virgin honey bee queens and its effect on acceptance during introduction. Acad J Entomol, 8, 174-82. McMenamin, AJ & Genersch, E (2015) Honey bee colony losses and associated viruses. Curr Opin Insect Sci, 8, 121-9. [http://dx.doi.org/10.1016/j.cois.2015.01.015] [PMID: 32846659] Meixner, MD, Francis, RM, Gajda, A, Kryger, P, Andonov, S, Uzunov, A, Topolska, G, Costa, C, Amiri, E, Berg, S, Bienkowska, M, Bouga, M, Büchler, R, Dyrba, W, Gurgulova, K, Hatjina, F, Ivanova, E, Janes, M, Kezic, N, Korpela, S, Conte, YL, Panasiuk, B, Pechhacker, H, Tsoktouridis, G, Vaccari, G & Wilde, J (2014) Occurrence of parasites and pathogens in honey bee colonies used in a European genotype-environment interactions experiment. J Apic Res, 53, 215-29. [http://dx.doi.org/10.3896/IBRA.1.53.2.04] Milchreit, K, Ruhnke, H, Wegener, J & Bienefeld, K (2016) Effects of an insect growth regulator and a solvent on honeybee (Apis mellifera L.) brood development and queen viability. Ecotoxicology, 25, 530-7. [http://dx.doi.org/10.1007/s10646-016-1611-4] [PMID: 26821233] Morse, RA & Calderone, NW (2000) The value of honey bees as pollinators of US crops in 2000. Bee culture 128, 1-15. Nazzi, F, Bortolomeazzi, R, Della Vedova, G, Del Piccolo, F, Annoscia, D & Milani, N (2009) Octanoic acid confers to royal jelly varroa-repellent properties. Naturwissenschaften, 96, 309-14. [http://dx.doi.org/10.1007/s00114-008-0470-0] [PMID: 19050844] Nazzi, F, Brown, SP, Annoscia, D, Del Piccolo, F, Di Prisco, G, Varricchio, P, Della Vedova, G, Cattonaro, F, Caprio, E & Pennacchio, F (2012) Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies. PLoS Pathog, 8e1002735 [http://dx.doi.org/10.1371/journal.ppat.1002735] [PMID: 22719246] Nelson, DL & Gary, NE (1983) Honey productivity of honey-bee colonies in relation to body weight, attractiveness and fecundity of the queen. J Apic Res, 22, 209-13. [http://dx.doi.org/10.1080/00218839.1983.11100589] Nelson, DL & Smirl, C (1977) The effect of queen-related problems and swarming on brood and honey production of honey bee colonies in Manitoba. Manitoba Entomol, 11, 45-9. Niño, EL, Tarpy, DR & Grozinger, CM (2013) Differential effects of insemination volume and substance on reproductive changes in honey bee queens ( Apis mellifera L.). Insect Mol Biol, 22, 233-44. [http://dx.doi.org/10.1111/imb.12016] [PMID: 23414204] Odemer, R & Odemer, F (2019) Effects of radiofrequency electromagnetic radiation (RF-EMF) on honey bee queen development and mating success. Sci Total Environ, 661, 553-62. [http://dx.doi.org/10.1016/j.scitotenv.2019.01.154] [PMID: 30682608] Oldroyd, BP, Goodman, RD & Allaway, MA (1990) On the relative importance of queens and workers to honey production. Apidologie (Celle), 21, 153-9. [http://dx.doi.org/10.1051/apido:19900209] Patel, A, Fondrk, MK, Kaftanoglu, O, Emore, C, Hunt, G, Frederick, K & Amdam, GV (2007) The making of a queen: TOR pathway is a key player in diphenic caste development. PLoS One, 2e509

106 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1371/journal.pone.0000509] [PMID: 17551589] Patrício, K & Cruz-Landim, C (2002) Mating influence in the ovary differentiation in adult queens of Apis mellifera L. (Hymenoptera, Apidae). Braz J Biol, 62, 641-9. [http://dx.doi.org/10.1590/S1519-69842002000400012] [PMID: 12659014] Peng, Y, Grassl, J, Millar, AH & Baer, B (2016) Seminal fluid of honeybees contains multiple mechanisms to combat infections of the sexually transmitted pathogen Nosema apis. Proc Biol Sci, 28320151785 [http://dx.doi.org/10.1098/rspb.2015.1785] Pettis, JS, Dietz, A & Eischen, FA (1989) Incidence rates of Acarapis woodi (Rennie) in queen honey bees of various ages. Apidologie (Celle), 20, 69-75. [http://dx.doi.org/10.1051/apido:19890107] Polsinelli, GA & Yu, HD (2018) Regulation of histone deacetylase 3 by metal cations and 10-hydroxy-E-decenoic acid: Possible epigenetic mechanisms of queen-worker bee differentiation. PLoS One, 13e0204538 [http://dx.doi.org/10.1371/journal.pone.0204538] [PMID: 30532259] Porporato, M, Grillone, G, Patetta, A, Manino, A & Laurino, D (2015) Survey of the health status of some honey bee queens in Italy. J Apic Res, 59, 27-36. Potts, SG, Biesmeijer, JC, Kremen, C, Neumann, P, Schweiger, O & Kunin, WE (2010) Global pollinator declines: trends, impacts and drivers. Trends Ecol Evol, 25, 345-53. [http://dx.doi.org/10.1016/j.tree.2010.01.007] [PMID: 20188434] Rangel, J & Fisher, A, II (2019) Factors affecting the reproductive health of honey bee (Apis mellifera) drones—a review. Apidologie (Celle), 50, 759-78. [http://dx.doi.org/10.1007/s13592-019-00684-x] Rangel, J, Keller, JJ & Tarpy, DR (2013) The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth. Insectes Soc, 60, 65-73. [http://dx.doi.org/10.1007/s00040-012-0267-1] Rangel, J & Tarpy, DR (2015) The combined effects of miticides on the mating health of honey bee ( Apis mellifera L.) queens. J Apic Res, 54, 275-83. [http://dx.doi.org/10.1080/00218839.2016.1147218] Rangel, J & R Tarpy, D (2016) In-hive miticides and their effect on queen supersedure and colony growth in the honey bee (Apis mellifera). J Environ Anal Toxicol, 61000377 [http://dx.doi.org/10.4172/2161-0525.1000377] Ratnieks, FW (1993) Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies. Behav Ecol Sociobiol, 32, 191-8. [http://dx.doi.org/10.1007/BF00173777] Ratnieks, FLW & Visscher, PK (1989) Worker policing in the honeybee. Nature, 342, 796-7. [http://dx.doi.org/10.1038/342796a0] Rhodes, JW, Somerville, DC & Harden, S (2004) Queen honey bee introduction and early survival? effects of queen age at introduction. Apidologie (Celle), 35, 383-8. [http://dx.doi.org/10.1051/apido:2004028] Ravoet, J, De Smet, L, Wenseleers, T & de Graaf, DC (2015) Vertical transmission of honey bee viruses in a Belgian queen breeding program. BMC Vet Res, 11, 61. [http://dx.doi.org/10.1186/s12917-015-0386-9] [PMID: 25889959] Reginato, R & Da Cruz-Landim, C (2002) Morphological characterization of cell death during the ovary differentiation in worker honey bee. Cell Biol Int, 26, 243-51. [http://dx.doi.org/10.1006/cbir.2001.0839] [PMID: 11991652] Reginato, RD & Cruz-Landim, C (2003) Ovarian growth during larval development of queen and worker of Apis mellifera (Hymenoptera: Apidae): a morphometric and histological study. Braz J Biol, 63, 121-7.

Factors and Disease

Polyandrous Queen Honey Bee 107

[http://dx.doi.org/10.1590/S1519-69842003000100016] [PMID: 12914423] Renner, M & Baumann, M (1964) �ber Komplexe von subepidermalen Dr�senzellen (Duftdr�sen?) der Bienenk�nigin. Naturwissenschaften, 51, 68-9. [http://dx.doi.org/10.1007/BF00603470] Richard, FJ, Schal, C, Tarpy, DR & Grozinger, CM (2011) Effects of instrumental insemination and insemination quantity on Dufour’s gland chemical profiles and vitellogenin expression in honey bee queens (Apis mellifera). J Chem Ecol, 37, 1027-36. [http://dx.doi.org/10.1007/s10886-011-9999-z] [PMID: 21786084] Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2e980 [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Ring, T (2009) Roberts, KE, Evison, SEF, Baer, B & Hughes, WOH (2015) The cost of promiscuity: sexual transmission of Nosema microsporidian parasites in polyandrous honey bees. Sci Rep, 5, 10982. [http://dx.doi.org/10.1038/srep10982] [PMID: 26123939] Ronai, I, Oldroyd, BP & Vergoz, V (2016) Queen pheromone regulates programmed cell death in the honey bee worker ovary. Insect Mol Biol, 25, 646-52. [http://dx.doi.org/10.1111/imb.12250] [PMID: 27321063] Rothenbuhler, WC (1964) Behaviour genetics of nest cleaning in honey bees. I. Responses of four inbred lines to disease-killed brood. Anim Behav, 12, 578-83. [http://dx.doi.org/10.1016/0003-3472(64)90082-X] Rueppell, O, Johnson, N & Rychtář, J (2008) Variance-based selection may explain general mating patterns in social insects. Biol Lett, 4, 270-3. [http://dx.doi.org/10.1098/rsbl.2008.0065] [PMID: 18364307] Sagili, RR, Metz, BN, Lucas, HM, Chakrabarti, P & Breece, CR (2018) Honey bees consider larval nutritional status rather than genetic relatedness when selecting larvae for emergency queen rearing. Sci Rep, 8, 7679. [http://dx.doi.org/10.1038/s41598-018-25976-7] [PMID: 29769574] Saiovici, M (1983) 9-Oxodecenoic acid and dominance in honey-bees. J Apic Res, 22, 27-32. [http://dx.doi.org/10.1080/00218839.1983.11100556] Schlüns, H, Moritz, RFA, Neumann, P, Kryger, P & Koeniger, G (2005) Multiple nuptial flights, sperm transfer and the evolution of extreme polyandry in honeybee queens. Anim Behav, 70, 125-31. [http://dx.doi.org/10.1016/j.anbehav.2004.11.005] Shehata, SM, Townsend, GF & Shuel, RW (1981) Seasonal physiological changes in queen and worker honey-bees. J Apic Res, 20, 69-78. [http://dx.doi.org/10.1080/00218839.1981.11100475] Skowronek, W, Bieñkowska, M & Kruk, C (2004) Changes in body weight of honey bee queens during their maturation. J Apic Sci, 48, 61-8. Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Smith, KM, Loh, EH, Rostal, MK, Zambrana-Torrelio, CM, Mendiola, L & Daszak, P (2013) Pathogens, pests, and economics: drivers of honey bee colony declines and losses. EcoHealth, 10, 434-45. [http://dx.doi.org/10.1007/s10393-013-0870-2] [PMID: 24496582] Snodgrass, RE (1956) Anatomy of the Honey Bee Spivak, M (1996) Honey bee hygienic behavior and defense against Varroa jacobsoni. Apidologie (Celle), 27, 245-60.

108 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1051/apido:19960407] Spivak, M & Gilliam, M (1998) Hygienic behaviour of honey bees and its application for control of brood diseases and varroa. Bee World, 79, 169-86. [http://dx.doi.org/10.1080/0005772X.1998.11099408] Tarpy, DR & Mayer, MK (2009) The effects of size and reproductive quality on the outcomes of duels between honey bee queens ( Apis mellifera L.). Ethol Ecol Evol, 21, 147-53. [http://dx.doi.org/10.1080/08927014.2009.9522503] Tarpy, DR, Gilley, DC & Seeley, TD (2004) Levels of selection in a social insect: a review of conflict and cooperation during honey bee ( Apis mellifera ) queen replacement. Behav Ecol Sociobiol, 55, 513-23. [http://dx.doi.org/10.1007/s00265-003-0738-5] Tarpy, DR, Hatch, S & Fletcher, DJC (2000) The influence of queen age and quality during queen replacement in honeybee colonies. Anim Behav, 59, 97-101. [http://dx.doi.org/10.1006/anbe.1999.1311] [PMID: 10640371] Tarpy, DR, Keller, JJ, Caren, JR & Delaney, DA (2011) Experimentally induced variation in the physical reproductive potential and mating success in honey bee queens. Insectes Soc, 58, 569-74. [http://dx.doi.org/10.1007/s00040-011-0180-z] Tarpy, DR (2003) Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth. Proc Biol Sci, 270, 99-103. [http://dx.doi.org/10.1098/rspb.2002.2199] [PMID: 12596763] Tarpy, DR, Keller, JJ, Caren, JR & Delaney, DA (2012) Assessing the mating ‘health’ of commercial honey bee queens. J Econ Entomol, 105, 20-5. [http://dx.doi.org/10.1603/EC11276] [PMID: 22420250] Tarpy, DR & Nielsen, DI (2002) Sampling error, effective paternity, and estimating the genetic structure of honey bee colonies (Hymenoptera: Apidae). Ann Entomol Soc Am, 95, 513-28. [http://dx.doi.org/10.1603/0013-8746(2002)095[0513:SEEPAE]2.0.CO;2] Tarpy, DR, Summers, J & Keller, JJ (2007) Comparison of parasitic mites in Russian-hybrid and Italian honey bee (Hymenoptera: Apidae) colonies across three different locations in North Carolina. J Econ Entomol, 100, 258-66. [http://dx.doi.org/10.1093/jee/100.2.258] [PMID: 17461045] Tarpy, DR, Nielsen, R & Nielsen, DI (2004) A scientific note on the revised estimates of effective paternity frequency in Apis. Insectes Soc, 51, 203-4. [http://dx.doi.org/10.1007/s00040-004-0734-4] Tarpy, DR, vanEngelsdorp, D & Pettis, JS (2013) Genetic diversity affects colony survivorship in commercial honey bee colonies. Naturwissenschaften, 100, 723-8. [http://dx.doi.org/10.1007/s00114-013-1065-y] [PMID: 23728203] Toth, AL, Bilof, KBJ, Henshaw, MT, Hunt, JH & Robinson, GE (2009) Lipid stores, ovary development, and brain gene expression in Polistes metricus females. Insectes Soc, 56, 77-84. [http://dx.doi.org/10.1007/s00040-008-1041-2] Traver, BE & Fell, RD (2012) Low natural levels of Nosema ceranae in Apis mellifera queens. J Invertebr Pathol, 110, 408-10. [http://dx.doi.org/10.1016/j.jip.2012.04.001] [PMID: 22546521] Van der Blom, J & Verkade, AJ (1991) Does kin recognition in honey bees, Apis mellifera L., influence the workers’ chances of becoming egg layers? Anim Behav, 42, 867-70. [http://dx.doi.org/10.1016/S0003-3472(05)80136-8] vanEngelsdorp, D, Hayes, J, Jr, Underwood, RM & Pettis, J (2008) A survey of honey bee colony losses in the U.S., fall 2007 to spring 2008. PLoS One, 3e4071 [http://dx.doi.org/10.1371/journal.pone.0004071] [PMID: 19115015]

Factors and Disease

Polyandrous Queen Honey Bee 109

vanEngelsdorp, D & Meixner, MD (2010) A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them. J Invertebr Pathol, 103 (Suppl. 1), S80-95. [http://dx.doi.org/10.1016/j.jip.2009.06.011] [PMID: 19909973] Velthuis, HHW (1967) On abdominal pheromones in the queen honey-bee. Proc XXI Int Beekeeping Congr, College Park, USA, Ed Apimondia, Bucharest, 58-9. Velthuis, HHW (1976) Egg laying, aggression and dominance in bees Proc XV Int Congr Entomol, 436-49. Velthuis, HHW & van Es, J (1964) Some functional aspects of the mandibular glands of the queen honeybee. J Apic Res, 3, 11-6. [http://dx.doi.org/10.1080/00218839.1964.11100076] Velthuis, HHW (1970) Queen substances from the abdomen of the honey bee queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 70, 210-21. a [http://dx.doi.org/10.1007/BF00297717] Velthuis, HHW (1970) b Velthuis, HHW (1985) The honey-bee queen and the social organization of her colony.Experimental Behavioural Ecology and Sociobiology, G Fischer Verlag, New York 343-37. Villa, JD & Danka, RG (2005) Caste, sex and strain of honey bees (Apis mellifera) affect infestation with tracheal mites (Acarapis woodi). Exp Appl Acarol, 37, 157-64. [http://dx.doi.org/10.1007/s10493-005-2647-y] [PMID: 16323047] Wheeler, DE, Buck, NA & Evans, JD (2014) Expression of insulin/insulin-like signalling and TOR pathway genes in honey bee caste determination. Insect Mol Biol, 23, 113-21. [http://dx.doi.org/10.1111/imb.12065] [PMID: 24224645] Wilkes, K & Oldroyd, B (2002) Oldroyd. 2002. Breeding hygienic disease resistant bees. A report for the Rural Industries Research and Development Corporation. RIRDC Publivation No:02/048 Williams, GR, Troxler, A, Retschnig, G, Roth, K, Yañez, O, Shutler, D, Neumann, P & Gauthier, L (2015) Neonicotinoid pesticides severely affect honey bee queens. Sci Rep, 5, 14621. [http://dx.doi.org/10.1038/srep14621] [PMID: 26459072] Willis, LG, Winston, ML & Slessor, KN (1990) Queen honey bee mandibular pheromone does not affect worker ovary development. Can Entomol, 122, 1093-9. [http://dx.doi.org/10.4039/Ent1221093-11] Wilson, EO (1971) The Insect SocietiesHarvard University Press, Cambridge, MA. Winston, ML & Slessor, KN (1992) The essence of royalty: Honey bee queen pheromone. Am Sci, 80, 37485. Winston, ML (1987) The biology of the honey beeHarvard University Press, Cambridge, Massachusetts. Wojciechowski, M, Lowe, R, Maleszka, J, Conn, D, Maleszka, R & Hurd, PJ (2018) Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development. Genome Res, 28, 1532-42. [http://dx.doi.org/10.1101/gr.236497.118] [PMID: 30135090] Woyke, J (1962) Natural and artificial insemination of queen honey-bees Woyke, J (1971) Correlations between the age at which honey-bee brood was grafted, characteristics of the resultant queens, and results of insemination. J Apic Res, 10, 45-55. [http://dx.doi.org/10.1080/00218839.1971.11099669] Yañez, O, Jaffé, R, Jarosch, A, Fries, I, Moritz, RFA, Paxton, RJ & de Miranda, JR (2012) Deformed wing virus and drone mating flights in the honey bee (Apis mellifera): implications for sexual transmission of a major honey bee virus. Apidologie (Celle), 43, 17-30. [http://dx.doi.org/10.1007/s13592-011-0088-7]

110 Polyandrous Queen Honey Bee

Lovleen Marwaha

Yin, L, Wang, K, Niu, L, Zhang, H, Chen, Y, Ji, T & Chen, G (2018) Uncovering the Changing Gene Expression Profile of Honeybee (Apis mellifera) Worker Larvae Transplanted to Queen Cells. Front Genet, 9, 416. [http://dx.doi.org/10.3389/fgene.2018.00416] [PMID: 30405683] Yue, C, Schröder, M, Gisder, S & Genersch, E (2007) Vertical-transmission routes for deformed wing virus of honeybees (Apis mellifera). J Gen Virol, 88, 2329-36. [http://dx.doi.org/10.1099/vir.0.83101-0] [PMID: 17622639] Zareie, R, Eubel, H, Millar, AH & Baer, B (2013) Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera. J Proteome Res, 12, 5180-8. [http://dx.doi.org/10.1021/pr4004773] [PMID: 24127784]

Polyandrous Queen Honey Bee, 2022, 111-137

111

CHAPTER 6

Differential Pheromone Secretion By Female Castes In Apis mellifera (Hymenoptera: Apidae) Abstract: The queen and worker caste of the honey bee exemplified the polyphenism phenomenon. In specific female caste, diversification of the same genomic (2n=32) expression ultimately induces plasticity in development, morphology, physiology, reproduction, division of labour, immunity, and life span. Physiological plasticity is remarkably highlighted through glandular secretion variation in female castes, as pheromonal queen glands ensure her reproductive monopoly and dominant hierarchy in the colony. In contrast, in workers, pheromonal profiling facilitates foraging, nursing, alarming, colony protection, pseudo-queen formation inhibition, and other social interactions. Queen's volatile bouquet emission contains biochemicals like 9-ODA, OLA, HVA, 9-HDA, 10-HDA, HOB, 10-HDAA, cetyl alcohol, coniferyl alcohol, linolenic acid, methyl oleate, and decyl decanoate. In contrast, workers' pheromones include predominantly; isopentyl acetate (IPA), butyl acetate, 1-hexanol, n-butanol, 1octanol, hexyl acetate, octyl acetate, n-pentyl acetate, and 2-nonanol, (Z)-11-eicosenol, 2-heptanone, geraniol, geranial, geranic acid, (E)-citral, nerolic acid, etc. Queen and workers secrete different pheromones following their role in the colony. This chapter provides insights into differential pheromonal secretion in queen and worker caste within the honey bee colony. The biochemical synthesis of pheromonal contents in both castes is elaborated on in the next chapter

Keywords: Associated Pheromones, Female Castes, Honey bees. 6.1. INTRODUCTION The polyphenism phenomenon is defined as differential gene expressions to form variant morphologies. In a honey bee colony, co-operative polyphenism exists, as both female castes are mutually complementary. The queen is disproportionately fertile with a hypertrophied reproductive system with intriguing functionality. At the same time, workers are sterile due to degeneration of the reproductive system under the influence of queen pheromones, hypo-secretion of developmental hormones, and genetic elements. Glandular plasticity in female castes of honey bees is correlated with differential development, physiology, and gene expression. Polyandrous queen honey bee secretes certain volatile chemicals like 9-ODA(2E-9-oxodecenoic acid), 9-HDA (9-hydroxy-(E)-2-decanoic acid), 10-HDA (10Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

112 Polyandrous Queen Honey Bee

Lovleen Marwaha

hydroxy-2-decanoic acid), HVA (4-hydroxy-3-methoxyphenylethanol), HOB(Methyl-p-hydroxybenzoate), 10-HDAA (10-hydroxydecanoic acid), OLA (oligolactide), methyl oleate, decyl decanoate, linolenic acid, coniferyl alcohol, cetyl alcohol, etc. from various glands like Mandibular gland, Salivary gland, Tergal gland, Koschevnikov, Dufour, Tarsal, Hypopharyngeal gland, etc. Similarly, worker honey bees secrete pheromones isopentyl acetate (IPA), butyl acetate, 1-hexanol, n-butanol, 1-octanol, hexyl acetate, octyl acetate, n-pentyl acetate, and 2-nonanol, (Z)-11-eicosenol, 2-heptanone, geraniol, geranial, geranic acid, (E)-citral, nerolic acid, etc. from Koschevnikov gland, Mandibular gland, Nasonov gland, Dufour's gland predominantly (Pain, 1961; Gary, 1962; Butler, 1967; Blum and Brand, 1972, Moritz and Hillesheim, 1985; Herburn et al., 1988; Pettis et al., 1989; Willis et al., 1990; Winston, 1992; Winston and Slessor, 1992; Blum 1992; Hartfelder, 1993; Herburn, 1994; Seeley, 1995; Hartfelder and Steinbruck, 1997; Richard et al., 2011; Collin and Pettis, 2003; Katzav-Gozansky et al., 2004).

Fig. (6a). Diagram depicting various pheromones secreted by queen honey bee and stimulations induced by concerned volatile chemicals. Multiple genetic factors, queen pheromones, and hormones associated with insect ecdysis suppress worker ovarian development. In addition, Queen pheromones influence swarming, workers' pheromonal release, wax production, retinue behaviour, drone attraction, etc.

Due to variant biochemical synthesis in queens and workers, the chemical composition of glandular secretion is different. Hence both castes possess differential social behaviour, hierarchy, and function in the colony. Queen pheromones induce regulation on retinue response, colonial caste interaction,

Differential Pheromone

Polyandrous Queen Honey Bee 113

developmental event, brood rearing, foraging, honey production, another queen rearing inhibition/induction, reproduction, protection, proper social colonial environment regulation, etc. In contrast, worker pheromones facilitate coordination and communication among hive residents for the sequential accomplishment of various processes comprising foraging, brood rearing, and protection from intruders who enter into honey bee colony to take nectar, honey, pollen, brood by stinging or through other deference systems (Fig. 6a - o). The appropriate level of queen pheromones inhibits another queen rearing, whereas low pheromone concentration in a hive accelerates another queen rearing (Velthuis, 1967; Velthuis, 1970a,b; Saiovici, 1983; Rangek et al., 2013;

Fig. (6b). Comparison of pheromones secreted by the virgin queen and mated queen. The above diagram illustrates the pheromonal difference between a virgin and a mated queen. There is a change in the composition of queen pheromones with mating.

Rangel and Trapy, 2015, 2016; Rangel and Fisher, 2019). The description of the queen and worker pheromones is elaborated in the following sections.

114 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (6c). Click depicting worker honey bees exchanging information with antennal contact at the hive's entrance. Hive honey bees exchange information with foragers through antennal contact, pheromone secretion, dances, and other clues.

Fig. (6d). Forager worker honey bee carrying pollen-filled basket to the colony. Unique pollen baskets on worker honey bees' metathoracic legs are present, facilitating quantitative transportation of pollen grains to the hive.

Differential Pheromone

Polyandrous Queen Honey Bee 115

Fig. (6e). Picture from the strong colony, reflecting considerable strength of worker bees. This colony is headed by a strong queen, which can be detected with many capped worker cells. Additionally, worker bees of different age groups can perform various duties.

Fig. (6f). Click of the queenless colony with poor colony strength and drone eggs. The colony had been maintained on the artificial diet in specific conditions before re-queening. In the absence of a queen, the probability of pseudo-queen formation increases, which can lay unfertilized eggs/Drone eggs.

116 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (6g). Image reflect guard honey bees at the hive's entrance for protection. Usually, a group of old worker honey bees remains at the entrance to check the bees entering the pack. In honey-filled conditions, colonies become more prone to be attacked by robbers bees. Guard honey bees discourage such introducers from fulfilling their ill covetousness.

Fig. (6h). Click specify a group of worker honey bees fanning at the hive's entrance. The specific behaviour was captured on a day with a temperature range of 35-40 0C when the hive was open for a short duration. Some workers were fanning on frames, while others were airing at the hive's entrance.

Differential Pheromone

Polyandrous Queen Honey Bee 117

Fig. (6i). Click from a strong colony with worker larvae and capped worker pupae. C-shaped white-coloured pupae of different instars can be easily detected in uncapped brood cells.

Fig. (6j). Click of same hive section from another frame with uncapped brood cells, capped brood cell, drone, and worker honey bee in a hive section. Few wax cells filled with unripe honey and pollen are left interspersed among brood cells; the specific act facilitates the brood rearing process of workers.

118 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (6k). Click depicting uncapped and capped honey cells for unripe and ripe honey storage. Concomitantly cells filled with pollen grains are also visible. Usually, in the upper portion, honey bees store honey in the brood chamber. The colony used frames of the super chamber for honey storage by adding the queen excluder.

Fig. (6l). Picture from a colony with a central Queen and a group of workers in a hive section. Due to secretion of Queen Substance, workers retinue her. Queen substance is a group of pheromones secreted by her using various glands.

Differential Pheromone

Polyandrous Queen Honey Bee 119

Fig. (6m). Worker Honey Bees at the entrance of hive, feeding on sugary powder. Click had been captured during experimentation about the food preferences of honey bees. It has been observed that workers get more attracted toward liquid sugary material than sugary powder.

Fig. (6n). Further in the same experimentation, observation indicated that workers preferred sugary powder over liquid sugary material when the former had been served on yellow background. Further analysis revealed that workers use visual clues about food more than the physical state of nutrition. In the above click, a comparatively high number of workers are attracted to yellow than the dim sky blue colour.

120 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (6o). Queen with workers in a section of the hive, whereas workers can be seen making antennal contact to exchange information. While inspecting different hives, the queen disperses various pheromones, discouraging workers from raising another queen, except during swarming. In addition, the queen usually destroys any other developing queen cell to ensure dominance.

Fig. (6p). Picture of the strong colony with many capped and uncapped cells containing pupal and larval worker honey bees. There are fewer chances of queen replacement if she is reproductively active with strong pheromonal secretion.

Differential Pheromone

Polyandrous Queen Honey Bee 121

6.2. QUEEN PHEROMONES Queen pheromones are secreted by the Mandibular gland, Salivary gland, Tergal gland, Koschevnikov, Dufour, Tarsal, Hypopharyngeal gland, Tergal, Renner, Baumann tarsal, etc. (Fig. 6q-r). Additionally, numerous big gland cells are scattered on tergite III to V appendages (Velthuis 1970, 1985). In the queenrighted colony, queen pheromones induced coordinated regulation of worker honey bees' various activities, development, physiology, genetic expression, reproduction, etc. However, in queen-less honey bee colonies, certain workers start acting as pseudo-queen by oviposition of unfertilized eggs and through secreting pheromones emulating to the queen to suppress ovary development of other workers and for fallacious dominance on nest dwellers. A brief description of the various glands of the queen is as follows.

Fig. (6q). Picture describing various glands responsible for pheromonal secretion in queen honey bee. The mandibular gland and Dufour's gland had been explored comparatively to a greater extent than other glands.

6.2.1. Queen Mandibular Gland Mandibular glands are a pair of saclike glands located inside the head above the base of the mandible. Ducts of the concerned gland open at the bottom of the mandible, carrying glandular secretion through a deep channel surrounded by hairs. The concerned gland secretes volatile chemicals that modulate social behaviour, including hive maintenance, swarming, mating behavior, and inhibition of ovary development in worker honey bees. Queen Mandibular Pheromones (QMP) first had been detected in 1950, and (E)-9-oxodec-2-e-

122 Polyandrous Queen Honey Bee

Lovleen Marwaha

oic-acid (9-ODA) had been detected foremost of all honey bee pheromones. In addition, the chemical composition of the mandibular gland of superior quality and the inferior quality queen was determined using GC-MS, and worker retinue response was recorded to measure the attractiveness of workers to the high-quality queen in comparison to the low-quality queen. Mandibular gland composition of superior quality and inferior quality honey bees exhibit significant differences regarding biochemical composition concomitantly in the induction of variant responses in nest mates. Due to mandibular gland secretion, workers display more remarkable retinue behaviour toward high-quality queens than low-quality queens. Larval experimentation demonstrates that the superiority or inferiority of a queen is negatively correlated with the age of the worker larva entering into the queen development pathway. Additionally, the QMPs of the existing Queen influence the quality of the developing Queen (Rangel et al., 2016). 6.2.1.1. Composition of QMPs and Variation in Accordance of Mating Status Queen mandibular pheromones can be carboxylic acid derivatives and aromatic compounds. Queen mandibular gland secretion includes (E)-9-oxo-2-decanoic acid (9-ODA), methyl 4-hydroxybenzoate (HOB), (R)- and (S)-(E)-9-hydroy-2-decanoic acid (9-HDA), 4-hydroxy- 3-methoxyphenylethanol (HVA), 10hydroxy-decanoic acid (10-HDAA) and 10-hydroxy-2 (E)-decanoic acid (10HDA) (Slessor et al., 1988; Rangel et al., 2016). The production and chemical composition of QMP are regulated by the queen's ontogeny and mating state. There is a variation between the chemical composition of QMP of virgin vs mated Queen, egg laying and non-egg laying, naturally mated vs artificially inseminated queen. Furthermore, queen inseminated with one drone or multiple drones; the queen was inseminated with a low vs high volume of semen or saline solution (Kocher et al., 2008). Available scientific literature witnesses the variation in the composition of QMP in different situations, simultaneously analysis of worker bees' responses to specific pheromones. According to Engels et al. (1990), there are three different patterns of QMP secretion in queens depending upon their mating status, as follows: • Premating Queens; with the significant secretion of oleic acid (OLA) • Mating Queen; with predominant component 9-ODA, OLA, and diminutive proportionality of 9-HDA • Post-Mating Dominant Queen; with high concentrations of 9-ODA combined

Differential Pheromone

Polyandrous Queen Honey Bee 123

with a medium proportion of 9-HDA and a partial amount of OLA, oxygenated aromates, HOB and HVA. On the contrary, Slessor et al. (1990) reported: • 9-ODA Pheromone: consistent level of a virgin and mated queen • 9-HDA Pheromone: mated queen> virgin queen • HOB and HVA Pheromone: older mated queen> virgin and young mated ones. Queen mandibular gland chemical profile is highly variable in accordance to her mating status, as in queen honey bees ratio of 9-ODA to 10 HDA can be either 1 or >1 in mated condition (Plettner et al., 1997). Further, if the ratio of 9-ODA/(-ODA + 10-HDA) is one, then the mandibular gland bouquet is more queen, or if close to zero means worker like (Moritz et al., 2000; Moritz et al., 2004; Schafer et al., 2006). Plettner et al. (1997) carried out a comparative study on the composition of QMP between 6 days old and 1-year-old mated egg-laying queens and reported that mated queens secrete higher levels of 9-ODA, 9-HDA, HOB, and HVA. In contrast, workers and virgin queen mandibular glands secrete higher concentrations of 10-HDA and 10-HDAA. After that, Strauss et al. (2008) analyzed mandibular gland compounds in virgin drone laying and mated queen. They concluded that in all cases, there was some concentration of 9-ODA, increasing engagement of (9-HDA, 10-HDA, 10HDAA, and HVA), except HOB, which suggests a positive correlation of pheromonal profiling enrichment with reproductive potential and ovarian activation in queen. Rangel et al., 2016 experimented on raising queens from two-day-old and sixday-old larvae. After that, they explored chemical QMP profiling concomitantly with retinue response and concluded that queens developed from younger larvae elicit a comparatively more robust response than queens produced from older larvae. Furthermore, composition screening of QMP revealed that the inferior quality queen's mandibular pheromone bouquet comprises a high relative amount of 10 HDA compared to the superior quality queen. Further, they analyzed the mandibular gland chemical composition of the virgin queen with a 1year old postmated queen. They concluded that the 10-HDA component is produced in higher proportionality by old virgin queens. 10-HDA is secreted in higher concentration by workers, whereas a mated queen has a high concentration of 9-HDA as the significant component of QMP. A lower ratio of 10-HDA to 9-HDA readily

124 Polyandrous Queen Honey Bee

Lovleen Marwaha

differentiate queen from workers (Plettner et al., 1997; Rangel et al., 2016).

Fig. (6r). Depicts pheromonal role of queen honey bee in the colony, including reproduction, maintenance of social communication, swarming regulation, worker ovarian suppression, etc.

Furthermore, in higher quality queens, a low ratio of 10-HDA to 9-HDA can be considered a good indicator of queen quality. In contrast, the percentage of 9ODA to 10-HDA tends to increase with queen age and mating status of queen. Further, in a queen-less colony, workers' mandibular gland secretes predominantly 10-HDA and 10-HDAA, but later on, mandibular gland secretion becomes dominated by 9-ODA. In A. mellifera and A. cerana, 9-ODA, HOB, HVA, 9HDA, 10-HDA, and 10-HDAA levels were detected high in the mandibular gland of egg laying workers than in non-egg Queen (Plettner et al., 1997). Eventually, 9-ODA to 9-ODA+10-HDA tend to indicate queen superiority. Few chemicals like Methyl oleate, Coniferyl alcohol, Cetyl alcohol, and α-Linolenic acid act synergistically with QMP to attract workers (Moritz et al.,2000; Moritz et al., 2004; Schäfer et al., 2006). 6.2.1.2. Secretion of QMP Available reports indicate that the queen, after emergence, secretes a considerable amount of QMP and other mandibular gland compounds and later on, there is the secretion of more complex and abundant mandibular gland chemical bouquets (Slessor et al., 1990; Pankiw et al., 1996; Engels et al., 1990; Plettner et al., 1997;

Differential Pheromone

Polyandrous Queen Honey Bee 125

Keeling et al., 2003). Furthermore, few studies indicate that mating status, which includes virgin, single drone insemination, multiple drones inseminated, or naturally mated queens, influences the composition of mandibular secretion (Slessor et al., 1990; Plettner et al., 1997; Keeling et al., 2003; Richard et al., 2007). 6.2.1.3. Colonial Regulation Imposed by QMP On mating and swarm: Gary 1962 observed that QMP, especially 9-ODA, elicits the attraction of drones from surrounding colonies toward virgin queens during the mating flight. Further, 9-ODA facilitate worker honey bees to recognize the queen's presence in the hive. Additionally, Tergal gland extract and 9-ODA exhibit additive effects, concurrently increasing mating tendency in drones. Therefore, several glandular sources co-operative for a more robust mating response. Plettner et al. 1997 reported that a combination of 9-HDA, 10-HDA, and HOB, when applied to a dummy queen, attracts more drones. They further concluded that 10-HDA production occurred more predominant in virgin queens than in the mated queen. Therefore, the specific pheromone regulates the mating behaviour of the virgin queen. (Slessor, et al., 1988; Winston et al. 1989). QMP elicits long-term physiological and short-term behavioural responses in workers, which vary following the genetic composition of the colony. Naumann et al. 1991 reported that retinue workers transfer queen pheromones to other worker honey bees, and the distribution of queen pheromones is influenced by the size and strength of the colony, which becomes the primary reason for swarming in the strong colony. As in stronger colonies, there is less pheromonal density per worker bee in a specific area, which arouse a new queen-rearing tendency in colony residents. (E)-9-Oxodec-2-enoic acid (9-ODA) pheromone inhibits queen rearing and ovarian development in worker honey bees. Pettis et al. 1996, reported that administration of QMP in queenless colonies inhibited the growth of queen cups when admitted within 24 hours after queen loss. Still, no effect had been observed when admitted after four days. QMP inhibits the production of the new queen, induces suppression in workers' ovaries development, and acts as a sex attractant for drones during mating. Furthermore, QMP concomitantly induces pollen and nectar foraging, delays age onset for foraging tasks, and lowers juvenile hormone titers in worker honey bees. (R, E)-9-Hydroxy-2-enoic acid (9-HDA), (S, E)-(+)-9-HDA, Methylparaben (HOB), 4-Hydroxy-3-methoxy phenyl ethanol (HVA) induce calming influence by promoting stability influence on swarm (Butler and Fairey, 1963; Slessor et al.,

126 Polyandrous Queen Honey Bee

Lovleen Marwaha

1988; Pankiw et al., 1998; Hoover et al., 2003; Slessor et al., 2005). 6.2.1.4. On Retinue A QMP elicits a retinue response, a behavioural suite in which workers surround the queen, antennate, groome, and lick her (Slessor et al., 1988; Pankiw et al., 1994; Pankiw et al., 1996, 1998). Functional properties of QMP have been considered due to its characteristic attraction tendency for worker bees toward the queen. Therefore, specific chemicals help in the regulation of the retinue behaviour of workers and the maintenance of swarm clusters (Winston et al. 1989). Retinue or court means a circle of worker honey bees that surround the queen honey, feed, palpate and lick her on a stationary comb. Usually, a retinue is composed of 8-10 workers. However, in Fig. (6s), which is from the more substantial colony, the number of workers retinue queen is more than 10.

Fig. (6s). Picture clicked from a strong colony in a section of a hive. Retinue behaviour of honey bees with the queen in the centre.

Several reports indicate that Queen mandibular pheromonal patterns are responsible for retinue formation (Free 1987; Plettner et al. 1996; Keeling et al. 2003; Rangel et al., 2016) and workers' attraction. Retinue workers pay more attention to the mated egg-laying queen, but the increasing age of the queen is negatively correlated with the lure of workers toward her. The attractiveness of workers toward queen is null for 0–1 day old, moderate for

Differential Pheromone

Polyandrous Queen Honey Bee 127

2–4 days old, and considerably high for five days to 18-month-old. Therefore, mating is a crucial factor for the development of the chemical signal of the queen and its stunning effects on workers. Keeling et al. (2003) identified other additional compounds, including coniferyl alcohol (CA), methyl oleate (MO), hexadecane-1-ol (PA), and linoleic acid (LA), which act synergistically with QMP to attract worker honey bees. In the mandibular gland of the queen and worker honey bees, caste-specific pheromonal secretion has been reported with 8and 10-carbon fatty acids as the main components. Queen honey bees without a mandibular gland can still attract worker honey bees, which specifies that other substances, in addition to QMP, facilitate the attraction of worker honey bees (Plettner et al. 1996). The de-mandibulated queen can exert some regulatory functions on workers, including retinue behaviour, inhibition of queen cup construction, and suppression of worker ovary development. In the de-mandibulated queen, the level of QMP was similar to that of control except for 9-ODA, which is secreted by the mandibular gland only, whereas the queen's body produces HOB and 9-HDA. Predominantly, 9-ODA pheromone is responsible for the induction of retinue behaviour in worker bees. A queen raised from first instar worker larvae elicited a more significant worker retinue response than a queen raised from third instar worker larvae. Scientific literature search reveals that OMPs regulate physiological and behavioural responses in workers (Slessor et al., 1988; Pankiw et al., 1998; Hoover et al., 2003; Richard et al., 2007; Strauss et al., 2008; Rangel et al., 2016). Richard et al. (2007) reported that queens inseminated with a single drone or multiple drones elicit a differential-sized retinue. Furthermore, Kocher et al. (2009) said that caged worker bees become more attracted to mandibular gland extract of the naturally mated queen than instrumentally inseminated queens or virgin queens. In contrast, Nino et al. (2012) observed that the retinue was more enormous in queens inseminated with a high versus a low volume of semen. Retinue size can increase due to other factors, including cuticle lipids, tergal gland secretion, and Dufour's gland secretion. Some studies indicate that queen retinue response is seen even in de-mandibulated queens. To keep or to replace the queen with queen supersedure can be one of the decisions of worker honey bees. Rangel et al. (2013) did not observe any difference in queen supersedure in colonies headed by queens raised from either young or old worker larvae. Queen reproductive quality is a complex function of queen age, mating state, initial rearing age, insemination volume, and other factors (Rangel et al., 2016).

128 Polyandrous Queen Honey Bee

Lovleen Marwaha

6.2.1.5. On Wax Secretion QMPs of reproductively active queens elicit worker honey bees to produce more wax compared to colonies headed by virgin queen honey bees or in the queen-less colony. The presence of a mated queen or strong QMP emission inhibits the production of a male brood. Furthermore, predominantly HVA and HOB affect wax production in the colony. QMPs elicit foraging in worker honey bees by stimulating quantitative pollen transportation. QMPs enhance calmness in workers by decreasing stinging responses in caged honey bees or inhibiting the defensive behaviour of worker honey bees (Vergoz et al. 2007). 6.2.1.6. On Ovarian Development Furthermore, queen and brood pheromones, especially ethyl palmitate and methyl linoleate, suppress the ovarian development of worker honey bees. During the development of worker honey bees, the low titer of juvenile hormone affects ovarian development (Hartfelder 1993, 1997). Hoover et al. (2003) demonstrated that synthetic QMP was able to suppress the development of ovarian development in worker honey bees. Hoover et al. (2003) showed that QMP alone is sufficient to stop the growth of ovaries in workers. QMP, along with Dufour's gland, inhibits ovarian development in worker honey bees but has been proved less effective than the live queen. 6.2.2. Dufour's Gland The specific gland was described by Dufour in 1841 as a tubular gland associated with sting apparatus. The concerned gland is drained through a duct that opens into the vagina. Therefore, egg marking pheromones are applied before egg deposition into hexagonal wax cells (Katzav-Gozansky et al. 1997). The concerned gland secretion acts as caste-specific egg discriminating pheromones, which help differentiate queen-laid or worker-laid eggs (Ratnieks and Visscher 1989; Ratnieks 1993). Queen honey bee Dufour gland exudate contains wax-type esters, with predominant component tetradecyl hexadecanoate and hexadecyl tetradecanoate. The specific ester could be a possible queen-specific signal for egg marking. Earlier, it had been considered that Dufour's gland secretion facilitates worker policing, in which workers kill eggs laid by fellow workers (Ratnieks 1993), but later on, this hypothesis was rejected. Like QMP, Dufour's gland secretion seems to attract worker honey bees. Furthermore, in egg-laying workers, Dufour's gland secretion attracts other worker honey bees in the colony. Worker honey bees get

Differential Pheromone

Polyandrous Queen Honey Bee 129

more drawn toward Dufour's gland secretion of an inseminated queen than a virgin queen. Richard et al. 2011 reported that worker honey bees are more attracted to multi-drone inseminated than single drone insemination. Furthermore, QMP and Dufour's gland secretion effectively inhibit the ovarian development of worker honey bees. QMPs and Dufour's gland collectively influence the developmental phase of worker honey bees' reproductive formation. Dufour's gland secretion acts as a fertility signal, whereas mandibular gland secretion is involved in establishing reproductive dominance. In the queen-less colony, workers with highly aggressive behaviour but undeveloped ovaries produce fewer queen-like pheromones in mandibular glands. In contrast, workers with low aggressive behaviour and larger oocytes have more queen-like pheromones in Dufour's gland (fertility signal). 6.2.3. Koschevnikov Gland Koschevnikov glands are present in queens and workers, but their secretion is according to the honey bee caste. The specific gland is located near the sting shaft and is responsible for producing alarm pheromones, mainly composed of acids, alcohols, alkanes, and alkenes. QKG is composed of 31 compounds including acids, alcohol, alkanes and alkenes as follows: 1,1,3-trimethyl cyclopentane, 5,5 dimethyl-2-hexane, 3,3-dimethyl hexane, octenal; methyl cyclodecane; pmenthane-9-ol; 4,5 -dimethyl nonane; 2-propyl-1-heptanol; 4,6,8-trimethyl1-nonene, nonanoic acid, decanoic acid, 1,12-tridecadience; 1,11-docecadience, cyclohexyl hexanol; ethyl decanoate; 2-methyl-1-dodecanol; dodecyl acetate; 6cyclohexyl undecane; hexadecanoic acid; ethyl tetradecanoate; methylester 2 methyl hexadecanoate; 2-(hexadecyl oxy)-ethanol; 2,6,10,15- tetramethyl heptadecane; 1-dotriacontanol; 1,7-pentatriacotene; 3,5,24-trimethyl tetracontane. 6.2.4. Tarsal Gland The specific glands are located on all castes' 5th tarsomere of prothoracic, mesothoracic, and metathoracic appendages. Worker tarsal gland known as footprint pheromones is deposited at the hive entrance by foragers and probably on visited flowers. Footprint pheromones act as a proximity signal, as it is effective for a short distance, whereas Nasonov pheromone is effective for higher distances. In workers, virgin queen, and mated Queen of A. m. capensis, tergal gland secretion contains significant components, including (Z)-9-octadecenoic acid, decyl decanoate, and longer chain-length esters of decanoic acid. Furthermore, the tergal gland of the virgin queen possesses an inhibitory effect on worker ovary development. Tarsal gland secretion by the mated queen inhibits the construction of queen cups. Inhibitory effects of Queen tergal gland secretion had

130 Polyandrous Queen Honey Bee

Lovleen Marwaha

been studied on worker ovarian development with honey bee races Apis mellifera capensis and Apis mellifera scutellate (Renner and Baumann, 1964; Velthuis,1967; Velthuis, 1970; Velthuis, 1977; Hepburn et al., 1991; Lensky et al., 1995). Virgin queen tergal gland extract of Apis mellifera capensis and Apis mellifera scutellate inhibits ovarian development in their own workers. In honey bee colonies, queen movement is restricted to the central part of the comb. Therefore, queen cells are constructed on the bottom edge. Queen honey bee, while moving on the comb, deposits tarsal gland secretion, which discourages worker honey bees from raising a new queen. 6.3. WORKERS' PHEROMONES Worker honey bees secrete diverse messages from exocrine glands in the form of pheromones involved in foraging, brood rearing, protecting, and variant social communications. Different pheromones secreted by worker honey bees include: Alarm pheromone Several glands are associated with the sting apparatus of worker honey bees which produce a specific combination of odours that alarm the colony. In addition, worker honey bees secrete two specific alarm pheromones in honey bees by blending secretions of Koschevnikov and the mandibular gland. 6.3.1. Koschevnikov Gland Koschewnikov gland had been demonstrated to produce some alarm pheromones components (Mauchamp and Grandperrin, 1982). According to Lensky et al., 1995, ethanolic extract of a specific gland can provoke aggressive behaviour in the colony, similar to guarding honey bees toward introducers. Concerned glands are paired structures located in the seventh abdominal segment. Koschewnikov gland near the sting shaft secrete pheromones, which include more than 40 different chemicals, including isopentyl acetate (IPA), butyl acetate, 1-hexanol, n-butanol, 1-octanol, hexyl acetate, octyl acetate, n-pentyl acetate, and 2-nonanol. Further, these pheromones possess high volatility and lower molecular weight (Collins and Blum, 1982, 1983; Pickett et al., 1982). The chemical composition of Worker Koschewnikow Gland (WKG) is different from Queen Koschewnikow Gland (QKG). Mauchamp and Grandperrin, 1982 detected isoamyl acetate, isoamyl alcohol, hexyl acetate, nonanol, benzyl acetate, and benzyl alcohol in workers' Koschewnikow gland. A volatile substance released by the Koschewnikow gland and sting sheath triggers the aggressive behaviour of worker honey bees (Lensky et al., 1995). Isopentyl acetate (IPA, or isoamyl

Differential Pheromone

Polyandrous Queen Honey Bee 131

acetate) acts as a principal active component of alarm pheromones. Pickett et al. (1982) identified less volatile component (Z)-11-eicosenol as another effective alarm pheromone. Furthermore, it has been reported that there is variation in the composition of alarm pheromones in different species, affecting honey bees' aggressiveness. Alarm pheromones of Africanized honey bees are mainly composed of a specific unsaturated derivative of IPA (3-methyl-2-butene-1-yl acetate, 3M2BA). The release of alarm pheromones occurs when a honey bee sting another animal and attracts other honey bees to a specific location. 6.3.2. Mandibular Gland The second pheromone, which acts as an alarm pheromone in worker honey bees, is secreted by the mandibular gland and mainly consists of 2-heptanone composed of a highly volatile substance. The specific gland possesses a repellent effect, which potential enemies and robber bees detect. The amount of 2-heptanone increases with the age of worker honey bees, and a specific chemical, i.e. 2heptanone, is implemented by foragers to mark recently visited flowers and the depleted foraging location. Furthermore, it has been detected that 2-heptanone is used to paralyze intruders. Further, worker honey bees produce 2-heptanone (2-H) through their mandibular gland, which induces problematic and stinging behaviour, similar to iso-pentyl acetate by the Koschewnikow gland (Lensky et al., 1995). Worker honey bees' mandibular gland secretion act as footprint pheromones to mark nest entrance and as a thermoregulatory pheromone, which induces muscle contraction in worker honey bees. 6.3.3. Brood Recognition Pheromones Brood pheromones prevent worker bees from bearing offspring in a colony. Larvae and pupae emit brood recognition pheromones, which inhibit ovarian development in worker bees and facilitate the recognition of worker larvae from drone larvae. The specific pheromone is a blend of fatty acid ester, which modify the adult caste ratio. The composition of brood pheromone varies with the age of developing bees. 6.3.4. Nasonov Gland The Nasonov gland is located beneath the intersegmental membrane, between worker honey bees' sixth and seventh tergites. Worker honey bees spread this

132 Polyandrous Queen Honey Bee

Lovleen Marwaha

pheromone by elevating the abdomen and wing fanning. The concerned gland secretion contains mainly geraniol, geranial, geranic acid, (E)-citral, nerolic acid, (Z)-citral, (E-E)-farnesol, and nerol, which influence foraging, swarming, house hunting, and marking in worker honey bees (Pickett et al. 1980). The specific pheromones facilitate worker honey bees for marking hive entrance, swarm clustering, and keeping foraging sources. Worker honey bees usually disperse specific pheromones at the hive's entrance to direct foragers of any particular colony. Further, Nasonov pheromones are implemented by worker honey bees to mark specific selected larval cells to attract other workers toward that brood cell for proper rearing. Additionally, worker honey bees use Nasonov gland secretion for water collection. Further, along with QMP, secretion from worker honey bee Nasonov gland functions facilitate swarm clustering. (Free 1987). The specific pheromone is involved in orientation and recruitment. 6.3.5. Dufour's Gland Pheromone The specific glands open into the dorsal vaginal wall. Alkaline product is realized into the vaginal cavity and is laid along with eggs, which allows worker bees to distinguish between eggs laid by the queen. The specific gland secretion differs in composition between queen-less colonies and queen-right colonies. In queenless colonies, worker honey bees secrete long-chain alkanes with an even number of carbon atoms, whereas, in queenright colonies, there is a long-chain alkane with an odd number of the carbon atom. 6.3.6. Footprint Pheromones The specific pheromones are secreted by worker honey bees and left by bees while walking. 6.3.7. Forager Pheromone The primary forager pheromone is an inhibitory factor, 'ethyl oleate,' which delays the onset of foraging age. Older forager worker bees secrete the concerned forager pheromone to delay the maturation of nurse bees. Therefore, the specific volatility acts as a distribution regulator for the ratio of nurse bees to forager bees in the balance. To produce particular compounds, honey bees perform biochemical derivation of ethanol obtained from fermented nectar. After that. the chemical is exuded to the exoskeleton, from where it is transmitted to other workers. Ethyl

Differential Pheromone

Polyandrous Queen Honey Bee 133

oleate has been detected in the pheromone blend of queens and brood, therefore classified as colony pheromone (Keeling and Slessor 2005; Slessor et al. 2005). 6.3.8. Other Pheromones Other pheromones include rectal gland pheromone, tarsal pheromone, wax gland, comb pheromone, and tergite gland pheromone, which help communicate with other residents of the colony. The conclusion for honey bee pheromones is that differential gene expression, larval diets, developmental patterns, hormonal secretions, physiological plasticity, and variant biochemical synthesis pathways eventually result in different compositions of pheromonal gland emissions. Queen honey bee volatile characteristics confirm her reproductive uni-rightness and colonial dominance through the modulation of workers' behaviour, development, physiology, reproduction, etc., on the contrary, workers' pheromonal profiling ensures harmonious conduct of various colonial tasks, including brood rearing, foraging, protection, and other colony dwellers' social interaction. The correlation between genomic expres sion, physiological plasticity, and reproduction is remarkably highlighted through pheromonal differentiation in female honey bees. CONCLUSION Queen honey bee secretes specific pheromones, which induce differential behaviour in worker honey bees, induce programmed cell death in workers' ovaries, and regulate overall coordination and co-operation in the honey bee colony. Queen honey bee secretes pheromones through different glands, which induce submissive behaviour in workers and confirm her dominance. REFERENCES Blum, MS (1992) Honey bee pheromones in the hive and the honey bee, Dadant and Sons 385-9. Blum, MS & Brand, JM (1972) Social insect pheromones: their chemistry and function. Am Zool, 12, 553-76. [http://dx.doi.org/10.1093/icb/12.3.553] Butler, CG (1967) Insect pheromones. Biol Rev Camb Philos Soc, 42, 42-84. [http://dx.doi.org/10.1111/j.1469-185X.1967.tb01339.x] Butler, CG & Fairey, EM (1963) The role of the queen in preventing oogenesis in worker honeybees. J Apic Res, 2, 14-8. [http://dx.doi.org/10.1080/00218839.1963.11100051] Collins, AM & Blum, MS (1982) Bioassay of compounds derived from the honeybee sting. J Chem Ecol, 8, 463-70. [http://dx.doi.org/10.1007/BF00987794] [PMID: 24414957] Collins, AM & Blum, MS (1983) Alarm responses caused by newly identified compounds derived from the honeybee sting. J Chem Ecol, 9, 57-65.

134 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1007/BF00987770] [PMID: 24408619] Collins, AM & Pettis, JS (2013) Correlation of queen size and spermathecal contents and effects of miticide exposure during development. Apidologie (Celle), 44, 351-6. [http://dx.doi.org/10.1007/s13592-012-0186-1] Engels, W, Kaatz, H, Zillikens, A, Simões, ZP, Trube, A, Braun, R & Dittrich, F (1990) Free, JB (1987) Pheromones of social bees, Comstock PubAssociates, Ithaca, NY. Gary, NE (1962) Chemical mating attractants in the queen honey bee. Science, 136, 773-4. [http://dx.doi.org/10.1126/science.136.3518.773] [PMID: 17752107] Hartfelder, K (1993) Structure and function of the prothoracic gland in honey bee ( Apis mellifera L.) development. Invertebr Reprod Dev, 23, 59-74. [http://dx.doi.org/10.1080/07924259.1993.9672294] Hartfelder, K & Steinbrück, G (1997) Germ cell cluster formation and cell death are alternatives in castespecific differentiation of the larval honey bee ovary. Invertebr Reprod Dev, 31, 237-50. [http://dx.doi.org/10.1080/07924259.1997.9672582] Hepburn, HR (1994) Reproductive cycling and hierarchical competition in Cape honeybees, Apis mellifera capensis Esch. Apidologie (Celle), 25, 38-48. [http://dx.doi.org/10.1051/apido:19940105] Hepburn, HR, Magnuson, P, Herbert, L & Whiffler, LA (1991) The development of laying workers in field colonies of the Cape honey bee. J Apic Res, 30, 107-12. [http://dx.doi.org/10.1080/00218839.1991.11101242] Hepburn, HR, Nefdt, RJC & Whiffler, LA (1988) Queen loss in the Cape honeybee: the interactions of brood, laying workers (false queens?) and queen cells. S Afr J Sci, 84, 778-80. Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Katzav-Gozansky, T, Soroker, V, Hefetz, A, Cojocaru, M, Erdmann, DH & Francke, W (1997) Plasticity of caste-specific Dufour’s gland secretion in the honey bee (Apis mellifera L.). Naturwissenschaften, 84, 238-41. [http://dx.doi.org/10.1007/s001140050386] Katzav-Gozansky, T, Boulay, R, Soroker, V & Hefetz, A (2004) Queen–signal modulation of worker pheromonal composition in honeybees. Proc Biol Sci, 271, 2065-9. [http://dx.doi.org/10.1098/rspb.2004.2839] [PMID: 15451697] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2008) Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera). BMC Genomics, 9, 232. [http://dx.doi.org/10.1186/1471-2164-9-232] [PMID: 18489784] Lensky, Y & Cassier, P (1995) The alarm pheromones of the queen and worker honey bees. Bee World, 76, 119-29. [http://dx.doi.org/10.1080/0005772X.1995.11099258] Mauchamp, B & Grandperrin, D (1982) Chromatographie en phase gazeuse des composés volatils des glandes a phéromones des abeilles: méthodes d’analyse directe. Apidologie (Celle), 13, 29-37. [http://dx.doi.org/10.1051/apido:19820104] Moritz, RFA & Hillesheim, E (1985) Inheritance of dominance in honeybees (Apis mellifera capensis Esch.). Behav Ecol Sociobiol, 17, 87-9. [http://dx.doi.org/10.1007/BF00299434]

Differential Pheromone

Polyandrous Queen Honey Bee 135

Moritz, RF, Lattorff, HMG & Crewe, RM (2004) Honeybee workers (Apis mellifera capensis) compete for producing queen-like pheromone signals. Proc Biol Sci, 271 (Suppl. 3), S98-S100. [PMID: 15101431] Moritz, RFA, Simon, UE & Crewe, RM (2000) Pheromonal contest between honeybee workers (Apis mellifera capensis). Naturwissenschaften, 87, 395-7. [http://dx.doi.org/10.1007/s001140050748] [PMID: 11091962] Pain, J (1961) Absence du pouvoir d’inhibition de la phéromone, I. Sur le développement ovarien des jeunes ouvrières d’abeilles. CRAS, 252, 2316-7. Pankiw, T, Huang, Z, Winston, ML & Robinson, GE (1998) Queen mandibular gland pheromone influences worker honey bee (Apis mellifera L.) foraging ontogeny and juvenile hormone titers. J Insect Physiol, 44, 685-92. [http://dx.doi.org/10.1016/S0022-1910(98)00040-7] [PMID: 12769952] Pankiw, T, Winston, ML, Plettner, E, Slessor, KN, Pettis, JS & Taylor, OR (1996) Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.). J Chem Ecol, 22, 605-15. [http://dx.doi.org/10.1007/BF02033573] [PMID: 24227572] Pickett, JA, Williams, IH & Martin, AP (1982) (Z)-11-eicosen-1-ol, an important new pheromonal component from the sting of the honey bee,Apis mellifera L. (Hymenoptera, Apidae.). J Chem Ecol, 8, 16375. [http://dx.doi.org/10.1007/BF00984013] [PMID: 24414592] Pettis, JS, Dietz, A & Eischen, FA (1989) Incidence rates of Acarapis woodi (Rennie) in queen honey bees of various ages. Apidologie (Celle), 20, 69-75. [http://dx.doi.org/10.1051/apido:19890107] Plettner, E, Slessor, KN, Winston, ML & Oliver, JE (1996) Caste-selective pheromone biosynthesis in honeybees. Science, 271, 1851-3. [http://dx.doi.org/10.1126/science.271.5257.1851] Plettner, E, Otis, GW, Wimalaratne, PDC, Winston, ML, Slessor, KN, Pankiw, T & Punchihewa, PWK (1997) Species-and caste-determined mandibular gland signals in honeybees (Apis). J Chem Ecol, 23, 36377. [http://dx.doi.org/10.1023/B:JOEC.0000006365.20996.a2] Rangel, J & Fisher, A, II (2019) Factors affecting the reproductive health of honey bee (Apis mellifera) drones—a review. Apidologie (Celle), 50, 759-78. [http://dx.doi.org/10.1007/s13592-019-00684-x] Rangel, J & Tarpy, DR (2015) The combined effects of miticides on the mating health of honey bee ( Apis mellifera L.) queens. J Apic Res, 54, 275-83. [http://dx.doi.org/10.1080/00218839.2016.1147218] Rangel, J & R Tarpy, D (2016) In-hive miticides and their effect on queen supersedure and colony growth in the honey bee (Apis mellifera). J Environ Anal Toxicol, 61000377 [http://dx.doi.org/10.4172/2161-0525.1000377] Rangel, J, Keller, JJ & Tarpy, DR (2013) The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth. Insectes Soc, 60, 65-73. [http://dx.doi.org/10.1007/s00040-012-0267-1] Ratnieks, FW (1993) Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies. Behav Ecol Sociobiol, 32, 191-8. [http://dx.doi.org/10.1007/BF00173777] Renner, M & Baumann, M (1964) �ber Komplexe von subepidermalen Dr�senzellen (Duftdr�sen?) der Bienenk�nigin. Naturwissenschaften, 51, 68-9. [http://dx.doi.org/10.1007/BF00603470]

136 Polyandrous Queen Honey Bee

Lovleen Marwaha

Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2e980 [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Richard, FJ, Schal, C, Tarpy, DR & Grozinger, CM (2011) Effects of instrumental insemination and insemination quantity on Dufour’s gland chemical profiles and vitellogenin expression in honey bee queens (Apis mellifera). J Chem Ecol, 37, 1027-36. [http://dx.doi.org/10.1007/s10886-011-9999-z] [PMID: 21786084] Saiovici, M (1983) 9-Oxodecenoic acid and dominance in honeybees. J Apic Res, 22, 27-32. [http://dx.doi.org/10.1080/00218839.1983.11100556] Schäfer, MO, Dietemann, V, Pirk, CWW, Neumann, P, Crewe, RM, Hepburn, HR, Tautz, J & Crailsheim, K (2006) Individual versus social pathway to honeybee worker reproduction (Apis mellifera): pollen or jelly as protein source for oogenesis? J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 192, 761-8. [http://dx.doi.org/10.1007/s00359-006-0112-y] [PMID: 16508763] Seeley, TD (2009) The wisdom of the hive: the social physiology of honey bee coloniesHarvard University Press. [http://dx.doi.org/10.2307/j.ctv1kz4h15] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Kaminski, LA, King, GGS & Winston, ML (1990) Semiochemicals of the honeybee queen mandibular glands. J Chem Ecol, 16, 851-60. [http://dx.doi.org/10.1007/BF01016495] [PMID: 24263600] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Strauss, K, Scharpenberg, H, Crewe, RM, Glahn, F, Foth, H & Moritz, RFA (2008) The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent? Behav Ecol Sociobiol, 62, 1523-31. [http://dx.doi.org/10.1007/s00265-008-0581-9] Velthuis, HHW (1967) On abdominal pheromones in the queen honeybee. Proc XXI Int Beekeeping Congr, College Park, USA, Ed Apimondia, Bucharest, 58-9. Velthuis, HHW (1970) Queen substances from the abdomen of the honey bee queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 70, 210-21. [http://dx.doi.org/10.1007/BF00297717] Velthuis, HHW (1970) Ovarian development in Apis mellifera worker bees. Entomol Exp Appl, 13, 377-94. [http://dx.doi.org/10.1111/j.1570-7458.1970.tb00122.x] Velthuis, HHW (1977) Egg laying, aggression and dominance in bees. Proceedings of International Congress of Entomology, 436-49. Velthuis, HHW & van Es, J (1964) Some functional aspects of the mandibular glands of the queen honeybee. J Apic Res, 3, 11-6. [http://dx.doi.org/10.1080/00218839.1964.11100076] (1985) The honeybee queen and the social organization of her colony. Fortschritte der Zoologie (Stuttgart), 31, 343-57. Vergoz, V, Schreurs, HA & Mercer, AR (2007) Queen pheromone blocks aversive learning in young worker bees. Science, 317, 384-6. [http://dx.doi.org/10.1126/science.1142448] [PMID: 17641204] Willis, LG, Winston, ML & Slessor, KN (1990) Queen honey bee mandibular pheromone does not affect

Differential Pheromone

Polyandrous Queen Honey Bee 137

worker ovary development. Can Entomol, 122, 1093-9. [http://dx.doi.org/10.4039/Ent1221093-11] Winston, ML (1987) The biology of the honey bee Harvard Univ. Press Cambridge, MA Google Scholar Winston, ML & Slessor, KN (1992)

138

Polyandrous Queen Honey Bee, 2022, 138-162

CHAPTER 7

Mandibular Pheromone Types, Functions, Synthesis, And Associated Genetic Elements In The Queen Honey Bee, Apis mellifera Abstract: Queen Mandibular Pheromones (QMP) include (E)-9-oxo-2-decanoic acid(9-ODA), (R)-and (S)-(E)-9-hydroxy-2-decanoic acid(9-HDA), methyl 4hydroxybenzoate(HOB), 10-hydroxy-decanoic acid (10-HDAA), 4-hydroxy-methoxyphenyl ethanol (HVA), and10-hydroxy-2 (E)-decanoic acid (10-HDA), whereas worker honey bees mandibular gland pheromones include mainly 10-hydrox-2 (E)-decanoic acid (10-HDA),10-hydroxydecanoic acid (10-HDAA), and 2- mainly 2-heptanone (2-H), traces of 9-hydroxy-2 (E)-decanoic acid (9-HDA) and 9-ODA. Biochemical modifications of stearic acid occur through hydroxylation of stearic acid at ω or ω-1 positions in worker honey bee and queen, synthesizing the primary pheromones listed above. 9-ODA pheromone influences alcohol dehydrogenase gene expression, and the specific enzyme is essential for converting 9-HDA to 9-ODA in worker honey bees. Further, the differential synthesis process is influenced by the gene expression of various cytochromes. QMPs impose differential influence on various developmental, functional, and behavioural regulations on nest mates, which include retinue behaviour, suppression of the development of worker honey bee ovaries, wax secretion, drone attraction, swarming, queen dominance regulation, general regulation, mating, and reproduction, juvenile hormone secretion in workers, foraging behaviour and the different submissive response of workers in the presence of the queen.

Keywords: Differential secretion, Genetic elements, Mandibular gland, Pheromones. 7.1. INTRODUCTION Queen Mandibular Pheromones (QMP), the volatile chemicals, are chemical messengers that modulate bees' social behaviour. Furthermore, specific pheromones also influence different caste interactions, mating, reproduction, honey production, swarming decision, and functional regulation of colony, in facultative reproducible worker honey bees (Fig. 7a). Further, QMPs influence development, normal physiology, submissive mating behaviour, and ovarian programmed cell death in workers. Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 139

Fig. (7a). Pheromonal components of queen honey bee and impact on nest mates.

Specific volatile chemicals can exert specific short-term and long-term effects on worker honey bees within a composite hive. Scientific literature search witnesses that QMP was detected first in 1950. (E)-9-oxodec-2-enoic-acid (9-ODA) had been identified first. The concerned pheromone is a biochemical product of two saclike glands positioned within the head, near the base of the mandible, of the concerned insect. A specific exogenous gland's duct opens at the mandible's basal end, and biochemical secretion is carried by a deeper channel surrounded by hairs (Slessor et al., 1988). Further, they analyzed four other biochemical products secreted by mandibular glands, which act synergistically with (E)-9-oxode-2-enoic-acid (9-ODA), and the volatile chemical group includes; methyl phydroxybenzoate (HOB), two enantiomers 9-hydroxydec-2-enoic acid (9-HDA), and 4-hydroxy-3-methoxy-phenyl ethanol (HVA). All specified volatile chemicals induced a synergistic effect than additive, i.e. all compounds together induce a more substantial impact than any single compound, alone or in combination (Fig. 7b).

140 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (7b). Comparison of Pheromonal composition of a virgin and mated queen.

Recent advances reflect that QMP is composed of (E)-9-oxo-2-decanoic acid(9ODA), (R)-and (S)-(E)-9-hydroxy-2-decanoic acid(9-HDA), methyl--hydroxybenzoate (HOB), 10-hydroxy-decanoic acid (10-HDAA), 4-hydroxy-methoxyphenylethanol (HVA), and 10-hydroxy-2 (E)-decanoic acid (10-HDA) (Slessor et al., 2015). QMP induces two types of responses: primer, physiological, and releaser, short-term behavioural responses (Slessor et al., 1988; Pankiw et al., 1994; Pankiw et al., 1995; Slessor et al., 2005). In addition, QMPs inhibit the development of a new queen in the colony (Pettis et al., 1995; Melathopoulos et al., 1996; Pettis et al., 1997), and inhibit the development of workers' ovaries (Butler and Fairey, 1963,1968; Hoover et al., 2003), attract drones during mating (Gary,1962; Brockmann et al., 2006), stimulate foraging in worker honey bee (Higo et al., 1992; Pettis et al., 1995; Pankiw et al., 1998), delay foraging onset in worker honey bees, lower juvenile hormone secretion, etc. (Pankiw et al., 1998). QMP is responsible for inducing retinue responses in workers, which means workers surround the queen while collecting QMP (Winston, 1987; Slessor et al., 1988; Kaminski et al., 1990; Pankiw et al., 1994; Pankiw et al., 1995). Passing QMP among workers facilitates recognition of the queen's presence in the colony without coming into her direct contact (Seeley, 1979). The chemical composition of QMP depends upon the queen's ontogeny and mating state. Scientific explorations indicated the difference in the chemical composition of QMP between virgin. They mated queens (Plettner et al., 1997), laying or non-laying mated queens (Kocher et al., 2008), and naturally mated or artificially mated queens (Kocher et al., 2009). Further, QMP composition differs in queens mated to one drone versus multiple drones (Richard et al., 2007) or queens mated with

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 141

low versus high volume of semen or saline solution (Nino et al., 2012). The chemical composition of QMP influences the attractiveness of the workers toward the queen. The queen produces more 9-HDA but less 10-HDA, whereas worker honey bees have more 10-HDA but less 9-HDA. Further, the virgin queen secretes the same amount of 9-ODA and10-HDA, but the mated queen secretes more 9-ODA (Plettner et al., 1997). Rangel et al. (2016) concluded the presence of a high concentration of 10-HDA in QMP in low-quality queens than in high-quality queens. Further, they analyzed that the specific observation is due to the influence of environmental factors. Furthermore, they reported that queen grafting age influenced the chemical profile of the mandibular gland. All components in the mated queen had been secreted in a higher concentration in the virgin queen, except for 10-HDA. Further, 10-HDA is secreted in high concentration by workers, but 9-HDA is secreted in high concentration by the Queen (Plettner et al., 1997). Superior quality queen secretes more 9-ODA to 10-HDA (Plettner, et al., 1997; Moritz, et al., 2000; Moritz, et al., 2004; Schafer, et al., 2006). Finally, Rangel et al. (2016) explored the detailed analysis of the QMP bouquet of colonies headed by a more substantial or better quality queen, with colonies having a comparatively weak queen, through GCMS. Chemical profiling of QMG indicates high-quality and low-quality queens, and specific profiling is an indirect indicator of retinue efficiency, which is the attractiveness of workers to the queen. The mandibular gland composition of high-quality and low-quality honey bees exhibit significant differences in the productivity of queen mandibular pheromone components HVA and 9-HDA. Due to mandibular gland secretion, workers are attracted to the high-quality queen more than the low-quality queen. In addition, the age of the larva, when it enters into the queen's developmental pathway further, influences the composition of the mandibular gland and worker behaviour (Rangel et al., 2016). 7.2. COMPOSITION OF QMP Queen mandibular pheromones can be carboxylic acid derivatives and aromatic compounds, which comprise (E)-9-oxo-2-decanoic acid (9-ODA), (R)-and (S)(E)-9-hydroxy-2-decanoic acid (9-HDA), methyl 4-hydroxybenzoate (HOB), 10hydroxy-2 (E)-decanoic acid (10-HDA) and 10-hydroxy-decanoic acid (10HDAA) (Slessor et al., 1988; Apoegaite et al., 1999; Keeling et al.,2003; Pirk et al., 2011; Yusuf et al., 2015; Rangel et al., 2016). The biochemical synthesis of QMP is dependent upon many factors, including the genomic integrity of the Queen and her mating state.9-HDA, 10-HDA, and HOB increase the attractancy

142 Polyandrous Queen Honey Bee

Lovleen Marwaha

of drones toward the queen but possess short duration effectively, whereas 9ODA is effective for higher distance (Brockmann et al. 2006). The virgin queen implements QMPs to attract drones during mating flights (Gary 1962). Further, according to Blum 1992, queen honey bees produce volatile chemicals through 15 pheromone glands, with the primary hormone QMP, which regulates the behaviour of worker honey bees. QMPs affect retinue formation, drone attraction, and workers' ovarian development and inhibit juvenile hormone formation in workers (Gary 1962; Kaminski et al. 1990; Hoover et al. 2003, 2005). 9-oxo-2(E)-decanoic acid (9-ODA) forms a significant component, commonly known as the queen substance, whereas worker honey bees secrete the two fatty acids 10-hydroxy-2(E)-decanoic acid (10-HDA) and 10hydroxydecanoic acid (10-HDAA) (Plettner et al. 1993). 9-HDA, 10-HDA, and HOB increase the attractancy of drones toward the queen but possess shortduration effectivity, whereas 9-ODA is effective for higher distances (Brockmann et al. 2006). On removal of the queen from the colony, the mandibular gland composition of worker honey bees changes, with the relative increase in 9-ODA and concomitantly a decrease in 10-HDAA and 10-HDA concentration (Simon et al. 2001). In such colonies, even egg-laying workers can elicit retinue behaviour in other nest mates (Crewe and Velthuis 1980). Furthermore, it has been reported that the pheromone chemical composition of the newly emerged virgin queen resembles worker honey bees (Crewe 1982). Therefore, there is a remarkable change in the design of QMP, with a shift from worker to queen physiology (Slessor et al. 1990; Engels et al. 1997; Apsegaite and Skirkevicius 1999). Furthermore, as workers are facultatively reproducible, with the change in fecundity status, there is also a change in mandibular gland pheromonal composition (Plettner et al. 1995, 1996). Furthermore, numerous reports have witnessed that there is a variation in the chemical composite of QMPs of virgin versus mated queens, egg-laying and nonegg-laying queens naturally mated versus artificially inseminated, queens inseminated with one drone or multiple drones, queen inseminated with low versus high volume of semen or saline solution (Plettner et al., 1997; Kocher et al., 2005; Richard et al., 2007; Kocher et al., 2008; Nino et al., 2012). A comparison of virgin and mated queens indicated that there is more secretion of 10-HDA in the former than latter (Plettner et al. 1997). Furthermore, Plettner et al. (1997) conducted a comparative study on the composition of QMPs between 6-day-old and 1-year-old mated egg-laying queens. They reported that mated queens secrete higher levels of 9-ODA, HOB,

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 143

9-HDA, and HVA, whereas workers and virgin queen mandibular glands secrete higher concentrations of 10-HDAA and 10-HDA. Furthermore, in higher quality queens, there is a further reduction of 10-HDA to 9-HDA. Therefore, it can be considered a good indicator of queen quality, whereas the ratio of 9-ODA to 10HDA tends to increase with queen age and the mating status of the Queen (Plettner et al., 1997). According to Engels et al. (1997), there are three different patterns of QMP secretion such as virgin premating queens with the primary secretion of oleic acid (OLA), mated queens with 9-ODA, OLA, and 9-HDA, and post-mating dominant queens with high concentrations of 9-ODA, medium proportions of 9-HDA, less OLA, HOB, and HVA. Further, Engels et al. (1997) concluded that HOB and the late-appearing HVA are significant signals of old egg-laying and dominant queens. In contrast, Slessor et al. (1990) reported consistent levels of 9-ODA in virgin and mated queens, 9-HDA levels were higher in mated than in virgin queens, and HOB and HVA levels were higher in the oldest mated queens than virgin and young mated ones. In addition, the pheromone level in mature, mated, and laying queens was relatively high. Queen mandibular gland chemical profile is highly variable. For example, in some queens, honey bees' ratio of 10-HDA to 9-HDA may be >1 or 1 in mated queens (Plettner et al., 1997). If the ratio of 9-ODA/(9-ODA + 10-HDA) is one then mandibular gland secretion is more queen like or if close to zero, it means worker like (Moritz, et al., 2000; Moritz, et al., 2004; Schäfer, et al., 2006). Furthermore, Simon et al. (2001) reported that the mandibular gland secretion of workers is composed of 10-HDA and 10-HDAA. However, after four days of emergence in the queenless colony, workers' mandibular gland secretion becomes dominated by 9-ODA. Further, Tan et al. (2012) reported that in A. mellifera and A. cerana, 9-ODA, HVA, HOB, 10-HDA, 9-HDA, and 10-HDAA levels are high in the mandibular gland of egg-laying workers than non-egg laying one. Keeling et al. 2003 reported that four additional compounds act synergistically with QMP, which include: methyl oleate (MO), hexadecane-1-ol (PA), coniferyl alcohol (CA), and linoleic acid (LA). Richards et al. (2007) noted that the mandibular gland extract of the inseminated queen was more attractive than the virgin queen. They compared a 7-day-old virgin with a mated queen, and it had been discriminated against that a mated queen secretes low quantities of 9-ODA, 9-HDA, and HVA than a virgin. However, mated queen mandibular gland extract is more attractive for worker honey bees than virgin queens.

144 Polyandrous Queen Honey Bee

Lovleen Marwaha

In other words, the mandibular gland secretion signals the entire reproductive status within the colony. Worker honey bees can mimic mandibular gland secretion to dominate the colony (Moritz et al. 2000, 2004). According to Rhodes et al. 2007, with the queen's age, there is a change in the chemical composition of mandibular gland secretion. They reported that 7-day-old virgin queens possess a higher amount of 9-ODA than mated queens. Therefore, the reproductive activeness of the queen is positively correlated with 9-HDA, 10-HDAA, 10HDA, and HVA, whereas 9-ODA acts as a significant signal for drone attraction. With the increase in the queen's age, there is an increase in the relative concentration of 10HDAA and 10-HDA. Like 9-ODA, 9-HDA also elicits retinue behaviour of workers, suppresses workers' ovarian development, and inhibits queen rearing (Slessor et al. 1988; Engels et al. 1997; Pettis et al. 1997; Hoover et al. 2003), whereas HVA concentration is influenced with ovary activation which occurs after mating (Slessor et al. 1990; Engels et al. 1997). It had been observed that HVA reduced dopamine levels in the honey bee brain. Dopamine accelerates ovarian activation. Therefore, somewhere, HVA influences ovarian retard activation in worker honey bees. HOB had been reported to influence mating behaviour (Plettner et al. 1997). Strauss et al. (2008) reported that virgin, drone laying, and mated queens secrete the same concentration of 9-ODA, whereas after mating queen secretes more 9HDA, 10-HDAA, 10-HDA, and HVA than the virgin. Furthermore, the obtained data indicated that high proportionality of 9-ODA in virgin queen, which points out that 9-ODA is involved in drone attraction whereas, in mated queen, high concentration of 9-HDA, 10-HDA, 10-HDAA, and HVA indicated involvement of specific pheromones in reproduction (Strauss et al. 2008). Rangel et al., 2016 experimented on raising a queen from two days and older worker larvae. Further, they carried out chemical analysis of mandibular glands components and measured the retinue response of workers to the queen. They observed that a queen developed from younger worker larvae could attract more workers than a queen set from older worker larvae. Furthermore, they observed that the chemical composition QMPs varies, as a low-quality queen shows a high relative amount of 10-HDA compared to a high-quality queen. They analyzed the mandibular gland pheromone chemical composition of the virgin queen with a 1year old post-mated queen. They concluded that the 10-HDA component is produced in a higher proportion by 6-day-old virgin queens. 10-HDA is present in higher concentrations in workers, whereas a mated queen makes a high concentration of 9-HDA as the significant component of QMP. A lower ratio of 10-HDA to 9-HDA readily differentiates queens from workers (Plettner et al., 1997; Rangel et al., 2016).

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 145

7.3. DIFFERENTIAL MANDIBULAR PHEROMONAL COMPOSITION IN QUEEN AND WORKER HONEY BEE A worker honey bee possesses a high amount of 10-HDA, 10-HDAA, HOB, and a low concentration of 9-ODA, and 9-HDA, whereas the mandibular queen gland has a high amount of 9-ODA, HOB, R, and S 9-HDA, and HVA (Moritz et al., 2000; Moritz et al., 2004). The differential synthesis of the different components occurs due to the activation of various genes (Richard et al., 2007; Le Conte and Hefetz, 2008; Malka et al., 2009; Maka et al., 2014). Further, the presence or absence of a queen affects pheromone secretion in worker honey bees (Crewe and Velthuis, 1980; Hepburn et al., 1996; Zheng et al., 2010; Yusuf et al., 2015). The pseudo queen produces a high concentration of 9-ODA by conversion of 9HDA to 9-ODA. The transformation is facilitated by the enzyme alcohol dehydrogenase (ADH) (Malka et al., 2009; Jarosch et al., 2011; Malka et al., 2014; Wu et al., 2017). ADH's expression is higher in queens than in worker honey bees (Malka et al., 2014; Wu et al., 2017). QMPs regulate the reproductive capacity of worker honey bees, and the absence of QMPs promotes workers to become reproductively active and lay eggs. Workers produce a high concentration of 10-HDA through mandibular glands (Plettner et al., 1996), an essential constituent of royal jelly (Genc et al., 1999). In the proper queen colony, workers secrete more 10-HDA, whereas queen-less workers secrete more 9-ODA (Plettner et al., 1996; Moritz et al., 2000). In the proper queen colony, biosynthetic conversion of 9-HDA into 9-ODA becomes halted in the worker mandibular gland due to the influence of queen pheromones (Malka et al., 2014). 7.4. SYNTHESIS OF QMPS QMPs are composed of 9-oxo-2 (E)-decanoic acid (9-ODA), methyl phydroxybenzoate (HOB), (S, E)-9-hydroxy-2-decanoic acid (9-HDA), 4-hydrox-3-methoxyphenylethanol (HVA), 10-hydroxy-2 (E)-decanoic acid (R, E)--hydroxy-2-decanoic acid (9-HDA), and 10-hydroxydecanoic acid (10-HDAA) (Crewe, and Velthuis, 1980; Winston and Slessor, 1998). QMPs, inhibit workers' ovaries activation, rearing of the new queen, delay behavioural maturity in workers, and influence the physiology of workers (Butler et al., 1959; Butler, 1961; Melathopoulos et al., 1996; Le Conte and Hefetz, 1996; Hoover et al., 2003; Slessor et al., 2005; Rangel et al., 2016). Biosynthesis of mandibular gland components occurs in a specific manner, as stearic acid is biochemically converted into primary mandibular gland pheromones through a bifurcated three-

146 Polyandrous Queen Honey Bee

Lovleen Marwaha

step process (Plettner et al., 1996). The first step involves hydroxylation of stearic acid at ω or ω-1 positions; after that, chain shortening of 18 and 17-hydroxy stearic acid through β-oxidation, and finally, the oxidation of ω and the ω-1 hydroxy group to form diacids and keto acids (Fig. 7c).

Fig. (7c). Biochemical synthesis of various pheromones in the mandibular gland of queen honey bee.

In honey bees, the single genome is responsible for the differential phenotype. That includes a single fertile monopolizing morphological large queen and opportunistically reproducible workers. The phenotypic plasticity is induced due to different diets at the early larval stage, which is responsible for differential development (Rembold et al., 1974; Corona et al., 2016). Differential gene expression is responsible for organ plasticity and caste-specific pheromone synthesis. Queen mandibular gland is responsible for the synthesis of QMP. The biochemical pathway consists of two ω-1-hydroxylated decanoic acids (9-oxo2-decanoic acid (9-ODA) and 9-hydroxy-2-decanoic acid (9-HDA) and two aromatic components, whereas worker honey bees produced ω-hydroxylated decanoic acids, including 10-hydroxy-decanoic acid (10-HDAA) and 10-hydrox-2-decanoic acid (10-HDA)(Barker et al., 1959; Isdorov et al., 2012; Kinoshilla et al., 2012; Li et al., 2013). For the synthesis of pheromones, stearic acid is the primary precursor that first gets hydroxylated at the 17th or 18th position. After that, the product undergoes carbon shortening via β-oxidation, which results in the formation of 10-carbon

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 147

hydroxyl acid. Which then is oxidized in queen components (ω-1-hydroxylated) or the worker components (ω-hydroxylated) (Plettner et al., 1996). The specific pheromonal biosynthesis is entirely plastic and can vary as per environmental conditions. For example, the virgin queen secretes both queen and pheromonal worker components. The type of caste and social and environmental conditions influence the synthesis of queen pheromones. Further, they compared mandibular pheromones of the mated queen, queenright workers, and queenless workers, and differential gene expression was detected in these conditions. About 34 members of the cytochrome P450 family are involved in the biosynthetic process of mandibular gland synthesis(Plettner et al., 1996: Pirk et al., 2011). Queen honey bee pheromones inhibit the formation of 9-ODA by influencing enzyme alcohol dehydrogenase synthesis in worker honey bees (Malka et al., 2014; Wu et al., 2017). In workers, 9-ODA production is high in queen-less colonies than in queen-right colonies (Plettner et al., 1996). Further, lower gene expression responsible for alcohol dehydrogenase in the proper queen colony specifies the mechanism for regulating 9-ODA concentration. Additionally, due to specific genetic restrictions, there is the accumulation of precursor 9-HDA (Malka et al., 2014; Wu et al., 2017). The amount of 9-ODA is high in virgin and mated queens due to the increased expression of the alcohol dehydrogenase enzyme responsible for the conversion of 9-HDA to 9-ODA. The specific event also enhances the life span of the queen honey bee (Slessor et al., 1990). In the queen's right colony, QMPs of the queen prevent hyperexpression of alcohol dehydrogenase enzyme required for conversion of 9HDA to 9ODA. QMP inhibits the biosynthetic pathway, which results in the ω-1 hydroxylation of stearic acid (Fig. 7d) (Mumoki et al., 2018). Mumoki et al., 2018, had analyzed the reproductive dominance of A. m. capensis parasitic workers by recording pheromonal profile and ovarian activation. Further, they reported the quantity of enzyme ADH, which induces conversion of 9-HDA to queen substance 9-ODA. QMP regulate the pheromonal biosynthetic pathways in workers. QMPs inhibit the proper development of worker honey bee ovaries. In fewer queen colonies, workers' ovaries activation occurs to III, IV and V levels, whereas in good queen colonies, workers' ovaries activation occurs only upto III stages. Further, significant factors influencing ovarian activation include pheromone secreted by the queen and brood (Butler., 1959; Mohammed et al., 1998; Hoover et al., 2003). Although the queen secretes various pheromones from various glands to suppress the development of the reproductive system of workers (Wossler and Crewe, 1999; Hoover et al., 2003; Okosun et al., 2017), the major gland that influences the development is the mandibular gland, and it secretes QMP, which exert primary suppressive agent (Strauss, 2008). QMP is composed of 9-ODA, and 9-HDA that act predominantly to inhibit ovarian activation in

148 Polyandrous Queen Honey Bee

Lovleen Marwaha

worker honey bees of Apis mellifera and Apis cerana (Tan et al., 2010).

Fig. (7d). Bio-chemical synthesis of mandibular pheromones in worker honey bees.

Additionally, tergal glands contribute to suppressing reproductive dominance in workers. Components 9-HDA, 9-ODA, and 10-HDA vary significantly with ovarian activation (Hepburn, 1992; Schafer et al., 2006). Honey bee brood pheromones, (E)-beta ocimene, had been detected to suppress ovarian activation in honey bee workers (Traynor et al., 2014). In fewer queen colonies, a high concentration of 9-ODA and 9-HDA is common in workers, whereas in queenright colonies, workers possess a high concentration of 9-HDA and 10-HDA. 10HDA is produced in high quantity in the queen-right colony than Queen less colony. 7.5. EFFECT OF QMP 7.5.1. On Mating and Swarm Gary observed that QMPs induced attraction of drones toward the virgin queen, especially 9-ODA, attracted drones more. Furthermore, 9-HDA, 10-HDA, and HOB attract more drones toward the dummy queen (Brockmann et al. 2006). It has been concluded that 10-HDA is produced more predominantly by virgin Queens than by mated queens. Therefore, it regulates the mating behaviour of the virgin Queen (Plettner et al. 1997). Renner and Vierling 1977 reported that tergal

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 149

gland extract, along with 9-ODA, increases mating behaviour. Therefore in the queen, several glandular sources act synergistically for a more robust mating response. Further, 9-ODA act as a strong sexual attractant for drones from other colonies on nuptial flight and also facilitate worker honey bees to recognize the presence of the queen in the hive (Seeley, 1979; Rutter and Hesse, 1981; Winston et al., 1982; Slessor et al., 1988; Winston et al., 1989; Pettis et al., 1995; Visscher 1996; Rhodes et al., 2007; Strauss et al., 2008; Malka et al., 2009; Nino et al., 2013; Villar et al., 2015; Okosun et al., 2017, Pirk, et al., 2017) Available reports indicate that the queen, after emergence, secretes a considerable amount of QMPs and later on, with further development, there is abundant secretion of more complex mandibular gland chemical bouquets (Slessor et al., 1990; Engels et al., 1997; Plettner et al., 1997; Keeling et al., 2001). Furthermore, few studies indicate that mating status, which includes virgin, single drone insemination, multiple drones inseminated, or naturally mated queens, influences the composition of mandibular secretion (Slessor et al., 1990; Plettner et al., 1997; Keeling et al., 2001; Richard et al., 2007). QMPs induce numerous long-term physiological and short-term behavioural responses in workers, which vary following the genetic composition of the colony. Naumann et al. 1991 reported that retinue workers transferred queen pheromones to other worker honey bees. The distribution of queen pheromones is influenced by the colony's size, which becomes the primary reason for swarming in the strong colony. Reducing queen pheromones in the queen-less colony stimulates worker honey bees to raise a new queen. (E)-9-Oxodec-2-enoic acid (9-ODA) suppresses another queen rearing and ovarian development in worker honey bees. Pettis et al. 1995, reported that the administration of QMP in queenless colonies inhibited the growth of queen cups when admitted within 24 hours after queen loss. QMPs inhibit the production of the new queen, induce suppression in workers' ovaries development and act as a sex attractant for drones during mating, but QMPs concomitantly induce pollen and nectar foraging, delay onset of age for foraging tasks, and lower juvenile hormone titers in worker honey bees (Gary, 1962; Butler and Fairey, 1963; Slessor et al., 1988; Higo et al., 1992; Pankiw et al., 1994; Pettis et al.,1995; Melathopoulos et al., 1996; Pettis et al., 1997; Pankiw et al., 1998; Hoover et al., 2003; Slessor et al., 2005; Brockmann et al., 2006). (R, E)-9-Hydroxy-2-enoic acid (9-HDA), (S, E)-(+)-9-HDA, Methylparaben (HOB), 4-Hydroxy-3-methoxy phenyl ethanol (HVA) induce calming influence by promoting stability influence on the swarm.

150 Polyandrous Queen Honey Bee

Lovleen Marwaha

7.5.2. On Retinue Winston et al. 1989; Kaminsky et al. 1990, observed that QMPs attract workers toward the queen. Therefore, the queen remained surrounded by a group of 8-10 workers known as retinue. Worker honey bees face the queen, feeding her, palpating, and licking her. QMP and its components are responsible for retinue formation (Free 1987). From development to mating, there is an increase in attention paid by workers to the queen honey bee, as the attractiveness of workers to the queen is relatively low at 0-1 day old, moderate from 2-4 days old and highest from 5-18 months old (De Hazan et al. 1989; Richards et al. 2007). Further, queen QMPs are required for keeping the swarm in the cluster through pheromonal signals, including QMP. Winston et al.1989 compared the effect of the queen, mandibular gland and five chemical blends and reported that the queen alone possesses the most substantial impact on the swarm.

Fig. (7e). QMP composition and attraction of worker honey bees.

QMP induces retinue response, a behavioural suite in which workers attend to the queen by surrounding her (Slessor et al., 1988;1990; Kaminski et al., 1990: Pankiw et al., 1994; Pankiw et al., 1995). Functional properties of QMP have

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 151

been considered due to its attractive properties toward worker honey bees, as it controls workers' retinual behaviour and maintains swarm clusters (Fig. 7e) (Winston et al. 1989; Kaminsky et al. 1990). Retinue or court means a circle of worker honey bees which surround queen honey, toward her, feed, palpate and lick her in a stationary comb. Several reports indicate that QMPs are responsible for retinue formation (Free 1987). Furthermore, workers' attraction toward the queen is correlated with modifying the QMP pattern. Retinue workers pay more attention to the mated queen, which lays eggs and decreases attention as the queen grows old. In retinue behaviour, younger worker honey bees incidentally surround the queen as long as she remains at a particle segment of the comb. After the queen's exit from a specific section, they execute their regular work as per their roles in the colony (Naumann 1991;1992). The particular retinual group receive and pass the received queen pheromones to the remaining nest mate through antennal contact, which results in the distribution of Queen substance to the remaining nest mate. Therefore all workers receive QMPs without direct contact with the Queen (Slessor et al. 2005). Removal of Queen or absence of pheromonal signals for 1224 hours elicited a behavioural change in worker honey bees. They start making new Queens (Winston et al. 1990; 1991; Naumann et al., 1993; Pettis et al. 1995). Pankiw et al., 1998, had studied the influence of QMP on worker’s foraging and JH titers. QMPs inhibit the biosynthesis of JH biosynthesis, which further influence worker foraging ontogeny. QMPs delay the age at which foraging occurs in worker honey bees by reducing JH titers in worker honey bees. QMP treatment decreases colony foraging activity. Huang and Robinson (1996) reported that QMPs have an inhibitory effect on worker honey bees in addition to the inhibition imposed by remaining older worker bees. Further, even Pankiw et al.,1998 reported that QMP influences the foraging behaviour of worker honey bees by lowering JH titers. Huang et al., 1994, 1991 said that JH secretion increase from middle age to foraging age. Buhler et al. (1983) reported that JH, in the fall, can occur due to a temperature change; further, during overwintering, there is a reduction of foragers' JH secretion titers. QMP delay the foraging ontogeny in worker honey bees, which is associated with lower JH titers (Pankiw et al., 1998). Mating is crucial for developing efficient chemical signals by the queen to attract workers. In the mandibular gland of the queen and worker honey bees, castespecific pheromonal secretion can be observed (Plettner et al. 1996). Maisonnasse et al. 2010 demonstrated that queen honey bees without mandibular glands can still attract worker honey bees, which specifies that other substances in addition to

152 Polyandrous Queen Honey Bee

Lovleen Marwaha

QMP attract worker honey bees. Further, they reported that even in demandibulated, the queen could exert some regulatory functions on workers, including inhibition of queen cup construction, retinue behaviour, and suppression of worker ovary development. In the demandibulated queen, the pheromone (QP) level was similar to that of control except for 9-ODA, as the mandibular gland only secretes the specific pheromone. In contrast, the queen's body produces HOB and 9-HDA. Mainly 9-ODA is responsible for retinual behaviour. Other reports also indicate that queen mandibular gland components regulate physiological and behavioural responses in workers (Slessor et al., 1988; Winston et al., 1989; Pettis et al.,1995; Melathopoulos et al., 1996; Pankiw et al., 1998; Hoover et al., 2003; Katzav-Gozansky et al.,2004: Katzav-Gozansky, et al., 2006; Katzav-Gozansky et al., 2007; Richard et al., 2007; Strauss et al., 2008; Nino et al., 2012; Villar et al., 2015). Richard et al., 2007 reported that a queen inseminated with a single drone or multiple drones shows different retinue sizes. Furthermore, Kocher et al., 2009 said that caged bees become more attracted to the mandibular gland extract of a naturally mated queen than instrumentally inseminated queen or virgin queens. In contrast, Nino et al., 2012 observed that retinue was more considerable in queen inseminated with high versus low volume of semen. An increase in the size of retinue can occur due to other factors, including cuticle lipids (Babis et al., 2014), tergal gland secretion (Wossler and Crewe, 1999), Dufour's gland secretion (Katzaf-Gozansky et al., 2001). Some studies indicate that queen retinue response is seen even in queens whose mandibular glands had been removed (Katzaf-Gozansky et al., 2001; Maisonnasse et al., 2010). Low queen attractiveness toward queen can be one decision of worker honey bees to keep or to replace queen by queen supersedure. Queen reproductive quality is a complex function of queen age, initial rearing age, mating state, insemination volume and other associated factors (Rangel et al., 2016). It had been reported by Rangel et al., 2016 that queens from first instar worker larvae induce larger worker retinues in comparison to the queen from third instar worker larvae. Rangel et al., 2016 reported that a queen raised from first-instar larvae elicited an enormous worker retinue response than a queen raised from the third instar. Further, QMPs regulate the physiological and behavioural responses in worker honey bees (Butler and Fairey, 1963; Slessor et al., 1988; Winston et al., 1989; Pettis et al., 1995; Melathopoulos et al., 1996; Pankiw et al., 1998; Hoover et al., 2003; Katzav et al., 2004; Katzav et al., 2006; Richard et al., 2007; Strauss et al., 2008; Nino et al., 2012; Villar et al., 2015). The retinue size is more significant in queens, which is inseminated by one drone versus several drones (Richard et al., 2007). Further, semen quality influence the retinue size (Nino et al., 2012).

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 153

Additionally, workers' attraction is influenced by cuticular lipid content, tergal gland secretion, and Dufour secretion (Rangel et al., 2016). On Wax Secretion: QMPs of mated Queen activate worker honey bees to produce more wax compared to workers headed by virgin queen honey bees or workers of the queen-less colony. The presence of a mated Queen or QMP inhibits the production of a male brood (Ledoux et al. 2001). Furthermore, components of QMPs, HVA and HOB seem to affect wax production in the colony (Ledoux et al. 2001). QMPs increase the number of foragers honey bees and the weight of pollen load on worker honey bees. Further, QMPs decrease stinging response and defensive behaviour in worker honey bees (Vergoz et al. 2007). QMPs also regulate the construction of hexagonal wax cell numbers and dimensions for the specific formation of worker cells, drone cells, honey cells, pollen cells, worker behaviour maturation, resistance to starvation, olfactory learning and memory (Winston et al., 1989; Winston et al., 1991; Panik et al., 1998; Ledoux et al., 2001; Fischer and Grozinger, 2008). In addition to the mandibular gland, other glands, including tergal, tarsal and Dufour's gland, stimulate retinal behaviour and suppress the worker's ovarian activation (Neurmann et al., 1991; Wossler et al., 1999; Katzav et al., 2001; Slessor and Winston, 2005; Le Conte and Hefetz, 2008). A de-mandibulated virgin queen is equally efficient in controlling colony functions (Maisonnasse et al., 2010). On Ovarian Development: Furthermore, Mohammedi et al. 1998, reported that queen and brood pheromones, especially ethyl palmitate and methyl linoleate, suppress ovarian development of worker honey bees. During the development of worker honey bees, the low titer of juvenile hormone affects ovarian development. Hoover et al. (2003) demonstrated that synthetic QMP could suppress the growth of ovarian development in worker honey bees. Katzav-Gozansky et al. (2006) observed that QMP, along with Dufour's gland, inhibit ovarian development in worker honey bees but had been proved less effective than the live queen. Sex Attractant: QMPs act as an attractant for drones as sex pheromones (Gary 1962; Free1987). 9-ODA elicit attractant efficacy at a more considerable distance, attracting drones from a greater distance (Free 1987; Winston and Slessor 1992; Brockmann et al. 2006). 9-hydroxy-2decenoic acid (9-HDA) and 10-hydroxy-2-decanoic acid (10-HDA) increase male attractiveness at close range. Other components of QMP act synergistically with 9-ODA to enhance male attraction (Brockmann et al. 2006). QMPs regulate the behaviour and physiology of worker honey bees (Slessor et al. 2005). After mating, there is a

154 Polyandrous Queen Honey Bee

Lovleen Marwaha

change in the composition of QMP, which possess a significant influence on worker honey bee behaviour and development (Slessor et al. 2005). Further, the influence of QMP on worker honey bee behaviour, physiology and development has been well documented (Free 1987; Winston and Slessor 1992; Slessor et al. 2005; Alaux et al. 2010). Change in composition of queen pheromone induces new queen formation before reproductive swarming. Even worker honey bees can synthesize all the concerning pheromones, but due to the influence of QMP, there is a change in biosynthetic pathways (Malka et al. 2009; Plettner et al. 1996). 10-HDA is produced in high concentration by the virgin queen, which is involved in mating (Slessor et al., 1988; Brockmann et al., 2006; Jarriault, Alison Mercer, 2012). 7.6. WORKERS' MANDIBULAR GLAND PHEROMONES The mandibular gland secretes pheromones, which act as alarm pheromones and mainly consist of 2-heptanone, a highly volatile substance. The specific gland possesses repellent effects, which potential enemies detect. The amount of 2heptanone secretion increases with the age of worker honey bees, and foragers use a particular chemical, i.e. 2-heptanone, to mark recently visited flowers and depleted foraging locations. Furthermore, it has been detected that 2-heptanone is used to paralyze intruders. The mandibular gland, along with other glands, helps to mark no longer abundant flowers, increasing the efficiency of foraging. Worker honey bees' mandibular gland secretion acts as footprint pheromone, used to mark nest entrance, and as a thermoregulatory pheromone, which induces muscle contraction in worker honey bees. Pankiw, in 2004, reported that substances extracted from the surface of foraging honey bee increase foraging tendency of bees. Worker honey bees produce 2-heptanone (2-H) through their mandibular gland, which induces alarming and stinging behaviour, similar to iso-pentyl acetate by the Koschewnikow gland. In addition, 2-H pheromone possesses repellent properties which also affect the foraging behaviour of honey bees. A maximum concentration of 2-H had been detected in guards and in foragers. Additionally, workers communicate through pheromonal and antennal contact (Fig. 7f - h).

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 155

Fig. (7f). Section of bee hive depicting drone cell and workers engaged in different duties.

Fig. (7g). Antennal communication between worker honey bees.

156 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (7h). Antennal communication between worker honey bee and forager carrying pollen filled in the pollen basket.

7.7. GENE ASSOCIATED WITH MANDIBULAR GLAND For the synthesis of QMPs, x-1-hydroxylation occurs in queen honey bees, whereas in worker honey bees, x-hydroxylation occurs. Malka et al., 2009, identified that two specific proteins, CYP4AA1 and CYP18A1, are essential for mandibular pheromones synthesis. They deeply analyzed the particular gene for queen and worker honey bees. It has been detected that CYP4AA1 (xhydroxylation) expression occurs high in workers than in the queen, whereas CYP18A1 (x-1-hydroxylation) is more hyper-expressed in the queen than in workers. According to Wu et al., 2017, in queen honey bees, about 1601 genes are expressed in mandibular glands, whereas 1028 genes are expressed in worker honey bees' mandibular glands. After the removal of the queen, 2382 genes were down-regulated, whereas 379 genes had been upregulated in worker honey bees' mandibular glands. For fatty acid hydroxylation, stearic acid is catalyzed by the CYP45025 product. After hydroxylation, fatty acid carbon chair shortening occurs by β-oxidation, forming decanoic and decanoic acids with the help of mitochondria and peroxisome (Wu et al.,2017). Moreover, they compared the mandibular gland secretion from the queen, queen-right, and queen-less workers using RNA sequence technology. It has been detected that about 46 genes are responsible for caste-specific biosynthesis of mandibular gland pheromones through β-oxidation and ω-oxidation. Further, they analyzed CYP6AS8 and CYP6AS11, the most hyper expressed gene in workers, respectively.

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 157

CONCLUSION Certain specific genes are responsible for the bifurcated biosynthesis of mandibular gland pheromones. Further, differential synthesis process is influenced by gene expression of CYP4AA1,CYP18A1,CYP45025,CYP6AS8,CYP6AS11, CYP4G11, CYP9S1, CYP9R1, CYP6AS5, CYP6AQ1, CYP9Q2, CYP9Q1, CYP9Q3, CYP305D1, CYP6AS12, CYP6AS8, LOC408291, CYP4AZ1, CYP315A1, CYP6BD1, CYP4AV1, CYP15A1, CYP6AS1, CYP6AS15, CYP6AS11, CYP6AS3, CYP6BE1, CYP6AS17, CYP302A1, CYP6AS13, CYP6BC1, CYP314A1, CPTI, LOC409712, LOC412025, LOC410325, LOC408291, lOC412020, lOC552757, LOC411202,LOC411140,Aldh, Aldh-III, in queen and worker honey bees. REFERENCES Apðegaitë, V & Skirkevièius, A (1999) Content of (E)-9-oxo-2-decanoic acid in pheromones of honeybee (Apis mellifera L.) queens. Pheromones, 6, 27-32. Barker, SA, Foster, AB, Lamb, DC & Jackman, LM (1959) Biological origin and configuration of 10hydroxy-2-decenoic acid. Nature, 184 (Suppl. 9), 634-4. [http://dx.doi.org/10.1038/184634a0] [PMID: 13796816] Brockmann, A, Dietz, D, Spaethe, J & Tautz, J (2006) Beyond 9-ODA: sex pheromone communication in the European honey bee Apis mellifera L. J Chem Ecol, 32, 657-67. [http://dx.doi.org/10.1007/s10886-005-9027-2] [PMID: 16586035] Butler, CG (1959) The source of the substance is produced by a queen honeybee (Apis mellifera L.) which inhibits the development of the ovaries of the workers of her colony. Proc R Entomol Soc Lond, Ser A Gen Entomol, 34, 137-8. [http://dx.doi.org/10.1111/j.1365-3032.1959.tb00249.x] Butler, CG (1961) The scent of queen honeybees (A. mellifera L.) that causes partial inhibition of queen rearing. J Insect Physiol, 7, 258-64. [http://dx.doi.org/10.1016/0022-1910(61)90076-2] Butler, CG & Fairey, EM (1963) The role of the queen in preventing oogenesis in worker honeybees. J Apic Res, 2, 14-8. [http://dx.doi.org/10.1080/00218839.1963.11100051] Crewe, RM & Velthuis, HHW (1980) False queens: A consequence of mandibular gland signals in worker honeybees. Naturwissenschaften, 67, 467-9. [http://dx.doi.org/10.1007/BF00405650] Conte, YL & Hefetz, A (2008) Primer pheromones in social hymenoptera. Annu Rev Entomol, 53, 523-42. [http://dx.doi.org/10.1146/annurev.ento.52.110405.091434] [PMID: 17877458] Corona, M, Libbrecht, R & Wheeler, DE (2016) Molecular mechanisms of phenotypic plasticity in social insects. Curr Opin Insect Sci, 13, 55-60. [http://dx.doi.org/10.1016/j.cois.2015.12.003] [PMID: 27436553] De Hazan, M, Lensky, Y & Cassier, P (1989) Effects of queen honeybee (Apis mellifera L.) ageing on her attractiveness to workers. Comp Biochem Physiol A Comp Physiol, 93, 777-83. [http://dx.doi.org/10.1016/0300-9629(89)90501-X] Engels, W, Rosenkranz, P, Adler, A, Taghizadeh, T, Lübke, G & Francke, W (1997) Mandibular gland volatiles and their ontogenetic patterns in queen honey bees, Apis mellifera carnica. J Insect Physiol, 43, 30713.

158 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1016/S0022-1910(96)00110-2] [PMID: 12769892] Free, JB (1987) Pheromones of Social BeesChapman and Hall, London. Gary, NE (1962) Chemicalmatingattractantsinthequeenhoneybee. Science, 136, 773-4. [http://dx.doi.org/10.1126/science.136.3518.773] [PMID: 17752107] Hepburn, HR (1992) Pheromonal and ovarial development covary in cape worker honeybees,apis meffera capensis. Naturwissenschaften, 79, 523-4. [http://dx.doi.org/10.1007/BF01135775] Hepburn, HR & Crewe, RM (1991) Portrait of the Cape honeybee, Apis mellifera capensis. Apidologie (Celle), 22, 567-80. [http://dx.doi.org/10.1051/apido:19910601] Hepburn, HR & Radloff, SE (1996) Morphometric and pheromonal analyses of Apis mellifera L along a transect from the Sahara to the Pyrenees. Apidologie (Celle), 27, 35-45. [http://dx.doi.org/10.1051/apido:19960105] Higo, HA, Colley, SJ, Winston, ML & Slessor, KN (1992) Effects of honey bee (Apis mellifera L.) queen mandibular gland pheromone on foraging and brood rearing. Can Entomol, 124, 409-18. [http://dx.doi.org/10.4039/Ent124409-2] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Huang, ZY, Robinson, GE, I & Borst, DW (1994) Physiological correlates of division of labor among similarly aged honey bees. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 174, 731-9. [http://dx.doi.org/10.1007/BF00192722] [PMID: 8014920] Isidorov, VA, Bakier, S & Grzech, I (2012) Gas chromatographic–mass spectrometric investigation of volatile and extractable compounds of crude royal jelly. J Chromatogr B Analyt Technol Biomed Life Sci, 885-886, 109-16. [http://dx.doi.org/10.1016/j.jchromb.2011.12.025] [PMID: 22245368] Jarosch, A, Stolle, E, Crewe, RM & Moritz, RFA (2011) Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers ( Apis mellifera ). Proc Natl Acad Sci USA, 108, 15282-7. [http://dx.doi.org/10.1073/pnas.1109343108] [PMID: 21896748] Kaminski, LA, Slessor, KN, Winston, ML, Hay, NW & Borden, JH (1990) Honeybee response to queen mandibular pheromone in laboratory bioassays. J Chem Ecol, 16, 841-50. [http://dx.doi.org/10.1007/BF01016494] [PMID: 24263599] Kaatz, HH, Hildebrandt, H & Engels, W (1992) Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees. J Comp Physiol B, 162, 588-92. [http://dx.doi.org/10.1007/BF00296638] Katzav-Gozansky, T, Boulay, R, Soroker, V & Hefetz, A (2004) Queen–signal modulation of worker pheromonal composition in honeybees. Proc Biol Sci, 271, 2065-9. [http://dx.doi.org/10.1098/rspb.2004.2839] [PMID: 15451697] Katzav-Gozansky, T, Boulay, R, Soroker, V & Hefetz, A (2006) Queen pheromones were affecting the production of queen-like secretion in workers. A Neuroethology, sensory, neural, and behavioural physiology Katzav-Gozansky, T, Hefetz, A & Soroker, V (2007) Brain modulation of Dufour’s gland ester biosynthesis in vitro in the honeybee (Apis mellifera). Naturwissenschaften, 94, 407-11. [http://dx.doi.org/10.1007/s00114-006-0206-y] [PMID: 17235597] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987]

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 159

Kinoshita, G & Shuel, RW (1975) Mode of action of royal jelly in honeybee development. X. Some aspects of lipid nutrition. Can J Zool, 53, 311-9. [http://dx.doi.org/10.1139/z75-040] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2008) Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera). BMC Genomics, 9, 232. [http://dx.doi.org/10.1186/1471-2164-9-232] [PMID: 18489784] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2009) Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees. Behav Ecol, 20, 1007-14. [http://dx.doi.org/10.1093/beheco/arp090] [PMID: 22476212] Li, X, Huang, C & Xue, Y (2013) Contribution of lipids in honeybee (Apis mellifera) royal jelly to health. J Med Food, 16, 96-102. [http://dx.doi.org/10.1089/jmf.2012.2425] [PMID: 23351082] Maisonnasse, A, Alaux, C, Beslay, D, Crauser, D, Gines, C, Plettner, E & Le Conte, Y (2010) New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation? Front Zool, 7, 18. [http://dx.doi.org/10.1186/1742-9994-7-18] [PMID: 20565874] Malka, O, Karunker, I, Yeheskel, A, Morin, S & Hefetz, A (2009) The gene road to royalty - differential expression of hydroxylating genes in the mandibular glands of the honeybee. FEBS J, 276, 5481-90. [http://dx.doi.org/10.1111/j.1742-4658.2009.07232.x] [PMID: 19702829] Malka, O, Niño, EL, Grozinger, CM & Hefetz, A (2014) Genomic analysis of the interactions between social environment and social communication systems in honey bees (Apis mellifera). Insect Biochem Mol Biol, 47, 36-45. [http://dx.doi.org/10.1016/j.ibmb.2014.01.001] [PMID: 24486775] Melathopoulos, AP, Winston, ML, Pettis, JS & Pankiw, T (1996) Effect of Queen mandibular pheromone on initiation and maintenance of queen cells in the honey bee (Apis mellifera L.). Can Entomol, 128, 263-72. [http://dx.doi.org/10.4039/Ent128263-2] Mohammedi, A, Paris, A, Crauser, D & Le Conte, Y (1998) Effect of aliphatic esters on ovary development of queenless bees (Apis mellifera L.). Naturwissenschaften, 85, 455-8. [http://dx.doi.org/10.1007/s001140050531] Moritz, RFA, Simon, UE & Crewe, RM (2000) Pheromonal contest between honey bee workers. Naturwissenschaften, 87, 395-7. [http://dx.doi.org/10.1007/s001140050748] [PMID: 11091962] Moritz, RF, Lattorff, HMG & Crewe, RM (2004) Honeybee workers (Apis mellifera capensis) compete for producing queen-like pheromone signals. Proc Biol Sci, 271 (Suppl. 3), S98-S100. [PMID: 15101431] Mumoki, FN, Pirk, CWW, Yusuf, AA & Crewe, RM (2018) Reproductive parasitism by worker honey bees suppressed by queens through regulation of worker mandibular secretions. Sci Rep, 8, 7701. [http://dx.doi.org/10.1038/s41598-018-26060-w] [PMID: 29799016] Naumann, K, Winston, ML, Slessor, KN, Prestwich, GD & Webster, FX (1991) Production and transmission of honey bee queen (Apis mellifera L.) mandibular gland pheromone. Behav Ecol Sociobiol, 29, 321-32. [http://dx.doi.org/10.1007/BF00165956] Niño, EL, Malka, O, Hefetz, A, Teal, P, Hayes, J & Grozinger, CM (2012) Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology. J Insect Physiol, 58, 1082-9. [http://dx.doi.org/10.1016/j.jinsphys.2012.04.015] [PMID: 22579504] Niño, EL, Malka, O, Hefetz, A, Tarpy, DR & Grozinger, CM (2013) Chemical profiles of two pheromone glands are differentially regulated by distinct mating factors in honey bee queens (Apis mellifera L.). PLoS One, 8, e78637.

160 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1371/journal.pone.0078637] [PMID: 24236028] Okosun, OO, Pirk, CWW, Crewe, RM & Yusuf, AA (2017) Glandular sources of pheromones used to control host workers (Apis mellifera scutellata) by socially parasitic workers of Apis mellifera capensis. J Insect Physiol, 102, 42-9. [http://dx.doi.org/10.1016/j.jinsphys.2017.09.001] [PMID: 28889990] Okosun, OO, Yusuf, AA, Crewe, RM & Pirk, CWW (2015) Effects of age and reproductive status on tergal gland secretions in queenless honey bee workers, Apis mellifera scutellata and A. m. capensis. J Chem Ecol, 41, 896-903. [http://dx.doi.org/10.1007/s10886-015-0630-6] [PMID: 26384295] Pankiw, T, Huang, Z, Winston, ML & Robinson, GE (1998) Queen mandibular gland pheromone influences worker honey bee (Apis mellifera L.) foraging ontogeny and juvenile hormone titers. J Insect Physiol, 44, 685-92. [http://dx.doi.org/10.1016/S0022-1910(98)00040-7] [PMID: 12769952] Pankiw, T, Winston, ML & Slessor, KN (1994) Variation in worker response to honey bee (Apis mellifera L.) queen mandibular pheromone (Hymenoptera: Apidae). J Insect Behav, 7, 1-15. [http://dx.doi.org/10.1007/BF01989823] Pankiw, T, Winston, ML & Slessor, KN (1995) Queen attendance behavior of worker honey bees (Apis mellifera L.) that are high and low responding to queen mandibular pheromone. Insectes Soc, 42, 371-8. [http://dx.doi.org/10.1007/BF01242165] Pettis, JS, Higo, HA, Pankiw, T & Winston, ML (1997) Queen rearing suppression in the honey bee evidence for a fecundity signal. Insectes Soc, 44, 311-22. [http://dx.doi.org/10.1007/s000400050053] Pettis, JS, Winston, ML & Slessor, KN (1995) The behaviour of queen and worker honey bees (Hymenoptera: Apidae) in response to exogenous queen mandibular gland pheromone. Ann Entomol Soc Am, 88, 580-8. [http://dx.doi.org/10.1093/aesa/88.4.580] Pettis, JS, Winston, ML & Collins, AM (1995) Suppression of queen rearing in European and Africanized honey bees Apis mellifera L. by synthetic queen mandibular gland pheromone. Insectes Soc, 42, 113-21. [http://dx.doi.org/10.1007/BF01242447] Pirk, CWW, Sole, CL & Crewe, RM (2011) PheromonesIn:RadloffSE(ed.)HepburnR,pp207-214. HoneybeesofAsia. Springer, Heidelberg,Dordrecht Pirk, CW, Sole, CL & Crewe, R (2011) Honeybees of Asia in Pheromones, Springer 207-14. [http://dx.doi.org/10.1007/978-3-642-16422-4_9] Pirk, CWW, Crewe, RM & Moritz, RFA (2017) Risks and benefits of the biological interface between managed and wild bee pollinators. Funct Ecol, 31, 47-55. [http://dx.doi.org/10.1111/1365-2435.12768] Plettner, E, Otis, GW, Wimalaratne, PDC, Winston, ML, Slessor, KN, Pankiw, T & Punchihewa, PWK (1997) Species-and caste-determined mandibular gland signals in honeybees (Apis). J Chem Ecol, 23, 36377. [http://dx.doi.org/10.1023/B:JOEC.0000006365.20996.a2] Plettner, E, Slessor, KN, Winston, ML & Oliver, JE (1996) Caste-selective pheromone biosynthesis in honeybees. Science, 271, 1851-3. [http://dx.doi.org/10.1126/science.271.5257.1851] Rangel, J, Böröczky, K, Schal, C & Tarpy, DR (2016) Honey bee (Apis mellifera) queen reproductive potential affects queen mandibular gland pheromone composition and worker retinue response. PLoS One, 11, e0156027. [http://dx.doi.org/10.1371/journal.pone.0156027] [PMID: 27281328] Rembold, H, Lackner, B & Geistbeck, I (1974) The chemical basis of honeybee, Apis mellifera, caste

Mandibular Pheromone Types

Polyandrous Queen Honey Bee 161

formation. Partial purification of queen bee determinator from royal jelly. J Insect Physiol, 20, 307-14. [http://dx.doi.org/10.1016/0022-1910(74)90063-8] Renner, M & Vierling, G (1977) Die rolle des taschendrüsenpheromons beim Hochzeitsflug der Bienenkönigin. Behav Ecol Sociobiol, 2, 329-38. [http://dx.doi.org/10.1007/BF00299744] Rhodes, JW, Lacey, MJ & Harden, S (2007) Changes with age in queen honey bee (Apis mellifera) head chemical constituents (Hymenoptera: Apidae). Sociobiology, 50, 11-22. Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2, e980. [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Ruttner, F & Hesse, B (1981) Rassenspezifische unterschiede in ovarentwicklung und eiablage von weisellosen arbeiterinnen der honigbiene Apis mellifera L. Apidologie (Celle), 12, 159-83. [http://dx.doi.org/10.1051/apido:19810206] Schäfer, MO, Dietemann, V, Pirk, CWW, Neumann, P, Crewe, RM, Hepburn, HR, Tautz, J & Crailsheim, K (2006) Individual versus social pathway to honeybee worker reproduction (Apis mellifera): pollen or jelly as protein source for oogenesis? J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 192, 761-8. [http://dx.doi.org/10.1007/s00359-006-0112-y] [PMID: 16508763] Seely, TD (1979) Queen substance dispersal by messenger workers in honeybee colonies. Behav Ecol Sociobiol, 5, 391-415. [http://dx.doi.org/10.1007/BF00292527] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Kaminski, LA, King, GGS & Winston, ML (1990) Semiochemicals of the honeybee queen mandibular glands. J Chem Ecol, 16, 851-60. [http://dx.doi.org/10.1007/BF01016495] [PMID: 24263600] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Strauss, K, Scharpenberg, H, Crewe, RM, Glahn, F, Foth, H & Moritz, RFA (2008) The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent? Behav Ecol Sociobiol, 62, 1523-31. [http://dx.doi.org/10.1007/s00265-008-0581-9] Tan, K, Yang, M, Wang, Z, Radloff, SE & Pirk, CWW (2012) The pheromones of laying workers in two honeybee sister species: Apis cerana and Apis mellifera. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 198, 319-23. [http://dx.doi.org/10.1007/s00359-012-0710-9] [PMID: 22252612] Traynor, KS, Le Conte, Y & Page, RE, Jr (2014) Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers. Behav Ecol Sociobiol, 68, 2059-73. [http://dx.doi.org/10.1007/s00265-014-1811-y] [PMID: 25395721] Villar, G, Baker, TC, Patch, HM & Grozinger, CM (2015) Neurophysiological mechanisms underlying sexand maturation-related variation in pheromone responses in honey bees (Apis mellifera). J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 201, 731-9. [http://dx.doi.org/10.1007/s00359-015-1006-7] [PMID: 25840687] Visscher, PK (1996) Reproductive conflict in honey bees: a stalemate of worker egg-laying and policing. Behav Ecol Sociobiol, 39, 237-44. [http://dx.doi.org/10.1007/s002650050286] Winston, ML (1987) ThebiologyofthehoneybeeHarvardUniversityPress, Cambridge.

162 Polyandrous Queen Honey Bee

Lovleen Marwaha

Winston, ML & Slessor, KN (1998) Honey bee primer pheromones and colony organization: gaps in our knowledge. Apidologie (Celle), 29, 81-95. [http://dx.doi.org/10.1051/apido:19980105] Winston, ML, Slessor, KN, Smirle, MJ & Kandil, AA (1982) The influence of a queen-produced substance, 9HDA, on swarm clustering behavior in the honeybeeApis mellifera L. J Chem Ecol, 8, 1283-8. [http://dx.doi.org/10.1007/BF00987761] [PMID: 24414734] Winston, ML, Slessor, KN, Willis, LG, Naumann, K, Higo, HA, Wyborn, MH & Kaminski, LA (1989) The influence of queen mandibular pheromones on worker attraction to swarm clusters and inhibition of queen rearing in the honey bee (Apis mellifera L.). Insectes Soc, 36, 15-27. [http://dx.doi.org/10.1007/BF02225877] Wossler, TC & Crewe, RM (1999) Honeybee queen tergal gland secretion affects ovarian development in caged workers. Apidologie (Celle), 30, 311-20. [http://dx.doi.org/10.1051/apido:19990407] Wu, Y, Zheng, H, Corona, M, Pirk, C, Meng, F, Zheng, Y & Hu, F (2017) Comparative transcriptome analysis on the synthesis pathway of honey bee (Apis mellifera) mandibular gland secretions. Sci Rep, 7, 4530. [http://dx.doi.org/10.1038/s41598-017-04879-z] [PMID: 28674395] Yusuf, AA, Pirk, CWW & Crewe, RM (2015) Mandibular gland pheromone contents in workers and queens of Apis mellifera adansonii. Apidologie (Celle), 46, 559-72. [http://dx.doi.org/10.1007/s13592-014-0346-6] Zheng, HQ, Dietemann, V, Crewe, RM, Hepburn, R, Hu, FL, Yang, MX & Pirk, CWW (2010) Pheromonal predisposition to social parasitism in the honeybee Apis mellifera capensis. Behav Ecol, 21, 1221-6. [http://dx.doi.org/10.1093/beheco/arq131]

Polyandrous Queen Honey Bee, 2022, 163-174

163

CHAPTER 8

Retinue Behaviour of Worker Honey Bees Abstract: Different queen pheromones attract colonial workers who respond by forming a surrounding group around the stationary queen. This specific behaviour is considered retinue behaviour. Workers lick, groom, and antennate the queen to get pheromones which influence workers' behaviour, physiology, development, hormones, reproduction, etc. Various pheromonal glands like the Mandibular gland components, the Tergal gland, Dufour's gland, etc., influence the retinue. Primary pheromones which influence the retinue process include (E)-9-oxo-2-decanoic acid(9-ODA), methyl 4-hydroxybenzoate(HOB), (R)-and (S)-(E)-9-hydroxy-2-decanoic acid(9-HDA), 4hydroxy3-methoxyphenylethanol (HVA), 10-hydroxy-decanoic acid (10-HDAA) and10-hydroxy-2 (E)-decanoic acid (10-HDA), methyl oleate, coniferyl alcohol, palmityl alcohol, and linolenic acid. Furthermore, queen ester includes palmitates, oleates, ethyl stearate, ethyl, and methyl palmitoleate. Additionally, specific volatiles influence swarming, drone attraction, and general organization of the colony. This chapter comprehensively describes the retinue behaviour of workers, responsible elements, and the significance of retinue.

Keywords: Division of Labour, Pheromonal secretion, Reproduction, Queen Honey Bee. 8.1. INTRODUCTION The reproductive division of labour in eusocial insects is extreme, with the queen potentially fertile, workers facultatively fertile, and reproductively active drones (Wilson, 1971). Workers perform various tasks, including food collection, water collection, ventilation, temperature maintenance, wax cell construction, deference, and brood rearing (Wilson, 1971; Seeley, 1995; Crozier and Pamilo, 1996). Single queen monopolizes, with a multiple mating tendency which is usually mating with 10-28 drones, and she can lay about 1500 egg daily (Snodgrass, 1956; Tarpy et al., 2002; Tarpy et al., 2004; Kraus et al., 2005). Queen developmental path is entirely plastic, with major transformation during 3-5 instar, eventually forming either queen-like or worker-like phenotype. Specific developmental plasticity ultimately results in diversification of morphology, anatomy, physiology, reproduction, role in the colony, immunity, and life span(Kraus et al., 2005; Gilley et al., 2006). Due to pheromonal variation, the Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

164 Polyandrous Queen Honey Bee

Lovleen Marwaha

queen can attract workers toward her, which is known as retinue behaviour. Workers' attractiveness toward the queen can be modulated predominantly by mandibular gland components. Queen pheromones regulate task organization and productivity of the colony (Winston et al., 1989; Kaminsky et al., 1990; Slessor et al., 2005; Conte and Hefetz, 2008; Kocher et al., 2011). In the hive, the stationary queen remains surrounded by a group of workers called to court or retinue. The specific retinue comprises 8-12 workers with the central queen. Retinue behaviour includes court or attending behaviour (Ribbands, 1953; A Butler, 1954; Allen, 1955, 1960, Sakagami, 1958; Velthuis, 1972). Various research explorations witnessed that Queen Mandibular Pheromones (QMP) are responsible for retinue formation. By changing QMP profiling, the retinue pattern can be changed (Free, 1987; Conte and Hefetz, 2008; Kocher et al., 2011). In addition, workers pay more attention to the mated queen than the virgin queen, which increases with egg laying but decreases as the queen ages.

Fig (8a). Due to pheromonal profiling of the queen, she remains surrounded by a group of workers, and the concerning behaviour is known as retinue.

According to De Hazan et al. 1989, the attentiveness of workers to the queen is null to 0-1 day old queen, medium to 2 to the 4-day old queen, and reaches up to maximum in between 5 days to 18 months old. Slessor et al. (2005) reported the interaction between queens and workers. They concluded that queen retinue pheromones are responsible for the attraction of workers. Queen pheromones excite workers to lick and to antennate her.

Retinue Behaviour

Polyandrous Queen Honey Bee 165

Retinue behaviour ensures properly organized functionality of colonies. The queen secretes specific pheromones, which are passed from the queen to worker honey bees, according to Naumann et al., 1991. Similarly, wax comb also facilitates the transmission of pheromones to other workers, concomitantly with retinue bees. Retinal behaviour in workers is induced by secretion of the mandibular gland and Dufour's gland (Slessor et al., 1988; Kaminski et al.,1990; Pankiw et al., 1995; Katzav-Gozansky et al., 2003). Yang et al., 2010, reported the effect of mandibular gland secretion of species on the retinue behaviour of other species by considering two species, A. cerana and A. mellifera. They concluded that proportional values of mandibular gland pheromone differ in two species, which include 9ODA, 9-HDA, and 10-HDA. Apis cerana queen possesses significantly more components in QM than Apis mellifera, and Apis cerana responds less to the mandibular pheromones of Apis mellifera (Yang et al., 2010). 8.2. PHEROMONES FOR ATTRACTION OF NEST MATES Pheromones secreted through various glands induce retinue in workers and attract drones of other colonies (8a). Therefore, mandibular gland secretion is crucial for generating retinue behaviour in workers. QMP is composed of a blend of (E)-9-oxo-2-decanoic acid(9-ODA), methyl 4hydroxybenzoate(HOB), (R)-and (S)-(E)-9-hydroxy-2-decanoic acid(9-HDA), 4hydroxy3-methoxyphenylethanol (HVA), 10-hydroxy-decanoic acid (10-HDAA) and10-hydroxy-2 (E)-decanoic acid (10-HDA) (Slessor et al., 1988; Apðegaitë et al., 1999; Pirk et al.,2011; Yusuf et al., 2015). QMP suppresses the activation of worker ovaries, attracts drones, stimulates nectar foraging, and decreases juvenile hormone production (Gary et al., 1962; Butler and Fairey,1963; Higo et al., 1992; Pettis et al.,1995; Melathopoulos et al.,1996; Pettis et al., 1997; Pankiw et al., 1998; Hoover et al., 2003; Brockmann et al., 2006). QMP induces retinue response in workers, which includes surrounding the queen with workers who antennate, groom, and lick her to get QMP (Slessor et al., 1988; Pankiw et al., 1994; Pankiw et al.,1995). In addition, QMP regulates the physiology and behaviour of workers (Butler and Fairey, 1963; Slessor et al., 1988; Winston et al., 1989; Pettis et al., 1995; Melathopoulos et al., 1996; Pankiw et al., 1998; Hoover et al., 2003; Katzav et al., 2004; Villar et al., 2005; Katzav et al., 2006; Katzav et al., 2007; Richard et al., 2007; Strauss et al., 2008; Niño et al., 2012).

166 Polyandrous Queen Honey Bee

Lovleen Marwaha

8.2.1. Factors For Inducing Variation in Retinue Behaviour Various reports indicated that workers exhibited differential attraction toward the mandibular gland blend of different queens. Richard et al. (2007) reported that a queen inseminated by many drones could make a more considerable retinue than a queen inseminated by a single drone. Further, they concluded that mandibular gland extract of the inseminated queen is more effective than virgin QMP in acting as an attractant for the queen as workers can discriminate between the varying composition of queen pheromone of the virgin and mated queen. According to Kocher et al. (2009), the naturally mated queen possesses more substantial worker attractiveness than the artificial mated queen. Nino et al. (2012) reported that queens inseminated with lower semen volume possess weaker retinue behaviour than queens with higher-quality semen. It has been detected that workers can become attracted to the queen due to tergal gland secretion or Dufour's gland secretion (Katzaf et al., 2001), even toward the mandibulate queen (Velthuis et al., 1970; Butler et al., 1973; Maisonnasse et al., 2010). A queen raised from younger larvae possesses a more substantial colony and can increase a colony with enhanced productivity than a queen raised from older larvae. Further, queens raised from more immature larvae show more vital retinal behaviour than queens raised from older worker larvae. Low-quality queen shows a higher relative amount of10-HDA as compared to a high-quality queen. (Rangel et al., 2016). 8.2.2. Various Components of QMP and Retinue in Workers QMP is composed of a blend of chemicals, which elicit the retinual behaviour of workers toward the queen (Slessor et al. 1988). QMP, including 9-ODA, regulates workers' behaviour and the physiology of worker bees (Slessor et al. 2005). After mating, there is a change in the composition of QMP, which affects the response of younger worker bees. 9ODA possesses numerous behavioural and physiological effects (Free 1987; Winston and Slessor 1992; Slessor et al. 2005). According to Slessor and colleagues (1988), QMP comprises five components, which include 9ODA, −9HDA, +9HDA, HOB, and HVA. 10HDA is essential for mating but does not affect queen-worker interaction (Slessor et al. 1988; Brockmann et al. 2006). Maisonnasse et al. (2010) de-mandibulated queens without 9-ODA were found equally attractive as mandibulated queens. In 1992, Kaatz and colleagues reported that 9-ODA reduces the concentration of juvenile hormone (JH) biosynthesis. JH is necessary for bees' behavioural and physiological development (Fluri et al. 1982; Huang et al. 1991). The ORNs in

Retinue Behaviour

Polyandrous Queen Honey Bee 167

the antennae convey the signal to AL and, after that, to the higher centre of the insect brain. Behind mushroom bodies, large neurosecretory cells are located, which include corpora allata, responsible for the secretion of JH (Rachinsky and Hartfelder 1990; Tobe and Stay 1985). QMP reduces the secretion of JH biosynthesis, which affects foraging behaviour (Kaatz et al. 1992; Pankiw et al. 1998; Slessor et al. 2005; Beggs and Mercer 2009). QMP help in the transition of honey bees from nursing to foraging behaviour. All components of QMP act synergistically to elicit retinual response. Other compounds which act synergistic with QMP include Methyl oleate, coniferyl alcohol, palmityl alcohol, linolenic acid, and queen ester, including palmitates, oleates, ethyl stearate, ethyl, and methyl palmitoleate, ethyl palmitate acts as an inhibiter of worker ovarian development (Slessor et al., 2005). Keeling et al.2003, identified four synergistic chemicals like QMP, which include coniferyl alcohol (CA), methyl oleate (MO), hexadecane-1-ol (PA), and linoleic acid (LA). In addition, it has been tested that a queen deprived of a mandibular gland can still attract worker honey bees for retinue (Maisonnasse et al. (2010). 8.2.3. QMP Influence on Drone and Swarming Virgin queen secret specific pheromones attract drones for mating flight, and post-mating queen secretes specific pheromones attract workers (Brockmann et al., 2006). According to Gary 1962, QMP can attract drones toward the virgin queen. 9-HDA, 10-HDA, and HOB can attract drones toward the queen, whereas 9-HDA and 10-HDA can increase drone contracts. 9-HDA and 10-HDA are effective for short ranges, whereas 9-ODA is effective for long distances (Brockmann et al. 2006; Loper et al. 1996). 10-HDA is produced in high amounts by virgin queens. Therefore, it is considered a sex pheromone. Renner and Vierling 1977, reported that tergal gland extract alone with 9-ODA influences mating behaviour. 9-oxo-2-decanoic acid (9-ODA), (2E)-9-hydroxydecenoic acid (9-HDA), and (2E)-10-hydroxydecenoic acid (10-HDA) influence mating between queen and drones. Gilley et al., 2006 reported nine compounds emitted by honey bee queens and workers through solid-phase microextraction. Further, Queen pheromones can attract a swarm through pheromonal signals. QMP regulates the swarming behaviour of honey bees and inhibits the queenrearing behaviour of workers. Winston et al. 1989, compared the attractant potential of mandibular gland extract, five component blend, and queen alone and analyzed that queen alone showed the most substantial effect. Additionally,

168 Polyandrous Queen Honey Bee

Lovleen Marwaha

vitellogenin RNA had been detected in high concentrations in QMP-exposed worker honey bees. Pheromones secreted by tergal glands generally act as sex pheromones. The production of queen tergal gland alkenes starts after mating. Smith et al. (1993) demonstrated in their experiments that the production of tergal gland alkenes is stimulated by natural mating and not by experimental insemination. The beekeeping industry has long recognized that instrumentally inseminated queens are not as productive as naturally mated queens. Problems are observed with the initial introduction and acceptance of the inseminated queens to a colony. It had been reported that there is a rapid replacement of the introduced inseminated queen. The tergal gland alkenes may play a key role in the care and acceptance of the queen and her eggs by worker bees in the hive. 9-octadecenoic acid and decyl decanoate from the tergal gland facilitate communication between the sexes (Smith et al., 1993: Rhodes et al., 2007). The Queen honey bee also regulates swarming as Worker honey bees form a small cluster around the queen honey bee. Worker honey bees emit attraction pheromones from various Nasanov glands to attract other honey bees around the queen (Janson et al., 2005) to form a big cluster (Trhlin and Rajchard, 2011). 8.2.4. Colonial Transfer of Queen Pheromones Queen pheromones are essential for maintaining social harmony in a colony. The specific pheromones affect the recipient's behaviour (Le Conte and Hefetz, 2008) and inhibit ovarian development in workers (Naumann et al., 1991, 1992; Wanner et al., 2007). As the queen moves on the comb, workers extend their antennae and palpate her (Butler, 1954; Sakagami, 1958). Worker honey bees acquire pheromones by direct contact with retinue bees, which carry queen pheromones from the queen. Further, the grooming behaviour of workers also facilitates the transference of pheromones. Young workers pass queen pheromones to other nestmates through antennal contact and trophallaxis. Naumann (1991) reported that worker honey bees, by doing self-grooming, transfer pheromones from the mouthpart to their head, abdomen, and limbs of honey bee workers. Therefore, workers without contact still perceive the queen's presence (Seeley, 1979; Naumann et al., 1991; Pankiw et al.,1995). Queen mandibular pheromones also are communicated to colony workers through the comb as workers walk on the comb.

Retinue Behaviour

Polyandrous Queen Honey Bee 169

Fischer and Grozinger (2008) tested the effect of OMP inducing tolerance to starvation, lipid storage and gene expression of worker bees. QMP-treated worker honey bees showed stronger starvation resistance than non-exposed individuals. Retinue behaviour is essential for the regulation of functionality of the colony. Pheromones which help in retinue behaviour include secretion of the mandibular gland, Dufour's gland (Slessor et al., 1988; Kaminski et al.,1990; Pankiw et al., 1995; Katzav-Gozansky et al., 2003). As colonies grow, the worker population increases in size and the amount of QMP which reaches individual bees decreases due to a dilution effect and restricted movement. Consequently, worker bees are released from the inhibitory impact of QMP on queen rearing and begin to rear queens in preparation for swarming. Queen pheromones regulate the reproductive division of labour. Kocher et al. (2009) reported that queen blend pheromones had been modified as per the reproductive status of the queen. Worker honey bees exhibit more response to the queen with higher reproductive potential. Queen honey bees modulate the physiology, behaviour, and social organization of worker honey bees. As mentioned earlier, the discussion summary is due to the pheromonal bouquet emission of queen workers generally forming groups around her. The peculiar behaviour of workers is known as retinue. Workers receive queen pheromones by surrounding her through licking and antennating her. The received volatiles influence the general behaviour of workers, their development, physiology, reproduction, JH, life span, foraging, etc. Retinue behaviour is essential for the standard functionality of the colony, the queen's dominant hierarchy, colonial productivity, swarming resistance, and inhibition in new queen rearing. CONCLUSION Worker honey bees exhibit particular behaviour to surrounding queen honey bees due to her strong pheromone bouquet emission. The size of retinue varies as per the quality of queen pheromones. More vigorous queens get larger retinue sizes in comparison to weaker queens. Workers get queen pheromones by licking queen honey bee while exhibiting retinue behaviour. REFERENCES Allen, MD (1955) Observations on honeybees attending their queen. Br J Anim Behav, 3, 66-9. [http://dx.doi.org/10.1016/S0950-5601(55)80015-9] Allen, MD (1960) The honeybee queen and her attendants. Anim Behav, 8, 201-8. [http://dx.doi.org/10.1016/0003-3472(60)90028-2] Apðegaitë, V & Skirkevièius, A (1999) Content of (E)-9-oxo-2-decanoic acid in honeybee pheromones (Apis

170 Polyandrous Queen Honey Bee

Lovleen Marwaha

mellifera L.) queens. Pheromones, 6, 27-32. Beggs, KT & Mercer, AR (2009) Dopamine receptor activation by honey bee queen pheromone. Curr Biol, 19, 1206-9. [http://dx.doi.org/10.1016/j.cub.2009.05.051] [PMID: 19523830] Brockmann, A, Brückner, D & Crewe, RM (1998) The EAG response spectra of workers and drones to queen honeybee mandibular gland components: the evolution of a social signal. Naturwissenschaften, 85, 283-5. [http://dx.doi.org/10.1007/s001140050500] Brockmann, A, Dietz, D, Spaethe, J & Tautz, J (2006) Beyond 9-ODA: sex pheromone communication in the European honey bee Apis mellifera L. J Chem Ecol, 32, 657-67. [http://dx.doi.org/10.1007/s10886-005-9027-2] [PMID: 16586035] Butler, CG (1954) The method and importance of the recognition by a colony of honeybees (A. mellifera) of the presence of its queen. Trans R Entomol Soc Lond, 105, 11-29. [http://dx.doi.org/10.1111/j.1365-2311.1954.tb00773.x] Butler, CG, Callow, RK, Koster, CG & Simpson, J (1973) Perception of the queen by workers in the honeybee colony. J Apic Res, 12, 159-66. [http://dx.doi.org/10.1080/00218839.1973.11099744] Butler, CG & Fairey, EM (1963) The role of the queen in preventing oogenesis in worker honeybees. J Apic Res, 2, 14-8. [http://dx.doi.org/10.1080/00218839.1963.11100051] Conte, YL & Hefetz, A (2008) Primer pheromones in social hymenoptera. Annu Rev Entomol, 53, 523-42. [http://dx.doi.org/10.1146/annurev.ento.52.110405.091434] [PMID: 17877458] Crozier, RH & Pamilo, P (1996) Evolution of social insect oloniesOxfordUniversityPress, Oxford. De Hazan, M, Lensky, Y & Cassier, P (1989) Effects of queen honeybee (Apis mellifera L.) ageing on her attractiveness to workers. Comp Biochem Physiol A Comp Physiol, 93, 777-83. [http://dx.doi.org/10.1016/0300-9629(89)90501-X] Fischer, P & Grozinger, CM (2008) Pheromonal regulation of starvation resistance in honey bee workers (Apis mellifera). Naturwissenschaften, 95, 723-9. [http://dx.doi.org/10.1007/s00114-008-0378-8] [PMID: 18414825] Fluri, P, Lüscher, M, Wille, H & Gerig, L (1982) Changes in weight of the pharyngeal gland and haemolymph titres of juvenile hormone, protein and vitellogenin in worker honey bees. J Insect Physiol, 28, 61-8. [http://dx.doi.org/10.1016/0022-1910(82)90023-3] Free, JB (1987) Pheromones of Social BeesChapman and Hall, London. Gary, NE (1962) Chemical mating attractants in the queen honey bee. Science, 136, 773-4. [http://dx.doi.org/10.1126/science.136.3518.773] [PMID: 17752107] Gilley, DC, DeGrandi-Hoffman, G & Hooper, JE (2006) Volatile compounds emitted by live European honey bee (Apis mellifera L.) queens. J Insect Physiol, 52, 520-7. [http://dx.doi.org/10.1016/j.jinsphys.2006.01.014] [PMID: 16530783] Higo, HA, Colley, SJ, Winston, ML & Slessor, KN (1992) Effects of honey bee (Apis mellifera L.) queen mandibular gland pheromone on foraging and brood rearing. Can Entomol, 124, 409-18. [http://dx.doi.org/10.4039/Ent124409-2] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Huang, ZY, Robinson, GE, Tobe, SS, Yagi, KJ, Strambi, C, Strambi, A & Stay, B (1991) Hormonal regulation of behavioural development in the honey bee is based on changes in the rate of juvenile hormone biosynthesis. J Insect Physiol, 37, 733-41.

Retinue Behaviour

Polyandrous Queen Honey Bee 171

[http://dx.doi.org/10.1016/0022-1910(91)90107-B] Janson, S, Middendorf, M & Beekman, M (2005) Honeybee swarms: how do scouts guide a swarm of uninformed bees? Anim Behav, 70, 349-58. [http://dx.doi.org/10.1016/j.anbehav.2004.10.018] Kaatz, HH, Hildebrandt, H & Engels, W (1992) Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees. J Comp Physiol B, 162, 588-92. [http://dx.doi.org/10.1007/BF00296638] Kaminski, LA, Slessor, KN, Winston, ML, Hay, NW & Borden, JH (1990) Honeybee response to queen mandibular pheromone in laboratory bioassays. J Chem Ecol, 16, 841-50. [http://dx.doi.org/10.1007/BF01016494] [PMID: 24263599] Katzav-Gozansky, T, Boulay, R, Soroker, V & Hefetz, A (2004) Queen–signal modulation of worker pheromonal composition in honeybees. Proc Biol Sci, 271, 2065-9. [http://dx.doi.org/10.1098/rspb.2004.2839] [PMID: 15451697] Katzav-Gozansky, T, Boulay, R, Soroker, V & Hefetz, A (2006) Queen pheromones were affecting the production of queen-like secretion in workers. A Neuroethology, sensory, neural, and behavioural physiology. 192, 737-42. Katzav-Gozansky, T, Hefetz, A & Soroker, V (2007) Brain modulation of Dufour’s gland ester biosynthesis in vitro in the honeybee (Apis mellifera). Naturwissenschaften, 94, 407-11. [http://dx.doi.org/10.1007/s00114-006-0206-y] [PMID: 17235597] Katzav-Gozansky, T, Ibarra, F, Francke, W, Hefetz, A & Soroker, V (2001) Dufour’s gland secretion of the queen honeybee ( Apis mellifera ): an egg discriminator pheromone or a queen signal? Behav Ecol Sociobiol, 51, 76-86. [http://dx.doi.org/10.1007/s002650100406] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Kocher, SD & Grozinger, CM (2011) Cooperation, conflict, and the evolution of queen pheromones. J Chem Ecol, 37, 1263-75. [http://dx.doi.org/10.1007/s10886-011-0036-z] [PMID: 22083225] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2009) Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees. Behav Ecol, 20, 1007-14. [http://dx.doi.org/10.1093/beheco/arp090] [PMID: 22476212] Kraus, FB, Neumann, P & Moritz, RFA (2005) Genetic variance of mating frequency in the honeybee (Apis mellifera L.). Insectes Soc, 52, 1-5. [http://dx.doi.org/10.1007/s00040-004-0766-9] Loper, GM, Taylor, OR, Jr, Foster, LJ, Kochansky, J & Kochansky, J (1996) The relative attractiveness of queen mandibular pheromone components to honey bees (Apis mellifera L.) drones. J Apic Res, 35, 122-3. [http://dx.doi.org/10.1080/00218839.1996.11100923] Naumann, K, Winston, ML, Slessor, KN, Prestwich, GD & Latli, B (1992) Intra-nest transmission of aromatic honey bee queen mandibular gland pheromone components: movement as a unit. Can Entomol, 124, 917-34. [http://dx.doi.org/10.4039/Ent124917-5] Naumann, K, Winston, ML, Slessor, KN, Prestwich, GD & Webster, FX (1991) Production and transmission of honey bee queen (Apis mellifera L.) mandibular gland pheromone. Behav Ecol Sociobiol, 29, 321-32. [http://dx.doi.org/10.1007/BF00165956] Niño, EL, Malka, O, Hefetz, A, Teal, P, Hayes, J & Grozinger, CM (2012) Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology. J Insect Physiol, 58, 1082-9.

172 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1016/j.jinsphys.2012.04.015] [PMID: 22579504] Maisonnasse, A, Alaux, C, Beslay, D, Crauser, D, Gines, C, Plettner, E & Le Conte, Y (2010) New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation? Front Zool, 7, 18-25. [http://dx.doi.org/10.1186/1742-9994-7-18] [PMID: 20565874] Melathopoulos, AP, Winston, ML, Pettis, JS & Pankiw, T (1996) Effect of queen mandibular pheromone on initiation and maintenance of queen cells in the honey bee(Apis mellifera L.). Can Entomol, 128, 263-72. [http://dx.doi.org/10.4039/Ent128263-2] Pankiw, T, Huang, Z, Winston, ML & Robinson, GE (1998) Queen mandibular gland pheromone influences worker honey bee (Apis mellifera L.) foraging ontogeny and juvenile hormone titers. J Insect Physiol, 44, 685-92. [http://dx.doi.org/10.1016/S0022-1910(98)00040-7] [PMID: 12769952] Pankiw, T, Winston, ML & Slessor, KN (1994) Variation in worker response to honey bee (Apis mellifera L.) queen mandibular pheromone (Hymenoptera: Apidae). J Insect Behav, 7, 1-15. [http://dx.doi.org/10.1007/BF01989823] Pankiw, T, Winston, ML & Slessor, KN (1995) Queen attendance behavior of worker honey bees (Apis mellifera L.) that are high and low responding to queen mandibular pheromone. Insectes Soc, 42, 371-8. [http://dx.doi.org/10.1007/BF01242165] Pettis, JS, Higo, HA, Pankiw, T & Winston, ML (1997) Queen rearing suppression in the honey bee evidence for a fecundity signal. Insectes Soc, 44, 311-22. [http://dx.doi.org/10.1007/s000400050053] Pirk, CWW, Sole, CL & Crewe, RM (2011) Pettis, JS, Winston, ML & Collins, AM (1995) Suppression of queen rearing in European and Africanized honey bees Apis mellifera L. by synthetic queen mandibular gland pheromone. Insectes Soc, 42, 113-21. [http://dx.doi.org/10.1007/BF01242447] Pettis, JS, Winston, ML & Slessor, KN (1995) The behaviour of queen and worker honey bees (Hymenoptera: Apidae) in response to exogenous queen mandibular gland pheromone. Ann Entomol Soc Am, 88, 580-8. [http://dx.doi.org/10.1093/aesa/88.4.580] Rachinsky, A & Hartfelder, K (1990) Corpora allata activity, a prime regulating element for caste-specific juvenile hormone titre in honey bee larvae (Apis mellifera carnica). J Insect Physiol, 36, 189-94. [http://dx.doi.org/10.1016/0022-1910(90)90121-U] Rangel, J, Böröczky, K, Schal, C & Tarpy, DR (2016) Honey bee (Apis mellifera) queen reproductive potential affects queen mandibular gland pheromone composition and worker retinue response. PLoS One, 11e0156027 [http://dx.doi.org/10.1371/journal.pone.0156027] [PMID: 27281328] Renner, M & Vierling, G (1977) Die rolle des taschendrüsenpheromons beim Hochzeitsflug der Bienenkönigin. Behav Ecol Sociobiol, 2, 329-38. [http://dx.doi.org/10.1007/BF00299744] Rhodes, JW, Lacey, MJ & Harden, S (2007) Changes with age in queen honey bee (Apis mellifera) head chemical constituents (Hymenoptera: Apidae). Sociobiology, 50, 11-22. Ribbands, CR (1953) The behaviour and social life of honeybeesBee Research Association, London. Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2e980 [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Sakagami, SF (1958) The false-queen: fourth adjustive response in dequeened honeybee colonies1. Behaviour, 13, 280-95.

Retinue Behaviour

Polyandrous Queen Honey Bee 173

[http://dx.doi.org/10.1163/156853957X00056] Seely, TD (1979) Queen substance dispersal by messenger workers in honeybee colonies. Behav Ecol Sociobiol, 5, 391-415. [http://dx.doi.org/10.1007/BF00292527] Seeley, TD (1995) The wisdom of the hive: The social physiology of honey bee coloniesHarvard UniversityPress, Cambridge. [http://dx.doi.org/10.4159/9780674043404] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Smith, RK, Spivak, M, Taylor, OR, Jr, Bennett, C & Smith, ML (1993) Maturation of tergal gland alkene profiles in European honey bee queens,Apis mellifera L. J Chem Ecol, 19, 133-42. [http://dx.doi.org/10.1007/BF00987478] [PMID: 24248518] Snodgrass, R (1956) Anatomy of the honey beeCornell Paperbacks, Ithaca. Strauss, K, Scharpenberg, H, Crewe, RM, Glahn, F, Foth, H & Moritz, RFA (2008) The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent? Behav Ecol Sociobiol, 62, 1523-31. [http://dx.doi.org/10.1007/s00265-008-0581-9] Tarpy, DR & Nielsen, DI (2002) Sampling error, effective paternity, and estimating the genetic structure of honey bee colonies (Hymenoptera: Apidae). Ann Entomol Soc Am, 95, 513-28. [http://dx.doi.org/10.1603/0013-8746(2002)095[0513:SEEPAE]2.0.CO;2] Tarpy, DR, Nielsen, R & Nielsen, DI (2004) A scientific note on the revised estimates of effective paternity frequency in Apis. Insectes Soc, 51, 203-4. [http://dx.doi.org/10.1007/s00040-004-0734-4] Tobe, SS & Stay, B (1985) Structure and regulation of the corpus allatum. Adv Insect Physiol, 18, 305-432. [http://dx.doi.org/10.1016/S0065-2806(08)60043-0] Trhlin, M & Rajchard, J (2011) Chemical communication in the honeybee (Apis mellifera L.): a review. Vet Med (Praha), 56, 265-73. [http://dx.doi.org/10.17221/1543-VETMED] Velthuis, HHW (1970) Queen substances from the abdomen of the honey bee queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 70, 210-21. [http://dx.doi.org/10.1007/BF00297717] Velthuis, HHW (1972) Observations on the transmission of queen substances in the honey bee colony by the attendants of the queen. Behaviour, 41, 105-28. [http://dx.doi.org/10.1163/156853972X00239] Villar, G, Baker, TC, Patch, HM & Grozinger, CM (2015) Neurophysiological mechanisms underlying sexand maturation-related variation in pheromone responses in honey bees (Apis mellifera). J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 201, 731-9. [http://dx.doi.org/10.1007/s00359-015-1006-7] [PMID: 25840687] Wanner, KW, Nichols, AS, Walden, KKO, Brockmann, A, Luetje, CW & Robertson, HM (2007) A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid. Proc Natl Acad Sci USA, 104, 14383-8. [http://dx.doi.org/10.1073/pnas.0705459104] [PMID: 17761794] Wilson, EO (1971) The insect societiesHarvard University Press, Cambridge. Winston, ML (1987) The Biology of the Honey BeeHarvard University Press, Cambridge, MA.

174 Polyandrous Queen Honey Bee

Lovleen Marwaha

Winston, ML & Slessor, KN (1992) Winston, M.L. and Slessor, KN, 1992. The essence of royalty: honey bee queen pheromone. American scientist (USA). 80, 374-85. Winston, ML, Slessor, KN, Willis, LG, Naumann, K, Higo, HA, Wyborn, MH & Kaminski, LA (1989) The influence of queen mandibular pheromones on worker attraction to swarm clusters and inhibition of queen rearing in the honey bee (Apis mellifera L.). Insectes Soc, 36, 15-27. [http://dx.doi.org/10.1007/BF02225877] Yang, MX, Tan, K, Radloff, SE, Pirk, CWW & Hepburn, HR (2010) Hetero-specific queen retinue behavior of worker bees in mixed-species colonies of Apis cerana and Apis mellifera. Apidologie (Celle), 41, 54-61. [http://dx.doi.org/10.1051/apido/2009047] Yusuf, AA, Pirk, CWW & Crewe, RM (2015) Mandibular gland pheromone contents in workers and queens of Apis mellifera adansonii. Apidologie (Celle), 46, 559-72. [http://dx.doi.org/10.1007/s13592-014-0346-6]

Polyandrous Queen Honey Bee, 2022, 175-196

175

CHAPTER 9

Influence of Queen Pheromones on Worker Ovarian PCD in Apis mellifera (Hymenoptera: Apidae) Abstract: Queen honey bee imposes her reproductive dominance through the secretion of volatile chemicals, especially from the mandibular gland, tergal and defour glands. Further, queen pheromones and different larval diets; aggregately control the differential expression of specific genetic elements. The altered transcriptomic activity resulted in Programmed Cell Death (PCD) in the ovaries of worker honey bees. Furthermore, after the hatching of workers, the specific degenerative process remains continuous for a brief period, destroying numerous ovarioles. As a result, few facultative functional ovarioles remain active in worker honey bees' ovaries. Available literature also witnesses the formation of pseudo-queens or egg-laying workers. This chapter provides insight into responsible queen pheromones for induction of programmed cell death in worker honey bees' ovaries. The next chapter focuses on the genetic elements for queen pheromones's-induced ovarian PCD in workers.

Keywords: Pheromones, Programmed Cell Death and Differential Gene Expression. 9.1. INTRODUCTION In queen-less (QL) colony, worker honey bees possess activated ovaries, whereas, in queen righted (QR) colony, workers have degenerative ovaries (Lin et al., 1999; Schafer et al., 2006). Development of workers' ovarian system is suppressed by healthy, queen and workers' pheromonal and genetic controls. That ultimately fixes the anatomical division of reproduction (Butler, 1959; Mohammedi et al., 1998; Hoover et al., 2003). Queen secretes pheromones from various organs, which induce Programmed Cell Death (PCD) in workers' ovaries (Wossler, and Crewe,1999; Hoover et al., 2003; Okosun et al., 2017), with prominent suppressive influence from mandibular pheromones (Strauss et al., 2008). Programmed Cell Death is of two major types: apoptosis and autophagy based on the basic sequential processes involved. ApopLovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

176 Polyandrous Queen Honey Bee

Lovleen Marwaha

tosis mainly involves DNA fragmentation, chromatin condensation, cell shrinkage and chromatin condensation, which can be detected using techniques including terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) assay and electron microscopy (Wu et al., 2015). Differential diets given to larvae after the third day and other environmental factors ultimately result in different gene expressions (Dedejet al., 1998; Evans and Wheeler, 2001; Kucharski et al., 2008; Guo et al., 2013). Further caste development in honey bees is brought about by developmental hormones like ecdysone, juvenile hormone, TOR pathway, epigenetic alteration, royalactin, and insulin (Capella and Hartfelder, 1998; Pinto et al., 2002; Wheeler et al., 2006; Patel et al.,2007; Kucharski et al.,2008; Kamakura 2011; Leimaret al., 2012; Hartfelderet al., 2015). For example, on the third day of larval development, a high concentration of JH specifies queen caste development, whereas a low concentration of juvenile hormone induces worker caste development. 9.2. QUEEN PHEROMONES RELATED TO PCD IN WORKERS' OVARIES 9.2.1. General Information Queen pheromones (QP) are volatile chemical signals that help regulate the reproductive division of labour in honey bee colonies (Slessor et al., 2005; Le Conte and Hefetz, 2008; Van Oystaeyenet al., 2014; Lago et al., 2016; Wojciechowski et al., 2019). Further, specific chemicals elicit multiple other effects, including attraction of workers toward queen honey bee (Fig. 9a), aggressive or submissive behaviour in workers, Programmed Cell Death (PCD) in worker honey bees' ovaries (Slessor et al., 1988; Vergozet al., 2007; Smith et al., 2009; Van Oystaeyenet al., 2014; Smith et al., 2016; Wojciechowski et al., 2018). In other words, QP are chemical signals to the residents, which witness the presence of reproductive active individuals in colony (Fig. 9b - h), and consequently workers exhibit worker like morphology, physiology, anatomically and behavioural responses (Groot and Voogd, 1954; Erp, 1960; Butler, et al., 1961; Michener, 1969; Kubisova and Haslbachova, 1978;Hemmling, et al., 1979; Ruttner and Hesse 1981;Rembold, 1987; Slessor, et al., 1988; Visscher, 1989;Rachinsky, et al., 1990; Winston, et al., 1990;Naumann, et al., 1991; Ratnieks, 1993; Keller and Nonacs, 1993; Oldroyd, et al., 1994 ; Pankiw, et al., 1996; Visscher 1996; Katzav, et al., 1997; Pernal and Currie, 2000 ;Neumann and Hepburn 2002; Moritz, et al., 2004 ; Hemmling, et al., 2006; Lattorff, et al., 2007;Evans and Wheeler, 2001; Matin, et al., 2002; Colonello and Hartfelder, 2005; Hartels, et al., 2006; Vergoz, 2007; Smith, et al., 2009; Zheng, et al.,

Queen Pheromones

Polyandrous Queen Honey Bee 177

2010;Jarosch, et al., 2011; Amsalem, et al., 2015; Oi, et al., 2015; Peso et al., 2015; Yusuf, 2015; Oldroyd and Vergoz, 2016;Smith, 2016; Ronai, et al., 2016; Smith and Liebig, 2017; Nunes, et al., 2017; Smith and Liebig 2017;Holman, 2018)

Fig. (9a). shows the strong colony during regular inspection of healthy growth.

QP is a blend of volatile chemicals secreted by the mandibular gland and tergal glands with components including 9-ODA, R and S 9-HDA, HOB and HVA (Slessor et al., 2005).

Fig. (9b). Frame from the same colony, with the headship of the good quality queen of one year. On the upper few rows, ripe honey within capped cells can be easily distinguished, followed by unripe honey cells in uncapped conditions are visible. In addition, numerous capped brood cells can be recognized, which witness the quality of the queen.

178 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (9c). Photo click of ripe honey cells filled within capped wax cells from the same colony. Workers prefer to store honey on peripheral frames or the upper edges of central hives of the brood chamber, leaving ample space for brood storage.

Fig. (9d). Pictorial presentation of retinue behaviour of worker honey bees. In a specific section, the queen is surrounded by a group of workers.

Queen Pheromones

Polyandrous Queen Honey Bee 179

Fig. (9e). Queen honey bee inspecting wax cell for egg laying. If the dimension of the wax cell is not appropriate, in that case, the queen rejects that cell and starts searching for another cell of the desired size.

9.2.1.1. General Ovarian Structure In hymenopterans, female ovaries carry elongated tubular ovarioles (Fig. 3a) (Martins and Serrão 2004). Several ovarioles can be counted in a freshly dissected ovary by a stereomicroscope. The fecundity of the queen honey bee varies according to ovarioles count. Honey bees pollinate many crops. Therefore, they are essential for economic profit. According to Chaud-Netto and Bueno 1979; Reginato and Cruz-Landim 2003, in Apis mellifera L. (Hymenoptera: Apidae), there are 100 ovarioles per ovary in the queen. Workers possess ten ovarioles per ovary (refer to chapter 2 for details). Few other studies indicated that queen honey bees have a maximum ovariole number from 120-200. Whereas in worker honey bees, the number of ovarioles varies from 2-12 (Snodgrass 2018; Linksvayer et al. 2011). Experimental data shows that in queen honey bees, more ovarioles are present in the right than left ovary. Whereas in worker honey bees, left ovary possesses more ovarioles than the right ovary (Eckert 1934; Chaud- Netto and Bueno 1979). Further, according to Jackson et al., 2011, 7.5% of queens have less than 125 ovarioles per ovary. Furthermore, the ovariole count provides insight into the reproductive potential of the adult queen (Fig. 9a). During the development of workers, programmed cell death of ovarian tissue occurs. In Apis mellifera, the other number of ovarioles can vary according to the genotype of workers (Thuller

180 Polyandrous Queen Honey Bee

Lovleen Marwaha

et al. 1998; Hatch et al. 1999; Reginato and Cruz-Landim 2002; Linksvayer et al. 2009; Kocher et al. 2010; Linksvayer et al. 2011). In ovarioles, there are clear germ cells and somatic cell organization, which remain separated by peritoneal sheath cells. In primordial ovarian, germ cells can be differentiated with their peculiar characteristics, including large cells, with spherical nuclei (Zander 1916). The queens with ovarian abnormalities are less fertile (Fyg 1964; Camazine et al. 1998). In those mentioned above and queen-less colonies, worker honey bees with larger ovarioles per ovary usually tend to become egg layers (Makert et al. 2006). But as spermathecae are poorly developed in workers, they cannot lay fertilized eggs (Snodgrass 2018). Usually, colonies with poor queens exhibit poor growth and suffer more from colony losses (van Engelsdorp et al. 2008). 9.2.1.2. Larval Gonadial Development Sex determination in honey bees occurs by haplodiploid molecular mechanism (Beye et al. 2003; Hasselmann et al. 2008). During the embryonic development of honey bees, there is no apparent pole plasm factor like the fruit fly. However, most of the genetic elements which code for pole cell components in the fruit fly are present in honey bees (Dearden et al. 2006). Further, in honey bees, the mode of germ cell specification is quite distinct from Drosophila and is controlled by specific epigenetic factors. After the formation, germ cell migration occurs toward the primordial germline in somatic gonads. Larval gonads are located bilaterally in the fifth and anterior parts of the sixth abdominal segment. Gonads are connected, over the dorsal midline, beneath the dorsal blood vessel. From the basal side of gonads, strings emerge out, which bilaterally surround the midgut. The specific strings develop into the oviduct in female larvae. Further, string contraction occurs during metamorphosis, which rotates ovaries by 90° rotation. The apical portion of the ovary becomes constricted, and a terminal filament forms at the distal part of the ovary. 9.2.2. Main Pheromones Which Influence PCD Various queen and brood pheromones are responsible for the induction of PCD in workers' ovaries. A brief description of the same is as follows. Predominant volatiles for PCD are Queen Mandibular Pheromones (QMP), a blend of different volatile components responsible for the developmental pattern of the worker ovaries and the queen's reproductive dominance in the colony (de

Queen Pheromones

Polyandrous Queen Honey Bee 181

Groot and Voogd, 1954; Verheijen-Voogd, 1959; Butler, 1959; Butler and Fairey, 1963; Velthuis, 1970a,b; Lin, 1999: Lago, et al., 2016). QMP consists of pheromones, mainly methyl p-hydroxybenzoate (HOB), 9-oxo-2 (E)-decanoic acid (9-ODA), 4-hydroxy-3-methoxyphenylethanol (HVA), (R, E)9-hydroxy-2-decanoic acid (9-HDA), (S, E)-9-hydroxy-2-decanoic acid (9-HDA), 10-hydroxy-2 (E)-decanoic acid and 10-hydroxydecanoic acid (10-HDAA)(Crewe and Velthuis, 1980; Winston and Slessor, 1998; Mumoki et al., 2018). Earlier, Butler et al., 1959 reported that (E)-9Oxodec-2-enoic acid (9-ODA) from the queen mandibular gland suppressed the development of the ovary in caged worker honey bees. After that, Butler and Fairey, 1963, re-confirmed that (E)--oxodec-2-enoic acid (9-ODA) regulates ovary development in worker honey bees within a colony. Similarly, few other explorations concluded that OMP influence worker ovarian development, new queen rearing, early behavioural maturation in worker honey bees etc. (Butler, 1959; Butler, 1961; Melathopoulos et al., 1996; Pankiw et al., 1998; Hoover et al., 2003). QMP components 9-ODA and 9-HDA inhibit workers' ovarian development in Apis mellifera and Apiscerana (Tan et al., 2010). Further, pheromones consisting of 9-ODA, 9-HDA and 10-HDA are associated with queen ovarian activation and for her colonial reproductive dominance in Apis mellifera capensis. The comparative study indicated that caged queen-less workers exposed to QMP possess better ovaries than workers from the queenright col. In addition to QMP, some other components are also responsible for suppressing workers' ovaries. Honey bee workers kept within queen-righted colonies possess less developed ovaries than all other honey bees exposed to pheromone treatment in caged conditions. The further specific observation could be due to the large group size, exposure of the ovary to larval esters and poor nutrition in the colony (Mohammedi et al., 1998; Lin et al., 1999). Specific explorations indicated that it is not only QMP but certain other chemicals secreted by the queen that facilitate worker ovarian deactivation (Velthuis, 1970b; Willis et al., 1990; Keeling et al. (2003). However, artificially exposure of QPs was unable to induce an observable effect on workers in queenless (QL) colonies than in queenright (QR) colonies (Willis et al. 1990). Further, some studies confirm that the queen's abdomen alone or de-mandibulated queen was still able to suppress worker ovarian development upto some limit (Velthuis and Van, 1964; Velthuis, 1970b). Extracting whole queen body washes inhibited worker ovarian development (Butler, 1957; Verheijen-Voogd, 1959).

182 Polyandrous Queen Honey Bee

Lovleen Marwaha

Keeling et al. (2003) reported the occurrence of four additional queen-produced compounds, which work synergistically with QMP. Chemicals included: methyl oleate; methyl (Z)-octadec-9-enoate, coniferyl alcohol; (E)-3-(4-hydroy-3-methoxyphenyl)-prop-2-en-1-ol, hexadecan-1-ol, linolenic acid ; (Z9, Z12, Z15)-octadeca-9,12,15-trienoic acid. According to Schafer et al. in 2006, pheromonal dominance is influenced by food consumed by Apis mellifera capensis workers. Royal jelly helps in the activation of queen ovarian development and in laying eggs (Schafer et al., 2006). Honey bee brood pheromone, mainly (E)-beta ocimene, suppresses ovarian activation in honey bee workers (Traynor et al., 2014). In caged Apis mellifera scutellate, brood pheromones do not affect ovarian activation (Démares et al., 2017). Further, tergal gland secretions also inhibit the ovarian development of queen honey bee in two species, including Apis mellifacapensis and Apis mellifera scutellata (Wossler and Crewe, 1999; Okosun et al., 2015; Okosun et al., 2017). 9.2.3. Differential Pheromonal Synthesis in Queen and Workers Physiological plasticity empowers differential pheromonal secretion of specific glands in the female caste of honey bees as per respective roles in the colony. The mandibular gland of worker honey bee secretes high concentrations of 10-HDA, 10-HDAA, HOB, 9-ODA, and 9-HDA, whereas queen mandibular gland pheromones indicated higher concentration of 9-ODA, R and S 9-HDA, HOB and HVA (Plettner et al., 1997; Yusuf et al., 2015) Further, in queen less colony, workers secrete mainly 9-ODA and 9-HDA pheromones, whereas, in queen righted colony, workers secrete mainly 9-HDA and 10-HDA(Moritz et al., 2000). A queen's presence or absence strongly affects pheromones' production in worker honey bees (Katzav et al., 1997; Katzav et al., 2004). During mandibular pheromone synthesis, the conversion of stearic acid (octadecanoic acid) into various mandibular pheromones takes place by bifurcated step processes (Plettner et al., 1996). The first step is hydroxylation of stearic acid at either ω or ω-1 positions and subsequent shortening of 18- and 17hydroxystearic acids through β-oxidation. Caste Specific bifurcated pathway is achieved by differential gene expression in female castes (Conte and Hefetz, 2008; Malka et al., 2009; Malka et al., 2014). Further, queen pheromones influence fatty acid biosynthesis in workers' mandibular glands by inhibiting the ω-1 hydroxylation route in the biosynthesis of

Queen Pheromones

Polyandrous Queen Honey Bee 183

mandibular gland pheromones (Malka et al., 2009;2014). Additionally, the queen influences fatty acid biosynthesis in workers by halting the oxidation of 9-HDA into 9-ODA (Malka et al., 2009;2014). Queen inhibits concerned biochemical conversion by influencing the production of the enzyme alcohol dehydrogenase (Malka et al., 2014; Wu et al., 2017). The specific enzyme is present in higher concentrations in QL workers, which indicates the inhibition of 9-ODA production imposed by the queen honey bee. Thus, the higher concentration of 9ODA in QL than in the QR colony is due to the oxidative enzymatic reduction of 9-HDA to 9-ODA. A lower concentration of alcohol dehydrogenase in workers of QR colony suggests one possible mechanism used by queen honey bees to suppress the production of 9-ODA, which results in the accumulation of precursor 9-HDA in worker honey bees. Moreover, higher activity of alcohol dehydrogenase is present in the queen mandibular gland than in workers (Malka et al., 2014; Wu et al., 2017). There is an increase in the concentration of 9-ODA with the increase in age of the queen due to a rise in the activity of the alcohol dehydrogenase enzyme, which converts 9-HDA to 9-ODA (Plettner et al., 1996; Genç and Aslan, 1999; Reece et al., 2002; Mumoki et al., 2018).

Fig. (9g). Diagram depicting partial mechanism for low concentration of 9-ODA in workers. Under the influence of queen pheromones, the activity of alcohol dehydrogenase decreases in workers. The specific enzyme catalyzes the conversion of 9-HDA to 9-ODA (detail is elucidated in chapter 10).

Different comparative studies indicated that worker ovarian development exposed to 9-ODA, queen's Dufour's gland, other queen mandibular pheromones, 9-HDA (9-hydroxy-(E)2-decanoic acid) drastically reduce ovarian development (Butler et al., 1961; Butler and Fairey 1963; Velthuis and van Es 1964; Kaatzet al., 1992; Tan et al. 2015; Holman, 2019).

184 Polyandrous Queen Honey Bee

Lovleen Marwaha

9.3. QUEEN'S INFLUENCE ON PROGRAMMED CELL DEATH 9.3.1. General Information PCD acts as a conserved mechanism to modulate oogenesis in animals, including insects. The specific event occurs at two distinct stages during oogenesis, including the first stage: when the female insect is exposed to adverse environmental conditions, which results in mid-oogenesis death of germ cells, second stage: PCD of nurse cells during late oogenesis (Jenkins et al., 2013). During mid-oogenesis workers, ovarian germ cells exhibited morphological characteristics of PCD (Ronaiet al., 2016). Across taxa, the primary determinant for initiation and execution of PCD includes the conserved family of proteases, the caspases. The expression of caspase takes place constitutively, but their functions are tightly regulative. After activation, caspase carries out a sequential set of reactions. The caspase activity in the ovary indicates PCD, including apoptosis and autophagy. Differential Ovarian Development: In honey bees up to the fourth instar, there is no significant difference in the ovaries of queen and workers concerning the number and structure of ovarioles (Hartfelder and Steinbrück 1997). Upon entering the fifth instar larval phase, degenerative events become predominant in the ovarioles. At this particular moment, the reversal of development phases, which can control the conversion of worker honey bee larvae to queens, becomes difficult (Dedej et al. 1998). According to Lago et al. (2016), there is a difference in the size of the ovary of the queen and workers. Determination of ovary phenotype occurs during the final larval instar when massive programmed cell death results in degeneration of 9599% of ovarian analogen in workers. Juvenile hormones at higher levels in the queen protect ovaries against such a decline. Ovarian development of worker honey bee is influenced by queen honey bee primer pheromones and brood pheromones. In a queen-less colony, worker honey bees exposed to uncapped broods exhibit little ovarian development. Queen pheromones and brood pheromones influence workers' ovarian development, egglaying workers and pseudo queen pheromones (Crewe and Velthuis, 1980; Saiovici, 1983). Additionally, the suppression of worker ovarian development occurs due to the influence of environmental factors and at the genetic level. Malka et al. (2007) reported that queenless egg-laying workers exhibited apparent regression in ovarian development when introduced to queenright colonies, indicating that ovarian degeneration occurs due to the queen and her signals. Live

Queen Pheromones

Polyandrous Queen Honey Bee 185

queen is more effective in the suppression of ovarian growth of workers than either mandibular or tergal gland pheromones, which witness the involvement of other pheromones in the specific process (Ronia et al., 2016). In the absence of a queen, workers' ovaries get activated, and accomplishment of oogenesis occurs. However, workers can lay only unfertilized eggs due to the lack of well-developed spermathecae and stored sperm (Jay 1968; Velthuis et al. 1990). Some reports support the likelihood of younger workers developing into egg-laying workers in the queenless colony (Leonardo, 1985; Hepburn et al., 1991; Van der and Verkade, 1991). Whereas Delaplane and Harbo, (1987) found different behaviour as they observed 54-day-old worker honey bee acting as an egg layer. 9.3.2. Mechanism of PCD PCD event represents an important mechanism that generates plasticity in honey bees' ovarian phenotype formation. During Programmed Cell Death (PCD) in worker ovaries, certain degenerative events occur, in which worker honey bees lose more than 90% of ovarioles primordial during larval development. Low levels of juvenile hormone during workers' development induced breakdown of the actin cytoskeleton in germ cell clusters. The actin cytoskeleton is essential in controlling cell death in ovarian adult bees, as detected by TUNEL-labeled and pycnotic nuclei in actin agglomerates. The specific mechanism is common for caste-specific ovarian phenotype development and activation of adult bees' reproductive system (Schmidt et al., 1998). Additionally, bromodeoxyuridine (BrdU) labelling of S-phase nuclei revealed that worker ovariole cell death happened during the fifth instar larval stage (Schmidt et al., 1998). According to Buning, 1994, about 39-50 nurse cells contribute to their cytoplasm for oocyte growth. PCD is the prominent feature of nurse cells, which dump their cytoplasm into oocytes before the chorion formation (Mahajan and Cooley,1994; Foley and Cooley,1998). At the end of this cytoplasmic dumping event, nurse cell nuclei start showing signs of cell death and specific cells are phagocytosed by follicular epithelium (Mahajan and Cooley,1994). The cytoplasmic transfer occurs by myosin-based contraction of the subcortical actin network of nurse cells. Apoptotic cell formation occurs in the late feeding stage of larval growth in the central region of the ovariole. After that, specific events proceeded toward the apical and basal part of the ovariole, resulting in the death of entire ovarioles. Degeneration of worker ovary cells occurs by the disintegration of polysome, i.e. mitotically dividing sister germ cells connected cytoplasmically (Hartfelder and Steinbrück 1997). Polyfusomes remain well intact in queen ovarioles, but in

186 Polyandrous Queen Honey Bee

Lovleen Marwaha

worker honey bee ovary, degeneration occurs due to disintegration of actin/spectrin cytoskeleton (Capella and Hartfelder 2002). PCD events begin in the centre and proceed toward the apical and basal ends of the ovarioles. Furthermore, the Hemolymph level of the JH hormone influences the PCD event in the fifth instar ovary. During the transition from fourth to fifth instar larvae, queen larvae have a high juvenile hormone level. Reduced JH titer decreases mitotic activity in the worker ovary during the critical phase of PCD induction that prevents disintegration of the fusomal and cortical actin cytoskeleton (Schmidt and Hartfelder,1998; Schmidt and Hartfelder, 2002; Tanaka et al., 2014). During the larval spinning phase, the JH level drops to the basal level in queen and worker honey bee larvae. Applying synthetic JH to fourth instar worker larvae inhibited PCD even as detected by TUNEL-labeling and helped in the retention of intactness of actin/spectrin cytoskeleton in individual germ cell rosettes (Capella and Hartfelder 1998; Capella and Hartfelder 2002; Lago et al., 2016). Different nutrition causes caste-specific activation of corpora allata and prothoracic glands (Hartfelder,1993). Therefore fifth instar queen larvae possess higher JH titer than worker larvae (Rachinsky et al., 1990; Rembold,1987). In addition, the queen and worker development began with a similar number of ovariole primordia in each ovary, yet it is present at a higher number in the queen than in the worker honey bee (Hartfelder K, Steinbrück G (1997; Reginato RD, Cruz-Landim C (2002). Ronai et al., 2016 observed that the queen secretes specific pheromones that regulate workers' reproductive state in social insect colonies. They considered that programmed cell death plays a central role in inhibiting oogenesis. Further, they described a new method for studying programmed cell death and estimating the concentration of adhesive triphosphate in insect tissue. Worker honey bees exposed to queen pheromone possess high caspase activity than the unexposed workers. Further, they reported that the social environment of the honey bee colony influences the programmed cell death in tissue. Worker honey bees exposed to queen pheromones exhibit a higher incidence of PCD in germarium than unexposed worker honey bees. After entering from nurse bee to the forager bee stage, the incidence of PCD becomes more pronounced. Only tunica propria and peritoneal sheath remain in older honey bees in ovarioles.

Queen Pheromones

Polyandrous Queen Honey Bee 187

9.3.3. Detection of PCD in Workers' Ovaries Programmed cell death and live cell number can be detected by considering caspase activity and the amount of adenosine triphosphate (Ronaiet al., 2015). In worker honey bees' ovaries, there is an increase in caspase activity after exposure to queen pheromones. Further, Ronai et al., 2015, had reported that queen pheromones activate the process of programmed cell death at mid-oogenesis, which causes hindrance in the formation of the oocyte. Further, they conclusively suggested that the social environment affects normal development in the living organism. Queen pheromones induce abortion of mature oocytes (Ronai et al., 2015), and further PCD is responsible for induced sterility in honey bee workers (Gutierrez-Aguilar and Baines, 2013; Ronai et al., 2015; Ronaiet al., 2016). Incidentally, in some cases, even without queen pheromones, deactivation of worker ovaries occurs, and some caspase activity has been detected. The explanation for specific biochemical detection is sometimes in the absence of a queen. Worker honey bees can secrete queen-like pheromones, or pheromones of the pseudo queen can influence the process. Queen mandibular pheromone enhances caspase activity and PCD in worker ovary cells when exposed to 7 days old or 14 days old larvae. (Velthuis, 1970; Crewe and Velthuis, 1980:Ronai, et al., 2015: 2016). Somewhere, queen pheromones influence the intensity of caspase activity. Ronai et al., 2015 observed that some of the ovarioles of workers who had not been exposed to queen pheromones exhibit transition phenotypes between nonactivated and activated ovaries. In contrast, workers with exposed ovaries possess non-activated ovaries. Dying cells contain disorganized structures and healthy epithelial sheath cells. Ronai et al., 2016 suggested that there is another mechanism than limited caspase activity for protecting maturing oocytes from PCD. The deterioration process in the ovaries of worker larvae can be detected by techniques including TUNEL labelling, histological sectioning and ultrastructure analysis (Hartfelder and Steinbrück 1997; Capella and Hartfelder 1998; Reginato and Cruz-Landim2001). CONCLUSION For above-elaborated discussion is that volatiles from queen-induced programmed cell death in workers' ovaries during larval stages influence developmental hormone secretion and through differential genetic elements expression. The majestic development phenomenon of divergent ovarian development establishes the base for reproductive dominance and submissiveness of the same genome.

188 Polyandrous Queen Honey Bee

Lovleen Marwaha

The biphasic gene expression ability of the same genome is for the synchronous colonial organization. REFERENCES Amsalem, E, Orlova, M & Grozinger, CM (2015) A conserved class of queen pheromones? Re-evaluating the evidence in bumblebees ( Bombus impatiens ). Proc Biol Sci, 282, 20151800. [http://dx.doi.org/10.1098/rspb.2015.1800] Barchuk, AR, Bitondi, MMG & Simões, ZLP (2002) Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera. J Insect Sci (Ludhiana), 2, 1-8. [http://dx.doi.org/10.1673/031.002.0101] [PMID: 15455035] Beye, M, Hasselmann, M, Fondrk, MK, Page, RE, Jr & Omholt, SW (2003) The gene csd is the primary signal for sexual development in the honeybee and encodes an SR-type protein. Cell, 114, 419-29. [http://dx.doi.org/10.1016/S0092-8674(03)00606-8] [PMID: 12941271] Butler, CG & Fairey, EM (1963) The role of the queen in preventing oogenesis in worker honeybees. J Apic Res, 2, 14-8. [http://dx.doi.org/10.1080/00218839.1963.11100051] Butler, CG (1957) The control of ovary development in worker honeybees (Apis mellifera). Experientia, 13, 256-7. [http://dx.doi.org/10.1007/BF02157449] [PMID: 13447955] Butler, CG (1959) The source of the substance is produced by a queen honeybee (Apis mellifera L.) which inhibits the development of the ovaries of the workers of her colony. Proc R Entomol Soc Lond, Ser A Gen Entomol, 34, 137-8. [). Oxford, UK: Blackwell Publishing Ltd.]. [http://dx.doi.org/10.1111/j.1365-3032.1959.tb00249.x] Butler, CG (1961) The scent of queen honeybees (A. mellifera L.) that causes partial inhibition of queen rearing. J Insect Physiol, 7, 258-64. [http://dx.doi.org/10.1016/0022-1910(61)90076-2] Butler, CG, Callow, RK & Johnston, NC (1962) The isolation and synthesis of queen substance, 9-oxodectrans -2-enoic acid, a honeybee pheromone. Proc R Soc Lond B Biol Sci, 155, 417-32. [http://dx.doi.org/10.1098/rspb.1962.0009] Camazine, S, Crailsheim, K, Hrassnigg, N, Robinson, GE, Leonhard, B & Kropiunigg, H (1998) Protein trophallaxis and the regulation of pollen foraging by honey bees (Apis mellifera L.). Apidologie (Celle), 29, 113-26. [http://dx.doi.org/10.1051/apido:19980107] Capella, ICS & Hartfelder, K (1998) Juvenile hormone effect on DNA synthesis and apoptosis in castespecific differentiation of the larval honey bee (Apis mellifera L.) ovary. J Insect Physiol, 44, 385-91. [http://dx.doi.org/10.1016/S0022-1910(98)00027-4] [PMID: 12770156] Schmidt Capella, IC & Hartfelder, K (2002) Juvenile-hormone-dependent interaction of actin and spectrin is crucial for polymorphic differentiation of the larval honey bee ovary. Cell Tissue Res, 307, 265-72. [http://dx.doi.org/10.1007/s00441-001-0490-y] [PMID: 11845333] Chaud-Netto, J & Bueno, OC (1979) The number of ovarioles in workers of Apis mellifera adansonii and Apis mellifera ligustica: a comparative study. J Apic Res, 18, 260-3. [http://dx.doi.org/10.1080/00218839.1979.11099980] Colonello, NA & Hartfelder, K (2005) She’s my girl - male accessory gland products and their function in the reproductive biology of social bees. Apidologie (Celle), 36, 231-44. [http://dx.doi.org/10.1051/apido:2005012] Conte, YL & Hefetz, A (2008) Primer pheromones in social hymenoptera. Annu Rev Entomol, 53, 523-42.

Queen Pheromones

Polyandrous Queen Honey Bee 189

[http://dx.doi.org/10.1146/annurev.ento.52.110405.091434] [PMID: 17877458] Crewe, RM & Velthuis, HHW (1980) False queens: A consequence of mandibular gland signals in worker honeybees. Naturwissenschaften, 67, 467-9. [http://dx.doi.org/10.1007/BF00405650] de Groot, AP & Voogd, S (1954) On the ovary development in queenless worker bees (Apis mellifica L.). Experientia, 10, 384-5. [http://dx.doi.org/10.1007/BF02160554] [PMID: 13210347] Dearden, PK, Wilson, MJ, Sablan, L, Osborne, PW, Havler, M, McNaughton, E, Kimura, K, Milshina, NV, Hasselmann, M, Gempe, T, Schioett, M, Brown, SJ, Elsik, CG, Holland, PWH, Kadowaki, T & Beye, M (2006) Patterns of conservation and change in honey bee developmental genes. Genome Res, 16, 1376-84. [http://dx.doi.org/10.1101/gr.5108606] [PMID: 17065607] Dedej, S, Hartfelder, K, Aumeier, P, Rosenkranz, P & Engels, W (1998) Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee ( Apis mellifera carnica ). J Apic Res, 37, 183-90. [http://dx.doi.org/10.1080/00218839.1998.11100970] Démares, FJ, Yusuf, AA, Nicolson, SW & Pirk, CWW (2017) Effect of a brood pheromone on survival and nutrient intake of African honey bees (Apis mellifera scutellata) under controlled conditions. J Chem Ecol, 43, 443-50. [http://dx.doi.org/10.1007/s10886-017-0840-1] [PMID: 28455796] Eckert, JE (1934) Studies in the number of ovarioles in queen honeybees in relation to body size. J Econ Entomol, 27, 629-35. [http://dx.doi.org/10.1093/jee/27.3.629] Evans, JD & Wheeler, DE (2001) Gene expression and the evolution of insect polyphenisms. BioEssays, 23, 62-8. [http://dx.doi.org/10.1002/1521-1878(200101)23:13.0.CO;2-7] [PMID: 11135310] Foley, K & Cooley, L (1998) Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development, 125, 1075-82. [http://dx.doi.org/10.1242/dev.125.6.1075] [PMID: 9463354] Fyg, W (1964) Anomalies and diseases of the queen honey bee. Annu Rev Entomol, 9, 207-24. [http://dx.doi.org/10.1146/annurev.en.09.010164.001231] Genç, M & Aslan, A (1999) Determination of trans-10-hydroxy-2-decenoic acid content in pure royal jelly and royal jelly products by column liquid chromatography. J Chromatogr A, 839, 265-8. [http://dx.doi.org/10.1016/S0021-9673(99)00151-X] [PMID: 10327631] Guilford, T & Dawkins, MS (1991) Receiver psychology and the evolution of animal signals. Anim Behav, 42, 1-14. [http://dx.doi.org/10.1016/S0003-3472(05)80600-1] Gutiérrez-Aguilar, M & Baines, CP (2013) Physiological and pathological roles of mitochondrial SLC25 carriers. Biochem J, 454, 371-86. [http://dx.doi.org/10.1042/BJ20121753] [PMID: 23988125] Härtel, S, Neumann, P, Raassen, FS, Moritz, RFA & Hepburn, HR (2006) Social parasitism by Cape honeybee workers in colonies of their own subspecies (Apis mellifera capensis Esch.). Insectes Soc, 53, 18393. [http://dx.doi.org/10.1007/s00040-005-0857-2] Hartfelder, K & Steinbrück, G (1997) Germ cell cluster formation and cell death are alternatives in castespecific differentiation of the larval honey bee ovary. Invertebr Reprod Dev, 31, 237-50. [http://dx.doi.org/10.1080/07924259.1997.9672582] Hartfelder, K (1993) Structure and function of the prothoracic gland in honey bee ( Apis mellifera L.) development. Invertebr Reprod Dev, 23, 59-74.

190 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1080/07924259.1993.9672294] Hartfelder, K, Guidugli-Lazzarini, KR, Cervoni, MS, Santos, DE & Humann, FC (2015) Old Threads Make New Tapestry—Rewiring of Signalling Pathways Underlies Caste Phenotypic Plasticity in the Honey Bee, Apis mellifera L. Adv Insect Physiol, 48, 1-36. [http://dx.doi.org/10.1016/bs.aiip.2014.12.001] Hasselmann, M, Gempe, T, Schiøtt, M, Nunes-Silva, CG, Otte, M & Beye, M (2008) Evidence for the evolutionary nascence of a novel sex determination pathway in honeybees. Nature, 454, 519-22. [http://dx.doi.org/10.1038/nature07052] [PMID: 18594516] Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Hemmling, C, Koeniger, N & Ruttner, F (1979) Quantitative Bestimmung der 9-oxodecensäure im Lebenszyklus der Kapbiene (Apis mellifera capensis Escholtz). Apidologie (Celle), 10, 227-40. [http://dx.doi.org/10.1051/apido:19790302] Hepburn, HR (1992) Pheromonal and ovarial development covary in cape worker honeybees,apis meffera capensis. Naturwissenschaften, 79, 523-4. [http://dx.doi.org/10.1007/BF01135775] Holman, L (2018) Queen pheromones and reproductive division of labour: a meta-analysis. Behav Ecol, 29, 1199-209. Holman, L, Helanterä, H, Trontti, K & Mikheyev, AS (2019) Comparative transcriptomics of social insect queen pheromones. Nat Commun, 10, 1593. [http://dx.doi.org/10.1038/s41467-019-09567-2] [PMID: 30962449] Holman, L, van Zweden, JS, Oliveira, RC, van Oystaeyen, A & Wenseleers, T (2017) Conserved queen pheromones in bumblebees: a reply to Amsalem et al. PeerJ, 5, e3332. [http://dx.doi.org/10.7717/peerj.3332] [PMID: 28533978] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Jackson, JT, Tarpy, DR & Fahrbach, SE (2011) Histological estimates of ovariole number in honey bee queens, Apis mellifera, reveal lack of correlation with other queen quality measures. J Insect Sci, 11, 1-11. [http://dx.doi.org/10.1673/031.011.8201] [PMID: 21870968] Jarosch, A, Stolle, E, Crewe, RM & Moritz, RFA (2011) Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers ( Apis mellifera ). Proc Natl Acad Sci USA, 108, 15282-7. [http://dx.doi.org/10.1073/pnas.1109343108] [PMID: 21896748] Kaatz, HH, Hildebrandt, H & Engels, W (1992) Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees. J Comp Physiol B, 162, 588-92. [http://dx.doi.org/10.1007/BF00296638] Kamakura, M (2011) Royalactin induces queen differentiation in honeybees. Nature, 473, 478-83. [http://dx.doi.org/10.1038/nature10093] [PMID: 21516106] Karlson, P & Lüscher, M (1959) Pheromones’: a new term for a class of biologically active substances. Nature, 183, 55-6. [http://dx.doi.org/10.1038/183055a0] [PMID: 13622694] Katzav-Gozansky, T, Boulay, R, Soroker, V & Hefetz, A (2004) Queen–signal modulation of worker pheromonal composition in honeybees. Proc Biol Sci, 271, 2065-9. [http://dx.doi.org/10.1098/rspb.2004.2839] [PMID: 15451697] Katzav-Gozansky, T, Soroker, V, Hefetz, A, Cojocaru, M, Erdmann, DH & Francke, W (1997) Plasticity of

Queen Pheromones

Polyandrous Queen Honey Bee 191

caste-specific Dufour’s gland secretion in the honey bee (Apis mellifera L.). Naturwissenschaften, 84, 238-41. [http://dx.doi.org/10.1007/s001140050386] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Keller, L & Nonacs, P (1993) The role of queen pheromones in social insects: queen control or queen signal? Anim Behav, 45, 787-94. [http://dx.doi.org/10.1006/anbe.1993.1092] Kocher, SD, Ayroles, JF, Stone, EA & Grozinger, CM (2010) Individual variation in pheromone response correlates with reproductive traits and brain gene expression in worker honey bees. PLoS One, 5, e9116. [http://dx.doi.org/10.1371/journal.pone.0009116] [PMID: 20161742] Kubisova, S (1978) Effects of larval extracts on the development of ovaries in caged worker honeybees. Acta Entomol Bohemoslov, 75, 9-14. Kucharski, R, Maleszka, J, Foret, S & Maleszka, R (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science, 319, 1827-30. [http://dx.doi.org/10.1126/science.1153069] [PMID: 18339900] Lago, DC, Humann, FC, Barchuk, AR, Abraham, KJ & Hartfelder, K (2016) Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development. Insect Biochem Mol Biol, 79, 1-12. [http://dx.doi.org/10.1016/j.ibmb.2016.10.001] [PMID: 27720811] Lattorff, HMG, Moritz, RFA, Crewe, RM & Solignac, M (2007) Control of reproductive dominance by the thelytoky gene in honeybees. Biol Lett, 3, 292-5. [http://dx.doi.org/10.1098/rsbl.2007.0083] [PMID: 17412668] Leimar, O, Hartfelder, K, Laubichler, MD & Page, RE, Jr (2012) Development and evolution of caste dimorphism in honeybees - a modeling approach. Ecol Evol, 2, 3098-109. [http://dx.doi.org/10.1002/ece3.414] [PMID: 23301175] Lin, H (1999) Regulation of worker honey bee reproduction Apis mellifera L(Hymenoptera: Apidae)Simon Fraser University. Lin, H, Winston, ML, Haunerland, NH & Slessor, KN (1999) Influence of age and population size on ovarian development, and of trophallaxis on ovarian development and vitellogenin titres of queenless worker honey bee (Hymenoptera: Apidae). Can Entomol, 131, 695-706. [http://dx.doi.org/10.4039/Ent131695-5] Linksvayer, TA, Kaftanoglu, O, Akyol, E, Blatch, S, Amdam, GV & Page, RE, Jr (2011) Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism. J Evol Biol, 24, 1939-48. [http://dx.doi.org/10.1111/j.1420-9101.2011.02331.x] [PMID: 21696476] Mahajan-Miklos, S & Cooley, L (1994) Intercellular cytoplasm transport during Drosophila oogenesis. Dev Biol, 165, 336-51. [http://dx.doi.org/10.1006/dbio.1994.1257] [PMID: 7958404] Malka, O, Karunker, I, Yeheskel, A, Morin, S & Hefetz, A (2009) The gene road to royalty - differential expression of hydroxylating genes in the mandibular glands of the honeybee. FEBS J, 276, 5481-90. [http://dx.doi.org/10.1111/j.1742-4658.2009.07232.x] [PMID: 19702829] Malka, O, Niño, EL, Grozinger, CM & Hefetz, A (2014) Genomic analysis of the interactions between social environment and social communication systems in honey bees (Apis mellifera). Insect Biochem Mol Biol, 47, 36-45. [http://dx.doi.org/10.1016/j.ibmb.2014.01.001] [PMID: 24486775] Malka, O, Shnieor, S, Hefetz, A & Katzav-Gozansky, T (2006) Reversible royalty in worker honeybees (Apis mellifera) under the queen influence. Behav Ecol Sociobiol, 61, 465-73.

192 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1007/s00265-006-0274-1] Martin, SJ, Beekman, M, Wossler, TC & Ratnieks, FLW (2002) Parasitic Cape honeybee workers, Apis mellifera capensis, evade policing. Nature, 415, 163-5. [http://dx.doi.org/10.1038/415163a] [PMID: 11805832] Melathopoulos, AP, Winston, ML, Pettis, JS & Pankiw, T (1996) Effect of queen mandibular pheromone on initiation and maintenance of queen cells in the honey bee (Apis mellifera L.). Can Entomol, 128, 263-72. [http://dx.doi.org/10.4039/Ent128263-2] Michener, CD (1969) Comparative social behaviour of bees. Annu Rev Entomol, 14, 299-342. [http://dx.doi.org/10.1146/annurev.en.14.010169.001503] Mohammedi, A, Paris, A, Crauser, D & Le Conte, Y (1998) Effect of aliphatic esters on ovary development of queenless bees (Apis mellifera L.). Naturwissenschaften, 85, 455-8. [http://dx.doi.org/10.1007/s001140050531] Moritz, RFA & Hillesheim, E (1985) Inheritance of dominance in honeybees (Apis mellifera capensis Esch.). Behav Ecol Sociobiol, 17, 87-9. [http://dx.doi.org/10.1007/BF00299434] Moritz, RF, Lattorff, HMG & Crewe, RM (2004) Honeybee workers (Apis mellifera capensis) compete for producing queen-like pheromone signals. Proc Biol Sci, 271 (Suppl. 3), S98-S100. [PMID: 15101431] Moritz, RFA, Simon, UE & Crewe, RM (2000) Pheromonal contest between honeybee workers (Apis mellifera capensis). Naturwissenschaften, 87, 395-7. [http://dx.doi.org/10.1007/s001140050748] [PMID: 11091962] Mumoki, FN, Pirk, CWW, Yusuf, AA & Crewe, RM (2018) Reproductive parasitism by worker honey bees suppressed by queens through regulation of worker mandibular secretions. Sci Rep, 8, 7701. [http://dx.doi.org/10.1038/s41598-018-26060-w] [PMID: 29799016] Naumann, K, Winston, ML, Slessor, KN, Prestwich, GD & Webster, FX (1991) Production and transmission of honey bee queen (Apis mellifera L.) mandibular gland pheromone. Behav Ecol Sociobiol, 29, 321-32. [http://dx.doi.org/10.1007/BF00165956] Neumann, P & Hepburn, R (2002) Behavioural basis for social parasitism of Cape honeybees ( Apis mellifera capensis ). Apidologie (Celle), 33, 165-92. [http://dx.doi.org/10.1051/apido:2002008] Nunes, TM, Oldroyd, BP, Elias, LG, Mateus, S, Turatti, IC & Lopes, NP (2017) Evolution of queen cuticular hydrocarbons and worker reproduction in stingless bees. Nat Ecol Evol, 1, 0185. [http://dx.doi.org/10.1038/s41559-017-0185] Oi, CA, van Zweden, JS, Oliveira, RC, Van Oystaeyen, A, Nascimento, FS & Wenseleers, T (2015) The origin and evolution of social insect queen pheromones: Novel hypotheses and outstanding problems. BioEssays, 37, 808-21. [http://dx.doi.org/10.1002/bies.201400180] [PMID: 25916998] Okosun, OO, Pirk, CWW, Crewe, RM & Yusuf, AA (2017) Glandular sources of pheromones used to control host workers (Apis mellifera scutellata) by socially parasitic workers of Apis mellifera capensis. J Insect Physiol, 102, 42-9. [http://dx.doi.org/10.1016/j.jinsphys.2017.09.001] [PMID: 28889990] Okosun, OO, Yusuf, AA, Crewe, RM & Pirk, CWW (2015) Effects of age and reproductive status on tergal gland secretions in queenless honey bee workers, Apis mellifera scutellata and A. m. capensis. J Chem Ecol, 41, 896-903. [http://dx.doi.org/10.1007/s10886-015-0630-6] [PMID: 26384295] Ronai, I, Oldroyd, BP & Vergoz, V (2016) Queen pheromone regulates programmed cell death in the honey bee worker ovary. Insect Mol Biol, 25, 646-52.

Queen Pheromones

Polyandrous Queen Honey Bee 193

[http://dx.doi.org/10.1111/imb.12250] [PMID: 27321063] Oldroyd, BP, Smolenski, AJ, Cornuet, JM & Crozler, RH (1994) Anarchy in the beehive. Nature, 371, 749. [http://dx.doi.org/10.1038/371749a0] Pankiw, T, Huang, Z, Winston, ML & Robinson, GE (1998) Queen mandibular gland pheromone influences worker honey bee (Apis mellifera L.) foraging ontogeny and juvenile hormone titers. J Insect Physiol, 44, 685-92. [http://dx.doi.org/10.1016/S0022-1910(98)00040-7] [PMID: 12769952] Pankiw, T, Winston, ML, Plettner, E, Slessor, KN, Pettis, JS & Taylor, OR (1996) Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.). J Chem Ecol, 22, 605-15. [http://dx.doi.org/10.1007/BF02033573] [PMID: 24227572] Pernal, SF & Currie, RW (2000) Pollen quality of fresh and 1-year-old single pollen diets for worker honey bees ( Apis mellifera L.). Apidologie (Celle), 31, 387-409. [http://dx.doi.org/10.1051/apido:2000130] Peso, M, Elgar, MA & Barron, AB (2015) Pheromonal control: reconciling physiological mechanism with signalling theory. Biol Rev Camb Philos Soc, 90, 542-59. [http://dx.doi.org/10.1111/brv.12123] [PMID: 24925630] Plettner, E, Otis, GW, Wimalaratne, PDC, Winston, ML, Slessor, KN, Pankiw, T & Punchihewa, PWK (1997) Species-and caste-determined mandibular gland signals in honeybees (Apis). J Chem Ecol, 23, 36377. [http://dx.doi.org/10.1023/B:JOEC.0000006365.20996.a2] Plettner, E, Slessor, KN, Winston, ML & Oliver, JE (1996) Caste-selective pheromone biosynthesis in honeybees. Science, 271, 1851-3. [http://dx.doi.org/10.1126/science.271.5257.1851] Rachinsky, A & Hartfelder, K (1990) Corpora allata activity, a prime regulating element for caste-specific juvenile hormone titre in honey bee larvae (Apis mellifera carnica). J Insect Physiol, 36, 189-94. [http://dx.doi.org/10.1016/0022-1910(90)90121-U] Rachinsky, A, Strambi, C, Strambi, A & Hartfelder, K (1990) Caste and metamorphosis: Hemolymph titers of juvenile hormone and ecdysteroids in last instar honeybee larvae. Gen Comp Endocrinol, 79, 31-8. [http://dx.doi.org/10.1016/0016-6480(90)90085-Z] [PMID: 2354779] Ratnieks, FW (1993) Egg-laying, egg-removal, and ovary development by workers in queenright honey bee colonies. Behav Ecol Sociobiol, 32, 191-8. [http://dx.doi.org/10.1007/BF00173777] Reece, SL (2002) A scientific note on the ovarial and pheromonal development of drifted and non-drifted Cape honeybee workers ( Apis mellifera capensis ). Apidologie (Celle), 33, 213-4. [http://dx.doi.org/10.1051/apido:2002007] Reginato, R & Da Cruz-Landim, C (2002) Morphological characterization of cell death during the ovary differentiation in worker honey bee. Cell Biol Int, 26, 243-51. [http://dx.doi.org/10.1006/cbir.2001.0839] [PMID: 11991652] Rembold, H (1987) Caste specific modulation of juvenile hormone titers in Apis mellifera. Insect Biochem, 17, 1003-6. [http://dx.doi.org/10.1016/0020-1790(87)90110-7] Ronai, I, Barton, DA, Oldroyd, BP & Vergoz, V (2015) Regulation of oogenesis in honey bee workers via programed cell death. J Insect Physiol, 81, 36-41. [http://dx.doi.org/10.1016/j.jinsphys.2015.06.014] [PMID: 26119324] Ronai, I, Oldroyd, BP & Vergoz, V (2016) Queen pheromone regulates programmed cell death in the honey bee worker ovary. Insect Mol Biol, 25, 646-52. [http://dx.doi.org/10.1111/imb.12250] [PMID: 27321063]

194 Polyandrous Queen Honey Bee

Lovleen Marwaha

Ronai, I, Oldroyd, BP, Barton, DA, Cabanes, G, Lim, J & Vergoz, V (2016) Anarchy is a molecular signature of worker sterility in the honey bee. Mol Biol Evol, 33, 134-42. [http://dx.doi.org/10.1093/molbev/msv202] [PMID: 26416979] Ruttner, F & Hesse, B (1981) Rassenspezifische unterschiede in ovarentwicklung und eiablage von weisellosen arbeiterinnen der honigbiene Apis mellifera L. Apidologie (Celle), 12, 159-83. [http://dx.doi.org/10.1051/apido:19810206] Saiovici, M (1983) 9-Oxodecenoic acid and dominance in honeybees. J Apic Res, 22, 27-32. [http://dx.doi.org/10.1080/00218839.1983.11100556] Schäfer, MO, Dietemann, V, Pirk, CWW, Neumann, P, Crewe, RM, Hepburn, HR, Tautz, J & Crailsheim, K (2006) Individual versus social pathway to honeybee worker reproduction (Apis mellifera): pollen or jelly as protein source for oogenesis? J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 192, 761-8. [http://dx.doi.org/10.1007/s00359-006-0112-y] [PMID: 16508763] Schäfer, MO, Genersch, E, Fünfhaus, A, Poppinga, L, Formella, N, Bettin, B & Karger, A (2014) Rapid identification of differentially virulent genotypes of Paenibacillus larvae, the causative organism of American foulbrood of honey bees, by whole cell MALDI-TOF mass spectrometry. Vet Microbiol, 170, 291-7. [http://dx.doi.org/10.1016/j.vetmic.2014.02.006] [PMID: 24613082] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Smith, AA & Liebig, J (2017) The evolution of cuticular fertility signals in eusocial insects. Curr Opin Insect Sci, 22, 79-84. [http://dx.doi.org/10.1016/j.cois.2017.05.017] [PMID: 28805643] Smith, AA, Hölldober, B & Liebig, J (2009) Cuticular hydrocarbons reliably identify cheaters and allow enforcement of altruism in a social insect. Curr Biol, 19, 78-81. [http://dx.doi.org/10.1016/j.cub.2008.11.059] [PMID: 19135369] Smith, AA, Millar, JG & Suarez, AV (2016) Comparative analysis of fertility signals and sex-specific cuticular chemical profiles of Odontomachus trap-jaw ants. J Exp Biol, 219, 419-30. [http://dx.doi.org/10.1242/jeb.128850] [PMID: 26847561] Snodgrass, RE (2018) Anatomy of the honey beeCornell University Press. Sole, CL, Kryger, P, Hefetz, A, Katzav-Gozansky, T & Crewe, RM (2002) Mimicry of queen Dufour’s gland secretions by workers of Apis mellifera scutellata and A. m. capensis. Naturwissenschaften, 89, 561-4. [http://dx.doi.org/10.1007/s00114-002-0370-7] [PMID: 12536278] Strauss, K, Scharpenberg, H, Crewe, RM, Glahn, F, Foth, H & Moritz, RFA (2008) The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent? Behav Ecol Sociobiol, 62, 1523-31. [http://dx.doi.org/10.1007/s00265-008-0581-9] Tan, K, Wang, ZW, Li, H, Yang, MX, Pirk, CWW, Hepburn, HR & Radloff, SE (2010) Responses of queenright and queenless workers of Apis cerana to 9-keto-2(E)-decenoic acid, a pheromonal constituent of the mandibular gland. J Chem Ecol, 36, 966-8. [http://dx.doi.org/10.1007/s10886-010-9833-z] [PMID: 20644984] Tanaka, ED & Hartfelder, K (2004) The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropod Struct Dev, 33, 431-42. [http://dx.doi.org/10.1016/j.asd.2004.06.006] [PMID: 18089049] Thuller, RH, Malaspina, O, Bueno, OC & Netto, JC (1996) The number of ovarioles in workers descendent

Queen Pheromones

Polyandrous Queen Honey Bee 195

from crossings between Africanized and Italian honeybees (Apis mellifera L.): comparing stock, inbred and F1 colonies. An Soc Entomol Bras, 27, 237-43. [http://dx.doi.org/10.1590/S0301-80591998000200010] Traynor, KS, Le Conte, Y & Page, RE, Jr (2014) Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers. Behav Ecol Sociobiol, 68, 2059-73. [http://dx.doi.org/10.1007/s00265-014-1811-y] [PMID: 25395721] van Erp, A (1960) Mode of action of the inhibitory substance of the honeybee queen. Insectes Soc, 7, 207-11. [http://dx.doi.org/10.1007/BF02224491] vanEngelsdorp, D, Hayes, J, Jr, Underwood, RM & Pettis, JS (2010) A survey of honey bee colony losses in the United States, fall 2008 to spring 2009. J Apic Res, 49, 7-14. [http://dx.doi.org/10.3896/IBRA.1.49.1.03] Velthuis, HHW & van Es, J (1964) Some functional aspects of the mandibular glands of the queen honeybee. J Apic Res, 3, 11-6. [http://dx.doi.org/10.1080/00218839.1964.11100076] Velthuis, HHW (1970) Ovarian development in Apis mellifera worker bees. Entomol Exp Appl, 13, 377-94. a [http://dx.doi.org/10.1111/j.1570-7458.1970.tb00122.x] Velthuis, HHW (1970) Queen substances from the abdomen of the honey bee queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 70, 210-21. b [http://dx.doi.org/10.1007/BF00297717] Vergoz, V, Schreurs, HA & Mercer, AR (2007) Queen pheromone blocks aversive learning in young worker bees. Science, 317, 384-6. [http://dx.doi.org/10.1126/science.1142448] [PMID: 17641204] Verheijen-Voogd, C (1959) How worker bees perceive the presence of their queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 41, 527-82. [http://dx.doi.org/10.1007/BF00299267] Visscher, PK (1989) A quantitative study of worker reproduction in honey bee colonies. Behav Ecol Sociobiol, 25, 247-54. [http://dx.doi.org/10.1007/BF00300050] Visscher, PK (1996) Reproductive conflict in honey bees: a stalemate of worker egg-laying and policing. Behav Ecol Sociobiol, 39, 237-44. [http://dx.doi.org/10.1007/s002650050286] Willis, LG, Winston, ML & Slessor, KN (1990) Queen honey bee mandibular pheromone does not affect worker ovary development. Can Entomol, 122, 1093-9. [http://dx.doi.org/10.4039/Ent1221093-11] Lin, H & Winston, ML (1998) The role of nutrition and temperature in the ovarian development of the worker honey bee (Apis mellifera). Can Entomol, 130, 883-91. [http://dx.doi.org/10.4039/Ent130883-6] Winston, ML & Slessor, KN (1998) Honey bee primer pheromones and colony organization: gaps in our knowledge. Apidologie (Celle), 29, 81-95. [http://dx.doi.org/10.1051/apido:19980105] Winston, ML, Higo, HA & Slessor, KN (1990) Effect of various dosages of queen mandibular gland pheromone on the inhibition of queen rearing in the honey bee (Hymenoptera: Apidae). Ann Entomol Soc Am, 83, 234-8. [http://dx.doi.org/10.1093/aesa/83.2.234] Wojciechowski, M, Lowe, R, Maleszka, J, Conn, D, Maleszka, R & Hurd, PJ (2018) Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development. Genome Res, 28, 1532-42.

196 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1101/gr.236497.118] [PMID: 30135090] Wossler, TC & Crewe, RM (1999) Honeybee queen tergal gland secretion affects ovarian development in caged workers. Apidologie (Celle), 30, 311-20. [http://dx.doi.org/10.1051/apido:19990407] Wu, Y, Zheng, H, Corona, M, Pirk, C, Meng, F, Zheng, Y & Hu, F (2017) Comparative transcriptome analysis on the synthesis pathway of honey bee (Apis mellifera) mandibular gland secretions. Sci Rep, 7, 4530. [http://dx.doi.org/10.1038/s41598-017-04879-z] [PMID: 28674395] Yusuf, AA, Pirk, CWW & Crewe, RM (2015) Mandibular gland pheromone contents in workers and queens of Apis mellifera adansonii. Apidologie (Celle), 46, 559-72. [http://dx.doi.org/10.1007/s13592-014-0346-6] Zander, E, Loschel, F & Meier, K (1916) Zander E, Loschel F, Meier K (1916) Die Ausbildung des Geschlechtesbei der Honigbiene (Apis mellifera L.). Z Angew Entomo l 3, 1-74. Zheng, HQ, Dietemann, V, Crewe, RM, Hepburn, R, Hu, FL, Yang, MX & Pirk, CWW (2010) Pheromonal predisposition to social parasitism in the honeybee Apis mellifera capensis. Behav Ecol, 21, 1221-6. [http://dx.doi.org/10.1093/beheco/arq131]

Polyandrous Queen Honey Bee, 2022, 197-210

197

CHAPTER 10

Genetic Influence on Ovarian Development Plasticity In Apis mellifera (Hymenoptera: Apidae) Abstract: Variant genomic expression and proteomics ultimately induce plasticity in honey bees' ovarian development. The expression of the same genomic content in female castes is influenced by; the compositional difference between royal jelly and workers jelly, queen pheromones, hormones associated with metamorphosis and environmental cues. Various concerned genetic elements with diversified transcriptomics include Kr-h1,hsp, Cut-like protein gene, Ftz-F1, anti-apoptotic buffy, Incov, oat, Apaf-1, ark, Incov2, MAPK, FoxO, mTOR, Hedgehog, TGF-β, Wnt, Hippo, Toll, Imd, H3K4me3, H3K27ac, H3K36me3, etc. The specific genetic elements are responsible for the structural and functional activation of the queen ovary. In workers, the same genetic factors act as the primary criterion for induction Programme Cell Death (PCD). This chapter attributes to enlisting concerned genetic elements which serve as an inducer for divergent ovarian development. The next chapter describes the details of PCD in workers' ovaries.

Keywords: Associated genetic elements and queen, Ovarian developmental plasticity. 10.1. INTRODUCTION Differential larval diets, queen pheromones, brood-secreted volatiles, nestmate chemical emission and environmental factors influence the genetic expression in the female caste of honey bees. Eventually, genetic expression induces diversification in the ovarian development of female castes. Literature survey highlights variation in genetic expression in queens. It works as Severson et al., 1989 had generalized gene expression pathways in queens and workers during the pre-pupae and pupal phase of development. Similarly, Hartfelder et al. 1989 discriminated the gene expression pattern in ovaries of early fifth instar larvae and concluded that specific genetic variability is a foundation stone for the remarkable difference in queen and workers in terms of morphology, anatomy, development, physiology, reproduction, immunity, life span, behaviour and role in the colony(Cale, 1963; Avetisyan et al., 1967; Corbella and Goncalves, 1982; Casagrande-Jaloretto et al., 1984; Arnold et al., 1995; Crozier and Pamilo, 1996; Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

198 Polyandrous Queen Honey Bee

Lovleen Marwaha

Berg et al., 1997 AL-Abbadi, 2005; Al-Qarni et al., 2005; Cox-Foster et al., 2007; Akyol et al., 2008; Alghamdi et al., 2012; Cornman et al., 2012; Dainat et al., 2012; Abdulaziz et al., 2013) 10.2. DIFFERENTIAL GENE EXPRESSION IN QUEEN AND WORKERS According to Laidlaw, 1992, the food provided immediately before the beginning of worker-queen developmental pathway differentiation acts as the primary criterion for different molecular events at the genetic level. Evas and Wheeler 1999 described genomics and proteomics of seven differentiated loci belonging to five different groups, and among them, two are particularly promising as potential regulators of caste differentiation. One gene possesses homologies to widespread protein binding with lipids and hydrophobic ligands like retinoic acid. The second gene possesses similarities with the DNA binding domain in the Ets family of the transcription factor. Five genes among seven gene products express exclusively in worker larvae. In queen larva, four of these genes are entirely silent. Further, the protein product of six differentially expressed genes exhibited significant similarity to proteins in the GeneBank database concerning the amino acid sequence. Sequence similarity can suggest a possible functional correlation, as proteins can diverge into the new structure and new functions. In addition, specific proteins show similarity with transcription factors involved in multiple signalling pathways or with oxidoreductase. Further, they concluded that the considered proteins were similar to the following proteins. Storage Protein: Two caste differential specific proteins of honey bees exhibited structural similarity with hexameric storage proteins, present in haemolymph and fat bodies of insects (Evas and Wheeler, 1999). The concerned protein possesses the exact evolutionary origin of hemocyanin used for oxygen transport in arthropods (Burmester and Scheller, 1999) and the protein present in the hemolymph of fruit flies (Danty et al., 1998). During honey bee development, two hexameric protein proteins persisted longer in adult queens than in worker honey bees. Crystallin and Other Crystallins: Further, they reported structural similarity of one protein with the Crystallin protein in rabbits, having a catalytic function in metabolic processes and mechanical role in vertebrate eye (Tomarev and Piatigorsky, 1996).

Genetic Influence

Polyandrous Queen Honey Bee 199

Fatty-Acid Binding Proteins. Other proteins reported being similar to fatty acid binding proteins composed of 130 amino acids and are universally present in vertebrates, which bind with retinoic acid. Retinoids bear structural similarities with Juvenile hormones and steroid hormones. 10.3. BRIEF DESCRIPTION OF VARIOUS GENETIC REGULATORS OF PCD Differential diets during the larval phase induced differential gene expression, resulting in two specific caste development types in honey bees, i.e. queen and worker honey bees. In honey bee workers, programmed cell death occurs within ovaries, resulting from the expression of different genes. Further, the social environment of the colony affects gene expression in worker honey bees (Beggs and Mercer, 2009). The description of various genes which influence the process is as per the following sections (Fig. 10a - c)

Fig. (10a). Differential activation of different genetic elements during dimorphic development of female caste. In queen larvae, higher concentrations of juvenile hormone and ecdysone hormone influence the expression of certain genetic factors.

10.3.1. Anarchy Gene Anarchy or Buffy genes are responsible for PCD in worker honey bee ovaries. In the QL colony, the expression of the Anarchy gene decreases in worker honey bees between the age of 2 and 15 days old by two-fold. In contrast, in the QR

200 Polyandrous Queen Honey Bee

Lovleen Marwaha

colony, the expression remains continuous, which indicates that degenerative changes remain continuous in worker honey bees' ovaries. Therefore, the expression of Anarchy is per the presence and absence of the queen. Further, Queen pheromones regulate the anarchy gene expression in workers' fat bodies and brains (Grozingeret al., 2003). The expression of Anarchy is more stable in queen than in worker honey bees. Hence concerned gene is considered to control the activation of worker honey bee ovaries (Thompson et al., 2013; Ronai et al., 2016). Anarchy, Pdk1, S6k, and Ulk3 show differential expression in worker and queen honey bee ovaries. Anarchy gene is present in the OvA3 region. It belongs to the mitochondrial solute carrier protein family and located on the peroxisomal membrane (Thompson et al., 2006), whereas Pdk1, S6k, and Ulk3 are involved in cell growth, cell division and in ovarian development. Furthermore, Thompson et al., 2006 reported that the expression of Anarchy is associated with the termination of oogenesis. Induced inhibition of the expression of Anarchy in the ovary using RNA interference (RNAi) resulted in altered expression of Buffy gene, associated with programmed cell death. In addition, fluorescent in situ hybridization (MFish) analysis indicated Anarchy transcripts localized in degenerating oocytes within the ovary. Niu et al. 2014 studied the relationship between Anarchy gene expression and ovary activation. 10.3.2. Ark Gene Gene expression in queen and worker honey bee larva indicates a higher expression of the pro-apoptotic ark in worker larvae than in queen during the prepupal phase. Expression of the anti-apoptotic buffy gene increases earlier in queen ovaries at the spinning phase, whereas in workers ovaries, the specific gene expression increases during the prepupal phase. Further, the presence of ark RNA in fifth instar worker ovarioles had been detected in the PCD-prone germ cell region (Schmidt Capella and Hartfelder 1998). In contrast, buffy RNA occurs in the peritoneal sheath, which surrounds each ovariole in a queen larval ovary (Dallacqua and Bitondi 2014). 10.3.3. Miscellaneous Description of Various Genes Involved in Development According to Bourke 1988, Kin gene expression is correlated with the presence of a queen, and the concerned genetic element is associated with worker sterility.

Genetic Influence

Polyandrous Queen Honey Bee 201

Generally, genes related to ovarian activation are influenced by the presence and absence of a Queen in the colony. Ovary-specific transcriptome analyses revealed differentially expressed genes associated with PCD, including two long noncoding RNAs (Groot and Voogd,1954; Michener, 1969; Kubisova, 1978; Winston et al., 1990). In queen ovaries, overexpression of SDR gene takes, as detected by DDRT-PCR (Hepperle and Hartfelder 2001), and the concerned genetic element had been seen as strongly ecdysone responsive in worker ovaries (Guidugli et al. 2004).). Further, the AMP-binding enzyme gene is also differentially expressed in the caste-specific development of queens and workers (Barchuk et al., 2007). OvA3 gene, located on chromosome 1, is associated with ovary activation in Queen (Oxley et al., 2008). Further, Cardoen et al., 2011 reported that the same genetic element is involved in the deactivation of ovaries in worker honey bees. Gene Dnmt3 has a repressing effect on queen differentiation, and further, silencing specific genes results in queen development with fully formed ovaries(Kucharski et al., 2008). In worker honey bee larvae, there is an overexpressed gene, including the oat gene, which possesses ornithine -oxo-acid transaminase activity (Humann and Hartfelder 2011). lncov1 is over-expressed during Programmed Cell Death (PCD) in workers' ovaries, and its transcripts are localized in cytoplasmic granules (Humann et al., 2013). Specific diet affects the methylation of particular genetic elements in honey bees, which results in differential expression of genetic material. Further, they carried out high-resolution bisulphite sequencing of the complete genome from the brain of the queen and worker honey bees. More than 550 genes have been reported to be significantly differentially methylated between queen and worker honey bees. Further, a conclusive interpretation of the study states that methylation within part of the gene occurs at the splicing site, suggesting the splicing control of several versions of gene expression. Humann and Hartfelder 2011 employed the RDA approach to detect differential gene expression in honey bee ovaries of the fifth instar queen and worker larvae. As seen through suppression-subtractive hybridization libraries, the expression of 40 and 32 genes had been detected in queen and worker honey bee larvae, respectively. Further, they obtained two libraries by real-time PCR to confirm the differential expression of 16 genetic elements.

202 Polyandrous Queen Honey Bee

Lovleen Marwaha

Vitellogenin is expressed 11-fold lower in nonactivated ovaries than in activated ovaries (Cardoen et al., 2012). Further expression of S6k relates to a worker's reproductive state, but its expression is not sensitive to the queen's presence. Moreover, its expression is affected by the reproductive state of both workers and queens. Further, Dallacqua and Bitondi (2014) carried out a detailed analysis for castespecific degeneration of the number of ovarioles, and it was detected that responsible genes include apoptotic peptidase activating factor (Apaf) related killer gene (ark), pro-apoptotic mammalian Apaf-1, and buffy, encoded for B cell lymphoma 2 (Bcl-2) protein. Two specific genetic elements are associated with influencing role during PCD but are not responsible for PCD initiation (Hartfelder et al., 2018), and the specific genetic elements include long noncoding ovary 1 (lncov1) and long noncoding ovary 2 (lncov2), the homolog of fringe protein. Concerned proteins regulate the Notch signalling pathway, which is involved in the cell-cell interaction process in insect development, including honey bees (Duncan et al. 2016). Transcriptional analysis of the ovary in both queen and worker revealed a highly complex set of about 824 genes in both queen and worker during ovarian activation (Niu et al. 2014). Guo et al., 2015 reported differential expression of small RNAs in queen and worker honey bee larvae, which specify differential development of worker and queen bee larvae. Analysis of micro RNA indicates that queen and worker share a set of 19 differentiated expressed micro RNAs (Macedo et al. 2016). Guo et al., 2016 reported that 23 known miRNAs were significantly expressed in queen bee larvae. Santos et al., 2016 conducted a study to find out why three hypoxia genes overexpressed in worker larvae. Furthermore, they analyzed the difference in external oxygen level and mitochondrial number in the queen larval body. They carried out immunofluorescence and electron microscopy and concluded that queen larvae have a high mitochondrial density. Furthermore, they concluded that queen larvae possess higher TFB and TFB2 homologue, and the nutritional regulator ERR is overexpressed (Santos et al., 2016). IAP2 is also involved in the caste-specific differentiation of honey bees (Capella and Hartfelder, 1998; Guo et al., 2016). Yin et al., 2018, had carried out a study by transplanting worker larvae into a queen cell. They reported that developmental differences exist between queen and worker honey bees. Further, they said there are differential gene expressions in queen and worker honey bee larvae for insect hormone biosynthesis, longevity

Genetic Influence

Polyandrous Queen Honey Bee 203

regulation, dorsoventral axis formation, MAPK, FoxO, mTOR, Hedgehog, TGFβ, Wnt, Hippo, and Toll and Imd signalling pathways. mTOR pathway plays an essential role in cell differentiation into a queen or a worker. Therefore, any change in the specific path is sufficient to alter oogenesis (Patel et al., 2007; Kamakura, 2011; Mutti et al., 2011). It has been observed that in queen-destined larvae, if blocking the TOR pathway occurs, it results in the formation of the bee with worker morphology (Patel et al., 2007). Further, Ronai et al., 2016 suggested that the mTOR pathway is likely to be directly involved in regulating worker ovary activation. Similarly, Notch signalling regulates specific ovarian events of PCD and oogenesis. Notch signalling possesses a repressive effect on the early phase of oogenesis in the worker ovary(Duncan et al. 2016). Wojciechowski et al., 2019 observed caste-specific transcription of different genomic elements, including H3K4me3, H3K27ac, and H3K36me3. Further, they concluded that H3K27ac is the primary genetic element for chromatin modification. Finally, they demonstrated caste-specific enhancer elements in honey bees.

Fig. (10b). Diagrams depict the influences of queen pheromones on differential expression of genetic elements.

Further, queen pheromones suppress ovarian development in worker honey bees. It had been reported that in the absence of queen pheromone, there is a change in the expression of Major Royal Jelly Protein 3, which exhibits 89-fold higher expression in A. mellifera workers. Major Royal Jelly Protein 1 is secreted about

204 Polyandrous Queen Honey Bee

Lovleen Marwaha

33-fold in higher concentration than control. Royalactin of royal jelly induces queen-specific differentiation (Kamakura, 2011; Leimar et al., 2012). Specific proteins are essential for normal ovarian development (Holman et al., 2019). Other genes responsible for variant development include the doublesex(dsx), fruitless(fru), and loc552773 genes. In addition, caste-specific H3K4me3, H3K27ac, and H3K36me3 had been identified to express differentially during caste-specific development (Roth, 2019). 10.3.4. Hormonal Effect on Gene Expression The specific genetic element possesses differential gene expression, are influenced by various hormones, including Juvenile hormone (JH) and ecdysone (Fig. 10d).

Fig. (10c). Diagram depicts the influence of different diets on caste development in queen and worker honey bees.

Fig. (10d). Description of various hormones and proteins play an essential role in insect moulting.

Genetic Influence

Polyandrous Queen Honey Bee 205

Rachinsky and Hartfelder, 1990 observed a significant difference in ecdysteroid titer peak in spinning phase larvae and concluded specific hormone is responsible for the activation of many genes. Further, JH induced differential gene expression in larval queen and worker ovaries. Differential-display reverse transcription (DDRT)-PCR that in honey bee larvae, after the fourth instar stage, there is ecdysteroid-regulated gene expression of Ftz-F1 homolog and a Cut-like transcript. Fitz-F1 is involved in metamorphosis in honey bees, whereas Cut-like proteins are essential for transcription. The concentration of JH and ecdysteroid concentration influence the expression of heat shock proteins in queen larvae. Gene coded for juvenile hormone acid methyltransferase is differentially expressed in worker honey bees (Li et al., 2013). JH bound with dimeric JH receptor complex Met/Taiman and induces expression of Kruppel homolog-1 (Krh1) gene (Jindra et al., 2013; Hartfelder et al., 2015). Additionally, the JH hormone causes an increase in the expression of sdr and hsp90. SDR are a large and phylogenetically ancient family of NAD(P)(H)-dependent oxidoreductases and possess multiple functions (Feldlaufer et al., 1985; Guidugli et al., 2004; Lago et al., 2016). Few other studies also witness the effect of JH on PCD in worker larval ovary (Schmidt Capella and Hartfelder, 1998; Schmidt Capella and Hartfelder, 2002; Humann and Hartfelder, 2011; Mello et al., 2014). Lago et al., 2016 concluded that JH induces differential gene expression in larval and worker ovaries as assessed during dissection of ovaries of caste-specific larval stage. Further, they reported that JH induced up-regulation of dehydrogenase reductase and heat shock protein 90. In contrast, the expression of other genes, including hsp60 and hexamerin 70b, was significantly down-regulated. By RTqPCR, Lago et al., 2016 analyzed that apoLp-III, gpdh, hex70b, hsp60, hsp90, map k-3, oclp-1, 15-pgdh and sdr all had been reported to express differentially. CONCLUSION Genetic elements like Kr-h1,hsp, Cut-like protein gene, Ftz-F1, anti-apoptotic buffy, Incov, oat, Apaf-1, ark, Incov2, MAPK, FoxO, mTOR, Hedgehog, TGF-β, Wnt, Hippo, Toll, Imd, H3K4me3, H3K27ac, and H3K36me3 influence the developmental pattern in honey bees. Further, queen pheromones repress the overall developmental process in worker honey bees. REFERENCES Abdulaziz, SA, Balhareth, HM & Owayss, AA (2013) Queen morphometric and reproductive characters of Apis mellifera jemenitica, a native honey bee to Saudi Arabia. Bull Insectol, 66, 239-44. Akyol, E, Yeninar, H & Kaftanoglu, O (2008) The live weight of queen honey bees (Apis mellifera L.)

206 Polyandrous Queen Honey Bee

Lovleen Marwaha

predicts reproductive characteristics. J Kans Entomol Soc, 81, 92-100. [http://dx.doi.org/10.2317/JKES-705.13.1] (2005) Ahmed, A, Mohammad, A, Yehya, A & Nuru, A (2012) Morphometric diversity of indigenous Honeybees, Apis mellifera (Linnaeus, 1758), in Saudi Arabia. Zool Middle East, 57, 97-103. [http://dx.doi.org/10.1080/09397140.2012.10648968] Alqarni, AS, Balhareth, HM & Owayss, AA (2013) Queen morphometric and reproductive characters of Apis mellifera jemenitica, a native honey bee to Saudi Arabia. Bull Insectol, 66, 239-44. Al-Qarni, AS, Phelan, PL, Smith, BH & Cobey, S (2005) Tergal glandular secretions of naturally mated and instrumentally inseminated honeybee queens (Apis mellifera L.). Journal of King Saud University, 17, 12537. Alqarni, AS Alqarni, A.S. (1995). Morphometrical and biological studies on the native honeybee race, Apis mellifera L.; the Carniolan, A. m. carnica Pollmann and their F1 hybrid.- M.Sc. Thesis, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia.. Arnold, G, Le Conte, Y, Trouiller, J, Hervet, H & Chappe, B (1994) Avetisyan, GA, Rakhmatov, KK & Ziedov, M (1967) The influence of rearing periods on queen bees’ external and internal characteristics 277-84.The 21st International Apiculture Congress of Apimondia, College Park, Maryland, USA Barchuk, AR, Cristino, AS, Kucharski, R, Costa, LF, Simões, ZLP & Maleszka, R (2007) Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera. BMC Dev Biol, 7, 70. [http://dx.doi.org/10.1186/1471-213X-7-70] [PMID: 17577409] Beggs, KT & Mercer, AR (2009) Dopamine receptor activation by honey bee queen pheromone. Curr Biol, 19, 1206-9. [http://dx.doi.org/10.1016/j.cub.2009.05.051] [PMID: 19523830] Berg, S, Koeniger, N, Koeniger, G & Fuchs, S (1997) Body size and reproductive success of drones (Apis mellifera L). Apidologie (Celle), 28, 449-60. [http://dx.doi.org/10.1051/apido:19970611] Bourke, AF (1988) Worker reproduction in the higher eusocial Hymenoptera. Q Rev Biol, 63, 291-311. [http://dx.doi.org/10.1086/415930] Bruinsma, O, Kruijt, JP & van Dusseldorp, W (1981) Delay of the emergence of honey bee queens in response to tooting sounds. Burmester, T & Scheller, K (1999) Ligands and receptors: common theme in insect storage protein transport. Naturwissenschaften, 86, 468-74. [http://dx.doi.org/10.1007/s001140050656] [PMID: 10541655] Cale, GH (1963) The production of queens, package bees, and royal jelly, pp. 437-462. In: The hive and the honeybee (GROUT R. A., Ed.).- Dadant & Sons, Hamilton, Illinois, USA. Capella, ICS & Hartfelder, K (1998) Juvenile hormone effect on DNA synthesis and apoptosis in castespecific differentiation of the larval honey bee (Apis mellifera L.) ovary. J Insect Physiol, 44, 385-91. [http://dx.doi.org/10.1016/S0022-1910(98)00027-4] [PMID: 12770156] Schmidt Capella, IC & Hartfelder, K (2002) Juvenile-hormone-dependent interaction of actin and spectrin is crucial for polymorphic differentiation of the larval honey bee ovary. Cell Tissue Res, 307, 265-72. [http://dx.doi.org/10.1007/s00441-001-0490-y] [PMID: 11845333] Cardoen, D, Ernst, UR, Boerjan, B, Bogaerts, A, Formesyn, E, de Graaf, DC, Wenseleers, T, Schoofs, L & Verleyen, P (2012) Worker honeybee sterility: a proteomic analysis of suppressed ovary activation. J Proteome Res, 11, 2838-50. [http://dx.doi.org/10.1021/pr201222s] [PMID: 22483170]

Genetic Influence

Polyandrous Queen Honey Bee 207

Cardoen, D, Wenseleers, , Ernst, UR, Danneels, EL, Laget, D, De Graaf, DC, Schoofs, L & Verleyen, P (2011) Genome-wide analysis of alternative reproductive phenotypes in honeybee workers. Mol Ecol, 20, 4070-84. [http://dx.doi.org/10.1111/j.1365-294X.2011.05254.x] [PMID: 21902748] Casagrande-Jaloretto, DC, Correa, BO & Stort, AC (1984) Ovariole number in queens of Apis mellifera. Naturalia (Sao Jose Rio Preto), 9, 73-9. Corbella, E & Goncalves, LS (1982) Relationship between weight at emergence, number of ovarioles and spermathecal volume of Africanized honeybee queens (Apis mellifera L). Rev Bras Genet, 5, 835-40. Cornman, RS, Tarpy, DR, Chen, Y, Jeffreys, L, Lopez, D, Pettis, JS, vanEngelsdorp, D & Evans, JD (2012) Pathogen webs in collapsing honey bee colonies. PLoS One, 7, e43562. [http://dx.doi.org/10.1371/journal.pone.0043562] [PMID: 22927991] Cox-Foster, DL, Conlan, S, Holmes, EC, Palacios, G, Evans, JD, Moran, NA, Quan, PL, Briese, T, Hornig, M, Geiser, DM, Martinson, V, vanEngelsdorp, D, Kalkstein, AL, Drysdale, A, Hui, J, Zhai, J, Cui, L, Hutchison, SK, Simons, JF, Egholm, M, Pettis, JS & Lipkin, WI (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318, 283-7. [http://dx.doi.org/10.1126/science.1146498] [PMID: 17823314] Crozier, RH & Pamilo, P (1996) Evolution of Social Insect Colonies: Sex Allocation and Kin SelectionOxford University Press, New York, NY. Dainat, B, Evans, JD, Chen, YP, Gauthier, L & Neumann, P (2012) Predictive markers of honey bee colony collapse. PLoS One, 7, e32151. [http://dx.doi.org/10.1371/journal.pone.0032151] [PMID: 22384162] Dallacqua, RP & Bitondi, MMG (2014) Dimorphic ovary differentiation in honeybee (Apis mellifera) larvae involves caste-specific expression of homologs of ark and buffy cell death genes. PLoS One, 9, e98088. [http://dx.doi.org/10.1371/journal.pone.0098088] [PMID: 24844304] Danty, E, Arnold, G, Burmester, T, Huet, JC, Huet, D, Pernollet, JC & Masson, C (1998) Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera. Insect Biochem Mol Biol, 28, 387-97. [http://dx.doi.org/10.1016/S0965-1748(98)00011-3] [PMID: 9692239] de Groot, AP & Voogd, S (1954) On the ovary development in queenless worker bees (Apis mellifica L.). Experientia, 10, 384-5. [http://dx.doi.org/10.1007/BF02160554] [PMID: 13210347] Duncan, EJ, Hyink, O & Dearden, PK (2016) Notch signalling mediates reproductive constraint in the adult worker honeybee. Nat Commun, 7, 12427. [http://dx.doi.org/10.1038/ncomms12427] [PMID: 27485026] Evans, JD & Wheeler, DE (1999) Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proc Natl Acad Sci USA, 96, 5575-80. [http://dx.doi.org/10.1073/pnas.96.10.5575] [PMID: 10318926] Feldlaufer, MF, Herbert, EW, Jr, Svoboda, JA, Thompson, MJ & Lusby, WR (1985) Makisterone. Insect Biochem, 15, 597-600. [http://dx.doi.org/10.1016/0020-1790(85)90120-9] Gozansky, TK, Soroker, V & Hefetz, A (1997) The biosynthesis of Dufour’s gland constituents in queens of the honeybee (Apis mellifera). Invert Neurosci, 3, 239-43. [http://dx.doi.org/10.1007/BF02480380] Grozinger, CM, Sharabash, NM, Whitfield, CW & Robinson, GE (2003) Pheromone-mediated gene expression in the honey bee brain. Proc Natl Acad Sci USA, 100 (Suppl. 2), 14519-25. [http://dx.doi.org/10.1073/pnas.2335884100] [PMID: 14573707] Guidugli, KR, Hepperle, C & Hartfelder, K (2004) A member of the short-chain dehydrogenase/reductase

208 Polyandrous Queen Honey Bee

Lovleen Marwaha

(SDR) superfamily is a target of the ecdysone response in honey bee ( Apis mellifera ) caste development. Apidologie (Celle), 35, 37-47. [http://dx.doi.org/10.1051/apido:2003068] Guo, J, Wu, J, Chen, Y, Evans, JD, Dai, R, Luo, W & Li, J (2015) Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana. J Invertebr Pathol, 127, 110-4. [http://dx.doi.org/10.1016/j.jip.2015.03.010] [PMID: 25805518] Hartfelder, K & Engels, W (1989) The composition of larval food in stingless bees: Evaluating nutritional balance by Chemosystematic methods. Insectes Soc, 36, 1-14. [http://dx.doi.org/10.1007/BF02225876] Hartfelder, K, Tiberio, GJ, Lago, DC, Dallacqua, RP & Bitondi, MMG (2018) The ovary and its genes—developmental processes underlying the establishment and function of a highly divergent reproductive system in the female castes of the honey bee, Apis mellifera. Apidologie (Celle), 49, 49-70. [http://dx.doi.org/10.1007/s13592-017-0548-9] Hepperle, C & Hartfelder, K (2001) Differentially expressed regulatory genes in honey bee caste development. Naturwissenschaften, 88, 113-6. [http://dx.doi.org/10.1007/s001140000196] [PMID: 11402838] Holman, L, Helanterä, H, Trontti, K & Mikheyev, AS (2019) Comparative transcriptomics of social insect queen pheromones. Nat Commun, 10, 1593. [http://dx.doi.org/10.1038/s41467-019-09567-2] [PMID: 30962449] Humann, FC & Hartfelder, K (2011) Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary. Insect Biochem Mol Biol, 41, 602-12. [http://dx.doi.org/10.1016/j.ibmb.2011.03.013] [PMID: 21477651] Humann, FC, Tiberio, GJ & Hartfelder, K (2013) Sequence and expression characteristics of long noncoding RNAs in honey bee caste development--potential novel regulators for transgressive ovary size. PLoS One, 8, e78915. [http://dx.doi.org/10.1371/journal.pone.0078915] [PMID: 24205350] Jindra, M, Palli, SR & Riddiford, LM (2013) The juvenile hormone signaling pathway in insect development. Annu Rev Entomol, 58, 181-204. [http://dx.doi.org/10.1146/annurev-ento-120811-153700] [PMID: 22994547] Kamakura, M (2011) Royalactin induces queen differentiation in honeybees. Nature, 473, 478-83. [http://dx.doi.org/10.1038/nature10093] [PMID: 21516106] Kubisova, S (1978) Effect of larva extract on the development of ovaries in caged worker honey bee Kucharski, R, Maleszka, J, Foret, S & Maleszka, R (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science, 319, 1827-30. [http://dx.doi.org/10.1126/science.1153069] [PMID: 18339900] Lago, DC, Humann, FC, Barchuk, AR, Abraham, KJ & Hartfelder, K (2016) Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development. Insect Biochem Mol Biol, 79, 1-12. [http://dx.doi.org/10.1016/j.ibmb.2016.10.001] [PMID: 27720811] Laidlaw, HH (1992) Production of Queen and package bees, pp. 989-1042. In: The hive and the honey bee (GRAHAM J. H., Ed.).- Dadant & Sons, Hamilton, Illinois, USA. Leimar, O, Hartfelder, K, Laubichler, MD & Page, RE, Jr (2012) Development and evolution of caste dimorphism in honeybees - a modeling approach. Ecol Evol, 2, 3098-109. [http://dx.doi.org/10.1002/ece3.414] [PMID: 23301175] Li, Z, Chen, Y, Zhang, S, Chen, S, Li, W, Yan, L, Shi, L, Wu, L, Sohr, A & Su, S (2013) Viral infection affects sucrose responsiveness and homing ability of forager honey bees, Apis mellifera L. PLoS One, 8, e77354.

Genetic Influence

Polyandrous Queen Honey Bee 209

[http://dx.doi.org/10.1371/journal.pone.0077354] [PMID: 24130876] Mello, TRP, Aleixo, AC, Pinheiro, DG, Nunes, FMF, Bitondi, MÃMG, Hartfelder, K, Barchuk, AR & Simões, ZÃLP (2014) Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera). Front Genet, 5, 445. [http://dx.doi.org/10.3389/fgene.2014.00445] [PMID: 25566327] Michener, CD (1969) Comparative social behaviour of bees. Annu Rev Entomol, 14, 299-342. [http://dx.doi.org/10.1146/annurev.en.14.010169.001503] Mutti, NS, Dolezal, AG, Wolschin, F, Mutti, JS, Gill, KS & Amdam, GV (2011) IRS and TOR nutrientsignaling pathways act via juvenile hormone to influence honey bee caste fate. J Exp Biol, 214, 3977-84. [http://dx.doi.org/10.1242/jeb.061499] [PMID: 22071189] Niu, J, Meeus, I, Cappelle, K, Piot, N & Smagghe, G (2014) The immune response of the small interfering RNA pathway in the defense against bee viruses. Curr Opin Insect Sci, 6, 22-7. [http://dx.doi.org/10.1016/j.cois.2014.09.014] [PMID: 32846664] Oxley, PR, Thompson, GJ & Oldroyd, BP (2008) Four quantitative trait loci that influence worker sterility in the honeybee (Apis mellifera). Genetics, 179, 1337-43. [http://dx.doi.org/10.1534/genetics.108.087270] [PMID: 18562647] Patel, A, Fondrk, MK, Kaftanoglu, O, Emore, C, Hunt, G, Frederick, K & Amdam, GV (2007) The making of a queen: TOR pathway is a key player in diphenic caste development. PLoS One, 2, e509. [http://dx.doi.org/10.1371/journal.pone.0000509] [PMID: 17551589] Rachinsky, A & Hartfelder, K (1990) Corpora allata activity, a prime regulating element for caste-specific juvenile hormone titre in honey bee larvae (Apis mellifera carnica). J Insect Physiol, 36, 189-94. [http://dx.doi.org/10.1016/0022-1910(90)90121-U] Ronai, I, Oldroyd, BP & Vergoz, V (2016) Queen pheromone regulates programmed cell death in the honey bee worker ovary. Insect Mol Biol, 25, 646-52. [http://dx.doi.org/10.1111/imb.12250] [PMID: 27321063] Roth, A, Vleurinck, C, Netschitailo, O, Bauer, V, Otte, M, Kaftanoglu, O, Page, RE & Beye, M (2019) A genetic switch for worker nutrition-mediated traits in honeybees. PLoS Biol, 17, e3000171. [http://dx.doi.org/10.1371/journal.pbio.3000171] [PMID: 30897091] Santos, DE, Alberici, LC & Hartfelder, K (2016) Mitochondrial structure and dynamics as critical factors in honey bee ( Apis mellifera L.) caste development. Insect Biochem Mol Biol, 73, 1-11. [http://dx.doi.org/10.1016/j.ibmb.2016.04.001] [PMID: 27058771] Severson, DW & Erickson, EH, Jr (1989) Seasonal constraints on mating and insemination of queen honey bees in a continental climate. Apidologie (Celle), 20, 21-7. [http://dx.doi.org/10.1051/apido:19890103] Thompson, GJ, Yockey, H, Lim, J & Oldroyd, BP (2007) Experimental manipulation of ovary activation and gene expression in honey bee (Apis mellifera) queens and workers: testing hypotheses of reproductive regulation. J Exp Zool Part A Ecol Genet Physiol, 307A, 600-10. [http://dx.doi.org/10.1002/jez.415] [PMID: 17786975] Tomarev, SI & Piatigorsky, J (1996) Lens crystallins of invertebrates--diversity and recruitment from detoxification enzymes and novel proteins. Eur J Biochem, 235, 449-65. [http://dx.doi.org/10.1111/j.1432-1033.1996.00449.x] [PMID: 8654388] Winston, ML, Higo, HA & Slessor, KN (1990) Effect of various dosages of Queen mandibular gland pheromone on the inhibition of queen rearing in the honey bee (Hymenoptera: Apidae). Ann Entomol Soc Am, 83, 234-8. [http://dx.doi.org/10.1093/aesa/83.2.234] Wojciechowski, M, Lowe, R, Maleszka, J, Conn, D, Maleszka, R & Hurd, PJ (2018) Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development. Genome Res, 28, 1532-42.

210 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1101/gr.236497.118] [PMID: 30135090] Yin, L, Wang, K, Niu, L, Zhang, H, Chen, Y, Ji, T & Chen, G (2018) Uncovering the changing gene expression profile of honeybee (Apis mellifera) worker larvae transplanted to queen cells. Front Genet, 9, 416. [http://dx.doi.org/10.3389/fgene.2018.00416] [PMID: 30405683]

Polyandrous Queen Honey Bee, 2022, 211-231

211

CHAPTER 11

Drone Development, Biology, and And Interaction With The Queen in Apis mellifera Abstract: Drone honey bees develop from haploid/unfertilized/diploid eggs produced by parthenogenesis or from fertilized eggs having identical sex alleles, formed after sexual reproduction, with more probability when the queen mates with drones of the same hives. Nurse bees generally remove diploid drone larvae due to cannibalism hormones secreted by developing larvae. Further, the development of drones is influenced by colony temperature, hence can be completed within 24-25 days. Queen attracts drone honey bees toward herself with pheromones9-ODA,9-HAD and 10 HDA. Drone number depends upon the colony's environmental conditions and available food to the colony. The specific chapter provides deep insight into the development of drones, the biology of drones, the reproductive system and the mating behaviour of particular castes.

Keywords: Diploid Drones, Haploid Drones, Pheromones, Reproductive system. 11.1. INTRODUCTION Drones, the honey bee colony's male caste, possess an exclusive reproduction function, and the concerned caste does not forage, maintain the hive or defend the colony. The Polyandrous honey bee queen usually mates with 6-17 drones during nuptial flight (Peer et al., 1956; Renner and Baumann,1964; Adams et al., 1977; Santomauro et al., 2004), and after that, post-mating death of drone is certain (Witherell, 1956). Limited explorations are available in scientific literature as drones contribute little to agricultural pollination, apicultural production and colony protection. However, drones can improve the colony quality by increasing productivity, disease and swarm resistance. 11.2. GENERAL DEVELOPMENT Drones usually inherit maternal inheritance as they develop from unfertilized eggs laid in drone wax cells by the queen or egg-laying honey bee workers (Kerr, 1974a,b; Herrmann et al., 2005; Brutscher et al., 2019). Drones develop by the Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

212 Polyandrous Queen Honey Bee

Lovleen Marwaha

haplodiploid sex-determination system. Further, even a diploid drone can create from a fertilized egg in case of the queen mates with drones of the same colony (Page and Laidlaw, 1985). Even egg-laying workers can lay down diploid eggs, with two sets of chromosomes coming from one of the polar bodies and ovum. The specific process is known as thelytoky. Usually, workers eat diploid drones a few hours after the egg hatches out (Woyke, 1965). For the development of drones from egg to adult, about 24 days are required (Jay, 1963), whereas in the peripheral area of the hive, usually more time is needed, which could be upto 25 days (Fukuda and Ohtani, 1977). Drones' development correlates with brood nest temperature variations (Free, 1967; Jay, 1963; Fukuda and Ohtani, 1977; Santomauro et al., 2004). Each drone produces about 10 million male sperm cells, which are genetically identical. Further, sperm cells are genetically similar to the hive's full sisters. Drones solicit food from workers after hatching (Fig., 11a-11d). Drones possess a diurnal feeding rhythm, with maximum food consumption before the flight. As drones are unable to feed themselves and contribute to the colony's productivity, their number is dependent upon great colony conditions. Drones are produced when colonies require them for mating. Usually, drone rearing (Fig. 11a) precedes queen rearing as drone rearing takes longer than the queen. The appearance of the drone indicates that the colony is prepared for swarming or queen rearing. Adult drone prefers a temperature of 35 0C in the hive, although variation in thermal preference occurs with age, as younger drones choose the warmer part of the hive. In comparison, older drones prefer a cooler area of the hive (Fukuda and Ohtani, 1977). 11.3. DIPLOID DRONES Usually, drones develop from unfertilized eggs (Fig. 11a). Therefore, they are haploid. In contrast, some drones develop from diploid eggs formed by the fusion of an ovum with one of the polar bodies or from the fertilized egg, homologous at the sex locus (Woyke et al., 1966; Herrmann et al., 2005). Further, the diploid drone can develop from uniparental origin or biparental origin. Biparental origin diploid drones can create by matchmaking queens and drones with identical sex alleles. Brood-attending worker honey bees eliminate false diploid drones (Woyke, 1962; Woyke, 1965; Woyke, 1963 a, b, c, d; Herrmann et al., 2005). Under laboratory conditions, diploid drones can be reared by controlled hatching in an incubator and by controlled feeding. Diploid drones are comparatively more significant and heavier but with smaller testes, testicular tubules and fewer wing hooks (Woyke, 1974, 1977a, b, 1978, Herrmann et al., 2005). Additionally, diploid drones produce more cuticular hydrocarbons than workers (Santomauro et

Biology, and interaction

Polyandrous Queen Honey Bee 213

al., 2004). Diploid drones produce diploid spermatozoa, having twice DNA, with elongated heads.

Fig. (11a). Depicting Capped Worker cells, Capped Drone cells, Pollen cells, Unripe honey cells. Worker honey bees perform different duties like exchange of information, honey processing and adding worker jelly to developing worker larvae.

Woyke,1967 analyzed that diploid drone larvae secrete certain substances known as cannibalism substances, which act as a significant marker for diploid drones, whereas other authors are unable to detect this kind of substance (Woyke, 1967; Dietz, 1975; Dietz and Lovins, 1975; Bienefeld et al., 1994, 2000; Santomauro et al., 2004). According to Woyke (1969 a,b), diploid drones can be reared outside the colony and, after that, they can be re-introduced into the colony and accepted by the colonial residents. In Hymenopteran, drones are either haploid or diploid, with meiotic gametogenesis. Therefore, drones contain carbon copies of the maternal genomic content (Woyke and Skowronek, 1974). In spermatozoa, diploid drones are diploid with double nuclear content (Woyke, 1975; Fahrenhorst, 1977). Young drones also produce vitellogenin mainly two weeks after emergence (Trenczek et al., 1989; Engels et al., 1990; Piulachs et al., 2003) but in lower hemolymph titer

214 Polyandrous Queen Honey Bee

Lovleen Marwaha

(Santomauro et al., 2004). Herrmann et al., 2005 analyzed the developmental pattern of diploid and haploid drone larvae. Further, they conducted experiments to produce diploid drones. Diploid drones had been made by feeding diploid drone larvae with an artificial diet composed of sugar, vitamin and fungicides (Woyke,1973, 1974; Herrmann et al., 2005).

Fig. (11b). Sexual and parthenogenesis in the honey bee resulted in the formation of diploid and haploid drones.

11.4. LIFE SPAN There is a variation in the life span of drone, which is from 13-14 days to 21-24 days (Howell and Usinger,1933; Lavrek,1947; Kepena,1963; Witherell, 1965; Drescher, 1969; Fukuda and Ohtani, 1997; Herrmann et al., 2005). Further, other factors affecting drone life span include flight activity or geographical region (Fukuda and Ohtani, 1977). For example, the life span of the drone is comparatively shorter in summer than in autumn (Fukuda and Ohtani, 1977). According to Neukirch, 1982, a drone's life span depends upon flight performance and energy consumed during the flight. Further, different studies which had indicated different life spans of the drone is due to other environmental conditions, which vary due to geographical constraints and seasonal variation. For instance, cool temperatures, high wind or overcast sky reduce flight activity, increasing drone life span. Further, predation negatively affects the life span of drones. The number of drones in a colony depends upon the drone comb constructed, which regulates the production of drone brood.

Biology, and interaction

Polyandrous Queen Honey Bee 215

11.5. DRONE NUMBER The age and fecundity of the queen influence the construction of drone cells or their conversion to worker cells under the influence of pheromones from the queen's mandibular gland (Darchen, 1960; Chauvin, 1961). Further, the presence of a queen and worker brood can increase the number of drone comb (Free, 1967). However, the construction of a drone comb is limited by the colony's strength and the number of drone cells already present (Allen, 1958; Free and Williams, 1975). The queen regulates the laying of the drone brood. Further, worker honey bees control the number of drone brood by destroying and eating drone eggs, larvae and pupae (Fig. 11c-11e) (Free and Williams,1975; Fukuda and Ohtani, 1977). The survival of drone cells in the queenless and queenright colonies is the same in spring and summer, but in autumn, the drone brood survival rate is higher in queenless colonies than in queenright colonies (Wovke et al., 1965a). Drone brood production and emergence are dependent upon the queen, temperature, pollen grain and nectar storage of the colony (Gorbaczaw, 1961; Taber,1964; Louveux et al., 1973; Mesqum, 1976; Fukuda and Ohtani, 1977), therefore for large drone brood production, colonies must have high sugar syrup, pollen supply and queenless condition. In a larger colony, on average, about 1500 adult drones are present(Currie,1982).

Fig. (11c). Section of Hive with larvae filled wax cell, capped drone cells, adult drones and worker honey bees performing various duties.

216 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (11d). Developmental of drones within 24 days, with egg, larval, pupal and adult phases.

Fig. (11e). Section of Hive with Capped Drone cells and worker honey bees providing worker jelly to developing larvae.

Biology, and interaction

Polyandrous Queen Honey Bee 217

Workers regulate the number of drones in the colony by evicting drones from the colony during scarcity of nectar. Worker honey bees usually tend to force drones toward the periphery, then to walls, bottom boards and finally from the colony (Levenets, 1956). Even workers aggressively expel the drones from the colony by chewing, mauling, and pulling them out (Morse et al., 1967). The expulsion of drones is a gradual process that takes several weeks in the autumn. Usually, 10-15 drones are evicted from the colony daily (MorsE et al., 1967). The eviction process is significantly delayed in queenless colonies (Free and Williams,1975). Other factors which affect the eviction process include low temperature, age of queen, sealed and unsealed brood, the odour of drones, the activity of the colony, available food in the colony, available honey and genetic strains of bees (Levenets, 1951; Alber, 1955; Orosi, 1959; Morse et al., 1967; Free and Williams, 1975; Free, et al., 1977; Taber, 1982). Food collected and stored within the colony is an essential factor which regulates the eviction of drones within the colony (Free et al., 1975). 11.6. FLIGHT ACTIVITY Flight activity begins when drones are four days old, but usually, the first flight occurs when drones are 5-7 days old (Howell and Usinger,1933; Witherell, 1972). All drones make their first flights at about 18 days (Drescher et al., 1969). Generally, drone flights take place in concentrated periods during the afternoon and occur at the same time of the day in different geographical locations (Laverekhin, 1947; Taber, 1964). The time of flight of Apis mellifera drones is more than Apis cerana, Apis florea and Apis dorsata (Lavrekhin, 1947; Ruttner et al., 1972; Koeniger and Sekera, 1976). For example, in European honey bee flight, drones began between 11.00- 14.00 hr and usually ended between 16.0018.00 hr (Kurennoi, 1953; Oertel,1956; Ruttner, 1966; Drescher, 1969; Strang, 1970). A drone can fly as early as 09.00 and leave the swarm between 9.00-13.00 (Callow, 1964; Avitable and Kasinskas, 1977). Further, the time of flight is influenced by other environmental conditions, the time of the year, and the hive entrance's direction. The time at which peak flight activity occurs varies with the time of year. Drone flight activity began early in spring and autumn and then in summer. Flight activities are influenced by light perceived by a drone inside the colony regarding sunrise and sunset. Further, drone flight is affected by daily temperature, relative humidity, the colony's position along the direct of the sun and the length of the day. Additionally, circadian rhythm influences drone flight activity. Usually, the flight activity of the queen begins approximately 20 hours after sunset.

218 Polyandrous Queen Honey Bee

Lovleen Marwaha

Drones and queen use their biological clock to measure the time of their flight activity. In unfavourable environmental conditions, drones usually do not fly and take only short flights. Drones generally fly when the temperature is about 18 20°C. Although drones can also fly below this temperature range, such flights are usually short-timed. Drones can fly with wind speed upto 25 km/h, but mating generally takes place at a wind speed of less than 18 km/h. With a wind speed of 8 - 16 km/h, or at low temperatures or in heavy cloud cover, there is a reduction in drone activity. Drones fly for orientation, defecation and mating. Flight duration varies with the drone's age, as the oldest drones take the longest flight, and additional flight time varies with season. For example, drones usually take longer flights during summer than in spring. Generally, drones take 2-4 flights per day, but they can even take 17 flights per day. 11.7. DRIFT AND ORIENTATION OF DRONES' FLIGHT TO THE HIVE Drones can fly upto 7 km from their colony and can return successfully. Capture and release studies indicated that the number of captured and released drones decreases with increased distance from the hive. Further, the direction of drone release does not affect the rate of return, even with removed antennae. Drones can use landmarks, the sun or the magnetic compass of the earth, to locate a hive. However, drones generally make orientation errors and usually enter into wrong colonies, known as drifting. Drifting drones is likely to affect honey productivity as drones can carry many diseases and parasites, including sacbrood (virus), Nosema, Acarapis and Varroa, when they drift between hive and hive and between apiaries. Usually, drone drifts range from (0-12%) to upto 80%. Drone drifting is generally higher than workers, usually 2-3 times higher than workers. Apiaries, where hives are placed in a straight row, usually provide no orientation cues, possessing equal proportionally of drone and worker drifting. Worker drifting can be reduced by angled hive layout, horseshoe patterns, and by different colours of the hive. The horseshoe layout effectively reduces drone drift by only 10% more than in a straight row of the hive. Further, drone drift can be reduced by combining two or more orientations, which provide appropriate cues regarding orientation cues. Studies have indicated that drones of all ages show drone drift between more than one hive. Usually, drone drifting begins on 5-7days old drone, which coincides with the beginning of flight activity. In older drones, there is less drifting with the increase in age, which could be due to more flight experience. Younger drones make the highest flight errors than older ones. Drone drift is less observable in the area with more orientation cues. Drones drift is influenced by the position and apparent

Biology, and interaction

Polyandrous Queen Honey Bee 219

movement of the sun. Further, it has been suggested that the sun may influence flight to drone congregation areas. It has been reported that drones tend to drift toward the south with the entrance facing east and west, whereas drones tend to drift toward the west with north and south facing rows. The direction of workers and drone drift appears to be influenced by the sun's position in the sky. Based on drone drifting behaviour, it seems that drones use the sun as a cue to move near to hive. Further queen state can influence the drone drift between colonies. There is no significant drift between colonies headed by virgin queens or mated queens. However, the critical difference is between queenless and queen-righted colonies. Drone drift is influenced by pheromone 9-oxo-trans-2-decanoic acid. Therefore, the virgin queen attracts more drones toward the colony than the queerness or mated queen. Virgin queen secretes more 9-oxo-trans-2-decanoic acid, attracting more drifting drones. 11.8. ATTRACTION OF DRONES TO VIRGIN QUEEN Drones detect the queen by anemotaxis and detect pheromones secreted by the queen. Queen's pheromones attract drones up to 60 m downwind of her. If the pheromonal concentration is below the threshold, drones usually fly until they locate pheromones. Under the influence of sex pheromones, drones are attracted to dark, compact and moving objects. Drones generally fly and are attracted to queen pheromones at 10-40 m above ground. The height to which drones are attracted varies inversely with wind speed. On very windy days, drones can be found within 1-2 m of the ground. The Virgin queen secretes sex pheromones released from the queen's mandibular glands. Virgin queens could even mate if their mandibular glands were removed. Queen mandibular gland secretes 32 different compounds. Two significant components attract drones which include 9-oxo-trans-2-decanoic acid and 9-hydroxy-decanoic acid. Whole queen's mandibular gland extract is slightly more attractive to drones than two components, including 9-oxo-trans-2-decanoic acid and 9-hydrox-decanoic acid alone. Generally, drones detect pheromones through specific pore plate receptor sites on the antennae. Fatty acids produced somewhere in the queen's head may act as keeper substances to ensure the gradual release of the pheromones. The number of drones attracted toward the queen is proportional to the concentration of 9-oxo2-decanoic acid in the mandibular gland.

220 Polyandrous Queen Honey Bee

Lovleen Marwaha

Two of the main components of the queen pheromone, produced in the mandibular glands, are 9-keto-2 (E)-decanoic acid (9-ODA) and 9-hydroxy-2 (E)decanoic acid (9-HDA). The 9-ODA, in particular, was highly influential in maleattraction bioassays. Additionally, drones have become attracted by 9-ODA,9HDA, and 10-HDA (Fig. 11f) (Gary, 1962; Butler and Fairey, 1964; Metzand Tarpy, 2019; Rangel, 2019).

Fig. (11f). Drone attraction toward queen through various queen pheromones including 9-ODA, 9HDA, 10 HDA.

11.9. DRONE DEVELOPMENT Developmental phases of drones include eggs, first instar larva, larva, larva in the cell, spinning larvae, stretched larva, prepupa, late prepupa, and pupa (Fig. 11d 11h). Drones develop from unfertilized haploid/diploid eggs laid by queen /egglaying workers. The drone egg appears to be a pearly white, elongated structure with a curved cylinder rounded at both ends. Both ends of the drone egg possess differences in thickness, one end thick and the other thin. The eggs remain attached with their narrow ends to the cell wall. Eggs usually sag gradually to the

Biology, and interaction

Polyandrous Queen Honey Bee 221

bottom of the cell during development or are pushed by workers during inoculation to the bottom of the wax cell. For the complete development of drones, about 24 days are required; 3 days for egg hatching, six days for the larval phase and 15 days for pupation.

Fig. (11g). Uncapped larvae of honey bees in the hive and worker honey bees performing different duties.

Fig. (11h). Different instar larvae and unripe honey cells are highlighted in a section of the hive.

11.10. REPRODUCTIVE SYSTEM OF DRONES The internal male reproductive system of the drone consists of a pair of testes, a pair of vasa differentia, a pair of accessory sex glands and a median ejaculatory

222 Polyandrous Queen Honey Bee

Lovleen Marwaha

duct(Bishop, 1920; Snodgrass, 1956; Woyke, 1958; Simpson, 1960; Chen, 1984; Davey, 1985; Koeniger, 1986; Gillot, 1988; Koeniger et al., 1989; Paliwal, 1993; Chapman, 1998). Testes: The testes are creamy, oval-shaped bodies lying at the anterior side of the mucus glands and situated between the 2nd and 3rd abdominal segments. The numbers of the testicular follicle are seven in A.c. indica and are variable in number in other hymenopterans (Snodgrass, 1956; Wheeler and Krutzsch, 1992; Duchateau and Mariën, 1995; Ferreira et al., 2004). Testes are packed with seven tube-like coiled follicles. Each follicle comprises an inner layer of epithelial cells and an outer layer of muscle fibres. The inner side of the testes carries several cysts full of spermatogenic stages. Each follicle opens posteriorly into the vas deferens. Vas Deferens: VD is divided into three regions: the apical short coiled tube, middle cylindrical seminal vesicle (SV) and distal straight duct. The apical part of VD possesses an outer circular muscle layer, an inner epithelial layer, and a large lumen. VD's distal part opens into the mucus gland's basal region (MG). Seminal Vesicle: Seminal vesicle is represented by a sizeable sac-like region of the vas deferens. The seminal vesicle is composed of inner epithelial lined by outer muscle layers and externally covered with a thin peritoneal sheath. The epithelial layer is composed of tall columnar cells with a brush border luminal surface and lumen filled with sperm bundles. The muscle layers are composed of outer longitudinal and inner circular muscle layers. The epithelial cells are tall and columnar with a brush border towards the lumen. The lumen is filled with a large mass of sperm bundles, having their heads towards the wall and tails at the centre of the lumen. Mucous Glands: The paired mucus glands (MG) are large, kidney-shaped, saclike structures representing a particular type of male accessory glands in the bee. The wall of the MG is composed of an inner epithelial layer, an outer thick muscle coat and is externally covered with a thin peritoneal sheath. The muscle coat is formed of three sublayers, the outer longitudinal, middle circular and inner longitudinal muscle layer (Bishop, 1920; Snodgrass, 1956; Paliwal, 1993; Paliwal, 1993: Sawarkar and Tembhare, 2015). The mucus gland and the seminal vesicle are mesodermal in origin. The mucus glands open into a common ejaculatory duct via lateral ejaculatory ducts. In drones, spermatogenesis occurs during the pupal stage. Ejaculatory Duct: The ejaculatory duct is a long slender tube, with the wall of the ejaculatory duct differentiated into the outer broad epithelial layer and inner cuticular layer. The inner thin cuticular layer bears elongated spines in the lumen.

Biology, and interaction

Polyandrous Queen Honey Bee 223

The presence of the cuticular layer showed that the ejaculatory duct is ectodermal in origin. 11.11:MATING Drones secrete through their mandibular glands specific pheromones, which act as an attractant for drones from other colonies. Drones do not get attracted toward the virgin queen of their hive but to the virgin queen of other hives. Therefore, when the virgin queen leaves the hive for nuptial flight, she is not chased by the drones of her hive. It had been detected that drones do not chase queens below 10 m height or beyond the congregation area. There is some behavioural and physiological mechanism which help in the attraction of drones toward queen of other hives than queens of their hives. Drones can mate with a queen from other colonies up to 16.2 km away. Mating occurs in drone congregation areas or assemblies, where drones regularly assemble with or without the queen. Congregation areas are common in hilly or mountainous regions. Butler and Strang tried to create an artificial congregation area by using sex pheromones (Currie, 1989). For mating flight, the queen flies about 3 Kms away from her hive to congregation areas which could be approximately 5-40 m above the group (Ellis et al., 2015). Reports witnessed that the queen mates with 12 drones, whereas few reports seen that the queen mates with 34-77 (Winston, 1987; Kraus et al., 2005; Withrow et al., 2018). In mating, drones irreversibly revert their endophallus into a female and transfer semen into her oviduct (Woyke, and Ruttner, 1958; Ellis et al., 2015). After the nuptial flight, the queen returns to her hive and stores received sperm in spermatheca (Ellis et al., 2015). About 3-5 per cent of sperm from a single drone are stored for fertilization (Woyke, 1962; Winston, 1987; Schluns et al., 2005; Ellis et al., 2015). Queen can store about 5-6 million total sperms in her spermatheca (Koeniger, and Koeniger, 2000;Baer, 2005; Ellis, et al., 2015). Mandibular gland pheromones of queen inseminated with semen from one drone exhibit poor retinue than MQP from queen inseminated by many drones (Richard et al., 2007). Honey bee colonies headed by multiple drones inseminated queens exhibited more robust retinue response, possessed more vital wax cell construction ability, produced more honey, reproduced fast, collected more pollen grain, made more drones, and had more resistance to diseases than the colony headed by the queen inseminated by single drone (Tarpy., 2003; Seeley, and Tarpy, 2006; Richard et al., 2007; Seeley, and Tarpy, 2007; Mattila, and Seeley,2015).

224 Polyandrous Queen Honey Bee

Lovleen Marwaha

In exploration, it was concluded that the queen inseminated with 8 µL of semen exhibited decreased sexual receptivity and more pronounced ovarian activity than the queen inseminated with 1 µL of semen (Niño et al., 2013). Further, colonies headed by a queen inseminated with lower volume show more queen cell building/supersedure rate (Niño et al., 2012). More insemination volume also induced a greater retinue response toward mandibular gland extract (Nino et al., 2012; Nino et al., 2013). Seminal fluid possesses many proteins that help provide pathogen defence, and significant proteins include chitinases, Osiris 7, and heat shock proteins (Cornman et al., 2013; Brutscher et al.,2015; Brutscher et al., 2017; McMenamin et al., 2018). It has been reported that seminal fluid reduces the spore viability of the fungal pathogen Nosema apis (Kurze et al., 2016; Peng et al., 2016). Both protein and non-protein fractions in seminal fluid reduced Nosema apis spore germination (Kurze et al., 2016). In addition, seminal fluid proteins induce Nosema spore wall rupture, whereas the non-protein portion of seminal fluid decreases spore viability (Kurze et al., 2016). Further, honey bee seminal fluid exhibited antiviral activity due to the presence of heat shock proteins (Brutscher et al., 2015; Brutscher et al., 2017; McMenamin et al., 2018). As the queen mates with multiple drones, she is more susceptible to acquiring pathogens during ejaculation, despite having comparatively stronger resistance (Schmid et al., 2008; Collins and Pettis,2013; Rueppell et al., 2016; Brutscher et al., 2019). 11.12. INFLUENTIAL FACTORS FOR DRONE QUALITY The larval environment affects the reproductive health of drones. For example, colonies which rear drones on a protein-restricted diet usually have drones with lower body mass, thorax mass, and smaller ejaculate volumes compared to colonies that rear drones on a pollen-rich diet (Peso et al., 2013). Usually, drones possess high semen volume during the early breeding season (Rueppell et al.,2005; Brutscher et al., 2019; Metz and Tarpy, 2019). 11.13. DRONE AGING Drone honey bees' reproductive quality is affected by age, season and genetics. Ageing of drones negatively affects sperm viscosity, volume, and viability(WoykeandJasiński1978; Locke and Peng, 1993; Rhodes, 2002; Cobey, 2007; Rhodes et al., 2011; Czeko ńska et al., 2013; Stürup et al. 2013).In more aged drones, semen become dark and more viscous than in younger drones (Woyke and Jasiński 1978; Cobey 2007; Czekońska et al. 2013a). such semen form plug in oviduct and affect reproduction (Woyke and Jasiński 1978; Czekońska et al. 2013a). Locke and Peng (1993) detected that ageing also affects sperm viability, decreasing from 86% to 81% in 14 days and 20 days old drones,

Biology, and interaction

Polyandrous Queen Honey Bee 225

respectively. Stürup et al. (2013) analyzed that drones older than 20 exhibited 50% lower sperm viability than drones that survived less than 20 days. However, Metzand Tarpy(2019) recently analyzed that sperm viability remains constant throughout life. In contrast, Czekońska et al. (2013) studied that drones aged 15-30 days possess less semen volume and decreased sperm viability. Usually, drones between 14 and 21 days old produce higher semen volume than 35 days old drones. Sperm production reaches upto peak 20 days post-emergence. Rhodes et al. (2011) reported a seasonal increase in sperm count. Drones negatively correlate age with sperm viability and seminal volume (Woyke and Jasiński 1978; Locke and Peng 1993; Cobey 2007; Rhodes et al. 2011; Czekońska et al. 2013a; Metzand Tarpy 2019; Rangel 2019). CONCLUSION Drones, the male caste of the colony, develop from unfertilized eggs through parthenogenesis or from a diploid egg if the queen mates with drones of the same colony. During development, drone larvae are initially fed on royal jelly and honey and pollen grains. Drones perform the duty of mating with the queen honey bee when they get attracted to her. REFERENCES Adams, J, Rothman, ED, Kerr, WE & Paulino, ZL (1977) Estimation of the number of sex alleles and queen matings from diploid male frequencies in a population of Apis mellifera. Genetics, 86, 583-96. [http://dx.doi.org/10.1093/genetics/86.3.583] [PMID: 892423] Akyol, E, Yeninar, H & Kaftanoglu, O (2008) The live weight of queen honey bees (Apis mellifera L.) predicts reproductive characteristics. J Kans Entomol Soc, 81, 92-100. [http://dx.doi.org/10.2317/JKES-705.13.1] Alber, MA (1955) Siidwestdeurscher Imker, 7, 106-7. [Drone colonies]. [In German.]. Al-Lawati, H, Kamp, G & Bienefeld, K (2009) Characteristics of the spermathecal contents of old and young honeybee queens. J Insect Physiol, 55, 117-22. [http://dx.doi.org/10.1016/j.jinsphys.2008.10.010] [PMID: 19027748] Allen, DM (1958) Drone brood in honeybee colonies. J Econ Entomol, 51, 46-8. [http://dx.doi.org/10.1093/jee/51.1.46] Al-Qarni, AS, Phelan, PL, Smith, BH & Cobey, SW (2005) The influence of mating type and oviposition period on mandibular pheromone levels in Apis mellifera L. honeybee queens. Saudi J Biol Sci, 12, 39-47. Amiri, E, Meixner, MD & Kryger, P (2016) Deformed wing virus can be transmitted during natural mating in honey bees and infect the queens. Sci Rep, 6, 33065. [http://dx.doi.org/10.1038/srep33065] [PMID: 27608961] Avitabile, A & Kasinskas, JR (1977) The drone population of natural honeybee swarms. J Apic Res, 16, 1459. [http://dx.doi.org/10.1080/00218839.1977.11099876] Baer, B (2005) Sexual selection in Apis bees. Apidologie (Celle), 36, 187-200.

226 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1051/apido:2005013] Bishop, GH (1920) Fertilization in the honey-bee. I. The male sexual organs: Their histological structure and physiological functioning. J Exp Zool, 31, 224-65. [http://dx.doi.org/10.1002/jez.1400310203] Brutscher, LM, Baer, B & Niño, EL (2019) Putative drone copulation factors regulating honey bee (Apis mellifera) queen reproduction and health: A review. Insects, 10, 8. [http://dx.doi.org/10.3390/insects10010008] [PMID: 30626022] Brutscher, LM, Daughenbaugh, KF & Flenniken, ML (2015) Antiviral defense mechanisms in honey bees. Curr Opin Insect Sci, 10, 71-82. [http://dx.doi.org/10.1016/j.cois.2015.04.016] [PMID: 26273564] Brutscher, LM, Daughenbaugh, KF & Flenniken, ML (2017) Virus and dsRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense. Sci Rep, 7, 6448. [http://dx.doi.org/10.1038/s41598-017-06623-z] [PMID: 28127051] Callow, RK, Chapman, JR & Paton, AN (1964) Pheromones of the honeybee: chemical studies of the mandibular gland secretion of the queen. J Apic Res, 3, 77-89. [http://dx.doi.org/10.1080/00218839.1964.11100086] Chaud-Netto, J (1975) Sex determination in bees. II. Additivity of maleness genes in Apis mellifera. Genetics, 79, 213-7. [http://dx.doi.org/10.1093/genetics/79.2.213] [PMID: 1132678] Collins, AM & Pettis, JS (2013) Correlation of queen size and spermathecal contents and effects of miticide exposure during development. Apidologie (Celle), 44, 351-6. [http://dx.doi.org/10.1007/s13592-012-0186-1] Cornman, RS, Lopez, D & Evans, JD (2013) Transcriptional response of honey bee larvae infected with the bacterial pathogen Paenibacillus larvae. PLoS One, 8e65424 [http://dx.doi.org/10.1371/journal.pone.0065424] [PMID: 23762370] Currie, R. W. (1982). Some factors affect the orientation of drone honeybees—University of Manitoba, Canada: M.Sc. thesis. Darchen, R (1957) La reine d’Apis mellifica les ouvrières pondeuses et les constructions cirières. Insectes Soc, 4, 321-5. [http://dx.doi.org/10.1007/BF02224152] de Miranda, JR & Fries, I (2008) Venereal and vertical transmission of deformed wing virus in honeybees (Apis mellifera L.). J Invertebr Pathol, 98, 184-9. [http://dx.doi.org/10.1016/j.jip.2008.02.004] [PMID: 18358488] Drescher, W (1969) The flight activity of Apis melifera carnica and Apis mellifera ligustica drones in relation to age and weather. Z Bienenf, 9, 390-409. Ellis, J, Lawrence, JC, Koeniger, N & Koeniger, G (2015) Mating Biology of Honey Bees (Apis mellifera). Free, JB & Williams, IH (1975) Factors determining the rearing and rejection of drones by the honeybee colony. Anim Behav, 23, 650-75. [http://dx.doi.org/10.1016/0003-3472(75)90143-8] Free, JB (1967) The production of drone comb by honeybee colonies. J Apic Res, 6, 29-36. [http://dx.doi.org/10.1080/00218839.1967.11100157] Fukuda, H & Ohtani, T (1977) Survival and life span of drone honeybees. Popul Ecol, 19, 51-68. [http://dx.doi.org/10.1007/BF02510939] Garofalo, C. (1972). Behaviour and sexual maturity of Apis mellifera adansonii drones. Pp. 177-185. In Homenagem 11 Warwick E Kerr: oferecida pelos seus exalunos por ocasiiio de seu 50 aniversario. Rio Claro, Brazil : Faculdade de Filisofia, Ciencias Letras. In Portuguese 45.

Biology, and interaction

Polyandrous Queen Honey Bee 227

Gilley, DC, Tarpy, DR & Land, BB (2003) Effect of queen quality on interactions between workers and dueling queens in honeybee ( Apis mellifera L.) colonies. Behav Ecol Sociobiol, 55, 190-6. [http://dx.doi.org/10.1007/s00265-003-0708-y] Gorbaczaw, WK (1961) Colonies without drones--future beekeeping.]. Pszczearstwo, 12, 19-21. Gregorc, A & Škerl, MIS (2015) Characteristics of honey bee (Apis mellifera Carnica, Pollman 1879) queens reared in Slovenian commercial breeding stations. J Apic Sci, 59, 5-12. Haarmann, T, Spivak, M, Weaver, D, Weaver, B & Glenn, T (2002) Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations. J Econ Entomol, 95, 28-35. [http://dx.doi.org/10.1603/0022-0493-95.1.28] [PMID: 11942761] Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Herrmann, M, Trenzcek, T, Fahrenhorst, H & Engels, W (2005) Characters that differ between diploid and haploid honey bee (Apis mellifera) drones. Genet Mol Res, 4, 624-41. [PMID: 16475107] Howell, DE & Usinger, RL (1933) Observations on the flight and length of life of drone bees. Ann Entomol Soc Am, 26, 239-46. [http://dx.doi.org/10.1093/aesa/26.2.239] Jay, SC (1963) The development of honeybees in their cells. J Apic Res, 2, 117-34. [http://dx.doi.org/10.1080/00218839.1963.11100072] Johnson, JN, Hardgrave, E, Gill, C & Moore, D (2010) Absence of consistent diel rhythmicity in mated honey bee queen behavior. J Insect Physiol, 56, 761-73. [http://dx.doi.org/10.1016/j.jinsphys.2010.01.004] [PMID: 20116381] Kahya, Y, Gençer, HV & Woyke, J (2008) Weight at emergence of honey bee ( Apis mellifera caucasica ) queens and its effect on live weights at the pre and post mating periods. J Apic Res, 47, 118-25. [http://dx.doi.org/10.1080/00218839.2008.11101437] Katzav-Gozansky, T., Soroker, V. and Hefetz, A., (1997). The biosynthesis of Dufour's gland constituents in queens of the honeybee. Apis mellifera, pp.239-243. Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Kepena, L (1963) On the biology of drones of the Tatranka breed. Proceedings XIX International Beekeeping Congress, 307-8. Kerr, WE (1974) Advances in cytology and genetics of bees. Annu Rev Entomol, 19, 253-68. [http://dx.doi.org/10.1146/annurev.en.19.010174.001345] Kerr, WE & Silveira, ZV (1974) A note on the formation of honeybee spermatozoa. J Apic Res, 13, 121-6. [http://dx.doi.org/10.1080/00218839.1974.11099767] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2009) Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees. Behav Ecol, 20, 1007-14. [http://dx.doi.org/10.1093/beheco/arp090] [PMID: 22476212] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2008) Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera). BMC Genomics, 9, 232. [http://dx.doi.org/10.1186/1471-2164-9-232] [PMID: 18489784] Koeniger, N (1969) Experiments concerning the ability of the queen (Apis mellifera L.) to distinguish between the drone and worker cells. XXII International Beekeeping Congress Summit, 138.

228 Polyandrous Queen Honey Bee

Lovleen Marwaha

Koeniger, N & Koeniger, G (2000) Reproductive isolation among species of the genus Apis. Apidologie (Celle), 31, 313-39. [http://dx.doi.org/10.1051/apido:2000125] Kurennoj, NM (1953) [Flight activity and sexual maturity of drones.) Pche/ovodstvo 31pp. 24-28 In Russian Kurze, C., Dosselli, R., Grassl, J., Le Conte, Y., Kryger, P., Baer, B. and Moritz, R.F., 2016. Differential proteomics reveals novel insights into Nosema–honey bee interactions. Insect Biochem Mol Biol, 79, 42-9. Lavrekmn, F.A. (1960). Comparative observations on the flight activity of drones. Pche/ovodstvo 37 pp. 4345 In Russian. Levenets, IP (1956) Pchelovodstvo, 33, 28-9. [Observations on the expulsion of drones.]. [In Russian.]. Lodesani, M, Balduzzi, D & Galli, A (2004) Functional characterisation of semen in honeybee queen ( A.m.ligustica S.) spermatheca and efficiency of the diluted semen technique in instrumental insemination. Ital J Anim Sci, 3, 385-92. [http://dx.doi.org/10.4081/ijas.2004.385] Louveaux, J, Mesquida, J & Fresnaye, J (1972) OBSERVATIONS SUR LA VARIABILITÉ DE LA PRODUCTION DU COUVAIN DE MÂLES DANS LES COLONIES D’ABEILLES (Apis mellifica L.). Apidologie (Celle), 3, 291-307. [Observations on the variability of drone brood production by honeybee colonies.]. [http://dx.doi.org/10.1051/apido:19720401] Mackensen, O (1964) Mackensen, O. .1964 Relation of Semen Volume to Success inArtificial Insemination of QueenHoney Bees. JEcon Entomol 57, 581-3. Mazeed, AM & Mohanny, KM (2010) Some reproductive characteristics of honeybee drones in relation to their ages. Entomol Res, 40, 245-50. [http://dx.doi.org/10.1111/j.1748-5967.2010.00297.x] McMenamin, A, Daughenbaugh, K, Parekh, F, Pizzorno, M & Flenniken, M (2018) Honey Bee and Bumble Bee Antiviral Defense. Viruses, 10, 395-407. [http://dx.doi.org/10.3390/v10080395] [PMID: 30060518] (1976) Metz, B & Tarpy, D (2019) Reproductive Senescence in Drones of the Honey Bee (Apis mellifera). Insects, 10, 11. [http://dx.doi.org/10.3390/insects10010011] [PMID: 30626026] Morse, RA, Strang, GE & Nowakowski, J (1967) Fall death rates of drone honeybees. J Econ Entomol, 60, 1198-202. [http://dx.doi.org/10.1093/jee/60.5.1198] Niño, EL, Malka, O, Hefetz, A, Teal, P, Hayes, J & Grozinger, CM (2012) Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology. J Insect Physiol, 58, 1082-9. [http://dx.doi.org/10.1016/j.jinsphys.2012.04.015] [PMID: 22579504] Niño, EL, Malka, O, Hefetz, A, Tarpy, DR & Grozinger, CM (2013) Chemical profiles of two pheromone glands are differentially regulated by distinct mating factors in honey bee queens (Apis mellifera L.). PLoS One, 8e78637 [http://dx.doi.org/10.1371/journal.pone.0078637] [PMID: 24236028] Niño, EL, Tarpy, DR & Grozinger, CM (2013) Differential effects of insemination volume and substance on reproductive changes in honey bee queens ( Apis mellifera L.). Insect Mol Biol, 22, 233-44. [http://dx.doi.org/10.1111/imb.12016] [PMID: 23414204] Oertel, E (1956) Observations on the flight of drone honey bees. Ann Entomol Soc Am, 49, 497-500. [http://dx.doi.org/10.1093/aesa/49.5.497] Pál, ZÖ (1959) The Behaviour and nutrition of drones. Bee World, 40, 141-6.

Biology, and interaction

Polyandrous Queen Honey Bee 229

[http://dx.doi.org/10.1080/0005772X.1959.11096717] Page, RE, Jr & Laidlaw, HH, Jr (1985) Closed population honeybee breeding. Bee World, 66, 63-72. [http://dx.doi.org/10.1080/0005772X.1985.11098826] Page, RE, Jr & Peng, CYS (2001) Aging and development in social insects with emphasis on the honey bee, Apis mellifera L. Exp Gerontol, 36, 695-711. [http://dx.doi.org/10.1016/S0531-5565(00)00236-9] [PMID: 11295509] Pankiw, T, Winston, ML, Plettner, E, Slessor, KN, Pettis, JS & Taylor, OR (1996) Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.). J Chem Ecol, 22, 605-15. [http://dx.doi.org/10.1007/BF02033573] [PMID: 24227572] Peer, DF (1956) Multiple matings of queen honeybees. J Econ Entomol, 49, 741-3. [http://dx.doi.org/10.1093/jee/49.6.741] Peng, Y, Grassl, J & Millar, AH (2015) Seminalfluidofhoneybeescontainsmultiplemechanismstocombat infections of the sexually transmitted pathogen Nosema apis. Proc Biol Sci, 283, 1785. Peso, M, Niño, EL, Grozinger, CM & Barron, AB (2013) Effect of honey bee queen mating condition on worker ovary activation. Insectes Soc, 60, 123-33. [http://dx.doi.org/10.1007/s00040-012-0275-1] Rangel, J, Böröczky, K, Schal, C & Tarpy, DR (2016) Honey bee (Apis mellifera) queen reproductive potential affects queen mandibular gland pheromone composition and worker retinue response. PLoS One, 11e0156027 [http://dx.doi.org/10.1371/journal.pone.0156027] [PMID: 27281328] Renner, M & Baumann, M (1964) Über Komplexe von subepidermalen Drüsenzellen (Duftdrüsen?) der Bienenkönigin. Naturwissenschaften, 51, 68-9. [http://dx.doi.org/10.1007/BF00603470] Rhodes, J (2002) Drone Honey Bees-Rearing and MaintenanceNSW Agriculture, Orange, Australia. Richard, FJ, Schal, C, Tarpy, DR & Grozinger, CM (2011) Effects of instrumental insemination and insemination quantity on Dufour’s gland chemical profiles and vitellogenin expression in honey bee queens (Apis mellifera). J Chem Ecol, 37, 1027-36. [http://dx.doi.org/10.1007/s10886-011-9999-z] [PMID: 21786084] Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2e980 [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Roberts, KE, Evison, SEF, Baer, B & Hughes, WOH (2015) The cost of promiscuity: sexual transmission of Nosema microsporidian parasites in polyandrous honey bees. Sci Rep, 5, 10982. [http://dx.doi.org/10.1038/srep10982] [PMID: 26123939] Rueppell, O, Aumer, D & Moritz, RFA (2016) Ties between ageing plasticity and reproductive physiology in honey bees (Apis mellifera) reveal a positive relation between fecundity and longevity as consequence of advanced social evolution. Curr Opin Insect Sci, 16, 64-8. [http://dx.doi.org/10.1016/j.cois.2016.05.009] [PMID: 27720052] Rueppell, O, Fondrk, MK & Page, RE, Jr (2005) Biodemographic analysis of male honey bee mortality. Aging Cell, 4, 13-9. [http://dx.doi.org/10.1111/j.1474-9728.2004.00141.x] [PMID: 15659209] Ruttner, F, Woyke, J & Koeniger, N (1972) Reproduction in Apis cerana 1. Mating behaviour. J Apic Res, 11, 141-6. [http://dx.doi.org/10.1080/00218839.1972.11099714] Ruttner, F (1966) The life and flight activity of drones. Bee World, 47, 93-100. [http://dx.doi.org/10.1080/0005772X.1966.11097111] Sandrock, C, Tanadini, M, Tanadini, LG, Fauser-Misslin, A, Potts, SG & Neumann, P (2014) Impact of

230 Polyandrous Queen Honey Bee

Lovleen Marwaha

chronic neonicotinoid exposure on honeybee colony performance and queen supersedure. PLoS One, 9e103592 [http://dx.doi.org/10.1371/journal.pone.0103592] [PMID: 25084279] Santomauro, G, Oldham, NJ, Boland, W & Engels, W (2004) Cannibalism of diploid drone larvae in the honey bee ( Apis mellifera ) is released by odd pattern of cuticular substances. J Apic Res, 43, 69-74. [http://dx.doi.org/10.1080/00218839.2004.11101114] Sawarkar, AB & Tembhare, DB (2015) Histomorphological study of the male reproductive system in the Indian drone Honeybee, Apis cerana indica (Hymenoptera). National Conference on Advances in Bioscience & Environmental Science: Present & Future (ABES), 63. Schlüns, H, Moritz, RFA, Neumann, P, Kryger, P & Koeniger, G (2005) Multiple nuptial flights, sperm transfer and the evolution of extreme polyandry in honeybee queens. Anim Behav, 70, 125-31. [http://dx.doi.org/10.1016/j.anbehav.2004.11.005] Schmid, MR, Brockmann, A, Pirk, CWW, Stanley, DW & Tautz, J (2008) Adult honeybees (Apis mellifera L.) abandon hemocytic, but not phenoloxidase-based immunity. J Insect Physiol, 54, 439-44. [http://dx.doi.org/10.1016/j.jinsphys.2007.11.002] [PMID: 18164310] Seeley, TD & Tarpy, DR (2007) Queen promiscuity lowers disease within honeybee colonies. Proc Biol Sci, 274, 67-72. [http://dx.doi.org/10.1098/rspb.2006.3702] [PMID: 17015336] Slessor, KN, Kaminski, LA, King, GGS & Winston, ML (1990) Semiochemicals of the honeybee queen mandibular glands. J Chem Ecol, 16, 851-60. [http://dx.doi.org/10.1007/BF01016495] [PMID: 24263600] Strang, GE (1970) A study of honey bee drone attraction in the mating response. J Econ Entomol, 63, 641-5. [http://dx.doi.org/10.1093/jee/63.2.641] Taber, S, III (1964) Factors influencing the circadian flight rhythm of drone honey bees. Ann Entomol Soc Am, 57, 769-75. [http://dx.doi.org/10.1093/aesa/57.6.769] Tanaka, ED & Hartfelder, K (2004) The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropod Struct Dev, 33, 431-42. [http://dx.doi.org/10.1016/j.asd.2004.06.006] [PMID: 18089049] Tarpy, DR (2003) Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth. Proc Biol Sci, 270, 99-103. [http://dx.doi.org/10.1098/rspb.2002.2199] [PMID: 12596763] Tarpy, DR, Hatch, S & Fletcher, DJC (2000) The influence of queen age and quality during queen replacement in honeybee colonies. Anim Behav, 59, 97-101. [http://dx.doi.org/10.1006/anbe.1999.1311] [PMID: 10640371] Tarpy, DR & Seeley, TD (2006) Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queens. Naturwissenschaften, 93, 195-9. [http://dx.doi.org/10.1007/s00114-006-0091-4] [PMID: 16518641] de Oliveira Tozetto, S, Bitondi, MMG, Dallacqua, RP & Simões, ZLP (2007) Protein profiles of testes, seminal vesicles and accessory glands of honey bee pupae and their relation to the ecdysteroid titer. Apidologie (Celle), 38, 1-11. [http://dx.doi.org/10.1051/apido:2006045] Winston, ML (1987) The Biology of the Honey BeeHarvard University Press, Cambridge, MA, USA. Witherell, P C (1965) Survival of drones following eversion [http://dx.doi.org/10.1051/apido:19650405] Witherell, P C (1972) Witherell, P. C. 1972. Flight activity and natural monality of normal and mutant drone honeybees. J Apic Res 11, 65-75.

Biology, and interaction

Polyandrous Queen Honey Bee 231

Withrow, JM & Tarpy, DR (2018) Cryptic “royal” subfamilies in honey bee (Apis mellifera) colonies. PLoS One, 13e0199124 [http://dx.doi.org/10.1371/journal.pone.0199124] [PMID: 29995879] Woyke, J (1962) Natural and Artificial Insemination of Queen Honeybees. Bee World, 43, 21-5. [http://dx.doi.org/10.1080/0005772X.1962.11096922] Woyke, J (1962) The hatchability of “lethal” eggs in a two-sex allele fraternity of honeybees. J Apic Res, 1, 6-13. a [http://dx.doi.org/10.1080/00218839.1962.11100040] Woyke, J (1963) Rearing and viability of diploid drone larvae. J Apic Res, 2, 77-84. b [http://dx.doi.org/10.1080/00218839.1963.11100064] Woyke, J (1963) What happens to diploid drone larvae in a honeybee colony? J Apic Res, 2, 73-5. c [http://dx.doi.org/10.1080/00218839.1963.11100063] Woyke, J (1965) Study on the comparative viability of diploid and haploid larval drone honeybees. J Apic Res, 4, 12-6. [http://dx.doi.org/10.1080/00218839.1965.11100096] Woyke, J (1974) Genic balance, heterozygosity and inheritance of testis size in diploid drone honeybees. J Apic Res, 13, 77-91. [http://dx.doi.org/10.1080/00218839.1974.11099763] Woyke, J (1977) Cannibalism and brood-rearing efficiency in the honeybee. J Apic Res, 16, 84-94. [http://dx.doi.org/10.1080/00218839.1977.11099866] Woyke, J (1978) Comparative biometrical investigation on diploid drones of the honeybee. II. The thorax. J Apic Res, 17, 195-205. a [http://dx.doi.org/10.1080/00218839.1978.11099927] Woyke, J (1978) Comparative biometrical investigation on diploid drones of the honeybee. III. The abdomen and weight. J Apic Res, 17, 206-17. b [http://dx.doi.org/10.1080/00218839.1978.11099928] Woyke, J & Ruttner, F (1958) An Anatomical Study of the Mating Process in the Honeybee. Bee World, 39, 3-18. [http://dx.doi.org/10.1080/0005772X.1958.11095028] Woyke, J & Knytel, A (1966) The chromosome number is proof that drones can arise from fertilized eggs of the honeybee. J Apic Res, 5, 149-54. [http://dx.doi.org/10.1080/00218839.1966.11100148] Woyke, J, Knytel, A & Bergandy, K (1966) The presence of spermatozoa in eggs as proof that drones can develop from inseminated eggs of the honeybee. J Apic Res, 5, 71-8. [http://dx.doi.org/10.1080/00218839.1966.11100137] Wu-Smart, J & Spivak, M (2016) Sub-lethal effects of dietary neonicotinoid insecticide exposure on honey bee queen fecundity and colony development. Sci Rep, 6, 32108. [http://dx.doi.org/10.1038/srep32108] [PMID: 27562025] Yue, C, Schröder, M, Bienefeld, K & Genersch, E (2006) Detection of viral sequences in semen of honeybees (Apis mellifera): Evidence for vertical transmission of viruses through drones. J Invertebr Pathol, 92, 105-8. [http://dx.doi.org/10.1016/j.jip.2006.03.001] [PMID: 16630626]

232

Polyandrous Queen Honey Bee, 2022, 232-246

CHAPTER 12

Mating and Reproduction in Queen Honey Bee Abstract: Queen honey bee is polyandrous, per her mating tendency with multiple drones of other colonies in the drone congregation area. Post-hatching, the virgin queen takes a few short flights near the colony, and eventually, within two weeks of her posthatching, she takes 1-2 nuptial flights for mating. During mating, the queen receives ample sperm storage for her entire life. Virgin queen attracts drones through her characteristic pheromonal profiling, especially by mandibular gland pheromones. After mating, drones die, and the queen returns to the native hive to perpetuate the species. The queen can regulate the fertilization of eggs; therefore, a colony is the composite aggregation of bees of different patriline inheritance. Queens can lay two types of eggs, fertilized and unfertilized, which tend to develop into female and male castes. Therefore, the queen can potentially regulate the overall strength of the colony and caste ratio to direct the colony in a specific direction.

Keywords: Drone congregation area, Mating, Queen honey bee, Reproduction. 12.1. INTRODUCTION Queen honey bees lay down two types of eggs: fertilized and unfertilized. Fertilized eggs develop into queens or workers, depending upon larval feed given specifically during 4-9 days, whereas unfertilized/fertilized eggs develop into honey bee drones. For the colony to survive, the queen must be able to lay fertilized eggs. In addition, the colony needs workers to accomplish various tasks, including foraging, honey processing, storing, rearing of brood, feeding brood, ventilation, and temperature regulation (Fig. 12a - f). Each colony is ruled by a monopolous polyandrous queen, who mates during the early stages of her life cycle and possesses spermathecae, with a storage capacity of 5 million sperms. Workers bees replace a less fertile honey bee queen with a newly reared reproductively more active queen. The fecundity of the busy queen honey bee is exponentially high, as her egg-laying capacity can increase upto 2000 in a day. The egg of the honey bee measures about 1 to 1.5 mm long, with a similar appearance to rice grain. Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

Mating and Reproduction

Polyandrous Queen Honey Bee 233

Fig. (12a). Comprehensive depiction of various duties of worker bees in the honey bee colony. The worker honey bee starts performing the colonial task from the very beginning of hatching. Further, workers' life span depends upon the worker assigned to them. Therefore, excessive work, somewhere affect the longevity of worker honey bees.

Fig. (12b). Depicting antennal communication of worker honey bees at the hive's entrance. Honey bees exchange information with each other during the performance of different duties in the comb.

234 Polyandrous Queen Honey Bee

Lovleen Marwaha

Fig. (12c). Click highlighting Group of workers exchanging information on the hive. Workers exchange information by antennal contact, dances, volatile emission etc., for brood rearing, foraging, honey processing, protection etc.

Fig. (12d). elucidate uncapped unripe honey cells and covered ripe honey cells. Worker honey bees perform different duties in the hive.

Mating and Reproduction

Polyandrous Queen Honey Bee 235

Fig. (12e). Worker honey bees taking artificial diet at the hive entrance. Worker honey bees prefer an artificial diet provided on yellow-coloured paper.

Fig. (12f). Worker Honey bees perform different duties like adding worker jelly to larval cells and capping prepupal cells.

236 Polyandrous Queen Honey Bee

Lovleen Marwaha

During oviposition, the queen examines different cells and selects wax cells appropriately constructed. After that, she lays an egg in that specific cell within a few seconds. Further, the fecundity and egg-laying pattern vary per the queen's age. The young queen lays eggs in an organized way in the centre of the comb so that workers can apply royal jelly, honey and other food commodities per caste and developmental features of the particular stage. In contrast, the older queen honey bee lays eggs on the periphery in a less organized manner. Eggs hatch into larvae within three days and subsequently feed on honey, royal jelly and liquid from plants (Fig. 12g-j). Honey bee larvae are without legs, eyes, antennae or wings. Virgin queen is polyandrous as it mates with multiple drones during flight. Drone inseminates queen with his endophallus and post-mating. The endophallus remains attached to the queen's body. For the next male to mate, it removes the endophallus of the already mated male and then inseminates the queen. Drones can mate 7-10 times during the flight and die quickly.

Fig. (12g). Comparision of hive sections from two separate colonies to highlight egg laying pattern of different queen (7a) section from a weak colony headed by the poor fertile queen as most of the wax cells are empty (7b) section captured from colony governed by a strong queen, egg laying pattern indicate her exponential fecundity.

Mating and Reproduction

Polyandrous Queen Honey Bee 237

Fig. (12h). Photographic click at the entrance of hive showing worker honey bees carrying pollens, filled pollen basket on metathoracic legs.

Fig. (12j). Section of the hive with uncapped larvae, uncapped unripe honey cells and with few worker honey bees.

238 Polyandrous Queen Honey Bee

Lovleen Marwaha

Virgin queen mates early in life, during the mating flight. Queen can store about 100 million sperms in her oviduct, but she can retain about 5 million sperms in her spermathecae. Further, the queen uses only a fraction of that for fertilization during her entire life. Queen honey bees can control the sex of offspring by regulating the fertilization of eggs, as unfertilized eggs develop into males, whereas fertilized eggs can develop into queens or workers. Queen larvae are fed on royal jelly during complete larval development. Worker larvae are fed on royal jelly for three days; after that, worker larvae are fed on worker jelly. For the development of the male caste, about 24 days are required, whereas for the development of queen and workers honey bees, about 16 and 21 days are needed, respectively (Fig. 12i).

Fig. (12i). representing uncapped larvae and capped larvae. Additionally, worker honey bees are engaged in adding worker jelly in a particular section of the hive.

12.2. MATING AND FREQUENCY OF OCCURRENCE The Queen honey bee is polyandrous, with a usual single mating frequency (Woyke, 1955; Tryasko, 1956; Taber, 1958; Laidlaw et al., 1984; Taber, 2014). Subsequently, mating occurs in flight in drone congregation areas (DCA) (Jean et al., 1957; Ruttner, 1962; Zmarlicki and Morse, 1963; Laidlaw et al., 1984). In the

Mating and Reproduction

Polyandrous Queen Honey Bee 239

drone congregation area, about 8000 to 15000 drones can be present during a specific time (Koeniger and Koeniger,2000; Koeniger et al., 2005). Queen usually carries out a few small flights before the nuptial flight (Fletcher and Tribe, 1977; Koeniger, 1986). There is multiple mating during single flight (Roberts, 1944; Ruttner, et al., 1954; Woyke, 1960; Woyke, 1964; Franck, et al., 2002; Schlüns, et al., 2005; Koeniger, and Koeniger, 2007). Usually, 6-26 male drones mate with the queen, with the average number corresponding to 12-14 (Estoup et al., 1994; Neumann et al., 1999; Tarpy et al., 2004). Some researchers consider that it is the influence of the insufficient number of stored spermatozoa in spermathecae and insufficient mating number, which act as a factor to promote the queen to make the additional mating flight or for oviposition (Woyke, 1964; Woyke, 1966; Schlüns et al., 2005). Whereas some of the researchers considered that the number of mating times is dependent upon mating behaviour and mate availability (Tarpy et al., 2000). Additionally, mating behaviour also depends upon the queen's age, temperature, wind and cloud cover (Alber et al., 1955; Oertel., 1956; Verbeek, 1976; Lensky and Demeter, 1985; Koeniger, 1986). Furthermore, the number of drones within the queen's flight range has been considered an essential factor influencing nuptial flights (Koeniger and Koeniger, 2007). Post-mating drones usually die, whereas the queen returns to the hive (Woyke,1958; Koeniger, 1990). Further, the number of mating drones and mating success can be assessed from the number of stored spermatozoa in spermathecae (Woyke, 1960; Woyke, 1964; Estoup et al., 1994; Neumann et al., 1999). It is challenging to observe the mating of queens and drones, despite the availability of advanced methods and techniques. Earlier, mating studies had been carried out on free-flying queens (Janscha et al., 1775; Langstroth, 1861; Demaree,1881; Shuck, 1882), whereas later on, studies had been carried out on wooden queen dummies coated with queen pheromones (Janscha, 1775; Shuck, 1861; Langstroth, 1861; Demaree,1881; Koeniger et al., 1990). 12.3. FACTORS AFFECTING MATING Mating in honey bees is influenced by queen quality, age of queen, pheromonal profile, temperature, wind speed, etc. According to Alber et al., 1955, mating is hampered at a temperature below 20 °C and with wind velocity above 30 km/h. Lensky, and Demeter, 1985, reported that high wind velocity with a corresponding range of 9–14 km/h and low temperature of 15 ° C–20 °C facilitate short-range queen flights, furtherHeidinger, et al., 2014, reported that with the decrease in temperature, there is also increase in flight frequency. Sometimes, the

240 Polyandrous Queen Honey Bee

Lovleen Marwaha

queen comes after the nuptial flight as unmated from DCA (Taber, 1954; Alber et al., 1955; Woyke, 1956; Heidinger et al., 2014). Further, mating is influenced by pheromonal profiling of queen honey bees. She possesses a sizeable glandular system responsible for the secretion of specific pheromones, which attract a male for mating. Honey bees gather in drone congregation areas, about 10-40m above ground, with an approximate diameter of about 30-200 m((Jean-Prost, 1957; Ruttner,1957; Ruttner and Ruttner, 1966; Ruttner and Ruttner,1972; Koeniger et al., 1979; Loper et al., 1987; Loper et al., 1992Michener, 2000; Ayasse et al., 2001; Danforth et al., 2006; Baer, 2005; Koeniger et al., 2014). A drone congregation can carry about 11,000 drones from different colonies (Free, 1987; Loper et al., 1992; Baudry et al., 1998; Koeniger et al., 2005). After drone departure, the queen leaves the hive about half an hour later (Jean-Prost, 1957; Ruttner and Ruttner, 1965; Koeniger and Koeniger, 2004). Furthermore, the specific congregation of drones has been studied by pheromone traps attached to helium-filled balloons (Koeniger, 1990; Koeniger et al., 2005). Male drones frequently fly before mating, whereas the queen takes comparatively few flights (Alber et al., 1955; Ruttner, 1956; Witherell, 1972). Drones take nuptial flight between 12:00-17:00 h, with peak activity between 13:00 and 16:00 hrs (Alber et al., 1955; Ruttner, 1956; Taber,1964; Drescher et al., 1969; Witherell, 1972; Verbeek, 1976; Bol'Shakova et al., 1978; Lensky, and Demter, 1985; Koeniger et al., 1989). Additionally, the mating behaviour is influenced by factors like temperature, wind and cloud (Oertel,1956; Verbeek,1976; Bol'Shakova, 1978; Lensky and Demter, 1985). 12.4. PHEROMONES Drone gets attracted to principal queen pheromones, including 9-oxo-2-decanoic acid (9-ODA). Queen is the only fertile female in the honey colony and imposes her reproductive dominance through the secretion of certain volatile chemicals, especially the mandibular gland. QMP regulates workers' Behaviour by suppressing the development of workers' ovarian development (Hoover et al., 2003). QMP comprises 9-oxo(E)-2-decanoic acid (9-ODA), two enantiomers of 9-ODA's biosynthetic precursor, (R)- and (S)-9-hydroxy-(E)-2-decanoic acid (9HDA), and two other compounds, methyl p-hydroxybenzoate (HOB) and 4hydroxy-3-methoxyphenylethanol (HVA). Virgin queen secretes more 9-ODA, whereas, in post-mating, there is a reduction in concerned pheromones (Pankiw et al., 1996; Plettner et al., 1997).9-ODA acts as a major attractant for drones toward virgin queen to congregation areas (Gary, 1962; Butler and Fairey, 1964; Loper et al., 1993). Further, it had been concluded that 9-ODAis not equally effective as complete queen extract. 9-HDA and 10-hydroxy-(E)-2-decanoic acid (10-HDA)

Mating and Reproduction

Polyandrous Queen Honey Bee 241

increase the number of mating contact of drones with dummy queens. Queen sex pheromones, therefore, are composed of a blend of different chemicals, with a clear role of 9-ODA. The formation of a congregation area is still not a very clear concept. The factors that induced drone and queen gathering near congregational area is not very clear to date. The different exact location of the drone congregation area year after year is not adequately explored (Jean-Prost, 1960; Ruttner and Ruttner, 1968; Ruttner, 1985; Koeniger and Koeniger, 2004). 12.5. DRONES CONGREGATION AREAS The drone congregation is the area outside the colony, where thousands of males gather, along with the reproductive queen, during afternoon hours. Mating in honey bees takes place in the air at a height which could be upto 15-60 m (Oertel 1956; Zmarlicki and Morse 1963; Loper et al.1992). All DCA events occur in the congregational area or on the in-flight path of drones (Laidlaw and Page 1984: Koeniger et al. 2005:Schlüns et al. 2005). The factors that serve as an attractant for drones and queens to the specific site are unknown, and it has been hypothesized that factors include landscape (Winston 1987). DCA is usually from about 500m to 5 km away from the colony (Ruttner 1976). According to the “behavioural DCA hypothesis, DCA could be formed due to the interaction of flying drones and queens (Loper et al. 1992). DCA forms per the geomagnetism of earth, as honey bees possess magnetic sense orientation. Additionally, sunlight affects the formation of DCA (Winston 1987; Soucy et al. 2003), and other factors, including wind direction and wind speed, influence DCA. Drone congregation areas remain constant yearly (JeanProst,1960; Ruttner and Ruttner, 1968; Ruttner, 1985). Small artificial congregation areas had been created by using a small number of queen pheromones, and the queen’s presence is not required to form a drone congregation (Strang, 1970). Drone congregation areas usually include the top of mountains, valleys and tree tops (Ruttner and Ruttner, 1966; 1972; Loper, 1985Ruttner, 1985; Pechhacker, 1994). Queens enter into congregation area about 1 hr after drones (Jean-Prost, 1957; Ruttner, 1985; Koeniger and Koeniger, 2004). As the virgin queen enters the area, many drones become attracted to her due to the secretion of 9-oxo-2-decanoic acid, 9-ODA and visualization (Gary, 1962; Gries and Koeniger, 1996). But the role of mandibular glands is debatable as it undergoes degeneration at 9 days, just before the nuptial flight (Ruttner, 1985; Lensky et al., 1985; Brandstaetter et al., 2014). The queen and drone move in a comet swarm, with each drone competing for an approximate position for mating (Gries and Koeniger, 1996). For 15-30

242 Polyandrous Queen Honey Bee

Lovleen Marwaha

minutes, the queen mate with 10-20 drones, which die after mating (Baudry et al., 1998; Palmer and Oldroyd, 2000; Schluns et al., 2005). Queen takes 1-2 nuptial flights and returns to the hive to start laying eggs (Koeniger et al., 2014). The queen is not attracted to drones within the pack (Le Conte and Hefetz,2008). About 1000 drones are required to stabilize the congregation area (Koeniger et al., 2014). Sometimes, the queen is not able to come back to the colony post-mating, which indicates the significant mating risk (Jean-Prost, 1957; Ruttner and Ruttner, 1965; Ruttner and Ruttner, 1966; Ruttner and Ruttner, 1972; Gerig,1972 Ruttner, 1985; 1988Lensky et al., 1985: Free, 1987; Pechhacker, 1994; Koeniger and Koeniger, 2004). In addition, drones can discriminate between the QMP of the virgin and mated queen. 12.6. DRONE ATTRACTION Additionally, the drones can become attracted to other drones due to specific chemicals (Brandstaetter et al., 2014). The drone produced an attractive odour substance, facilitating the formation of the drone congregation area. Furthermore, the drones secrete specific pheromones required for queen attraction. Drone pheromones serve as attractants only for certain particular females but not to workers of the same group. Further, the drone pheromones induce queens to travel a longer distance (Brandstaetter et al., 2014). Sex attractants are detected by antennae through olfactory receptors neuron, then to the primary olfactory centre, which includes the antennal lobe, and subsequently to the higher centre of the brain, including mushroom bodies and the lateral horn. CONCLUSION In short, mating in honey bees occurs during the nuptial flight in a drone congregational area, where drones from different colonies gather and secrete sex attractants from virgin queens of various colonies for mating. Drones die subsequent o post-mating, whereas the queen returns and stores sperms in her spermatheca for fertilization of eggs. REFERENCES Alber, M (1955) Von der paarung der honigbiene. Z Bienenforsch, 3, 1-28. Ayasse, M, Paxton, RJ & Tengö, J (2001) Mating behavior and chemical communication in the order Hymenoptera. Annu Rev Entomol, 46, 31-78. [http://dx.doi.org/10.1146/annurev.ento.46.1.31] [PMID: 11112163] Baer, B (2005) Sexual selection in Apis bees. Apidologie (Celle), 36, 187-200. [http://dx.doi.org/10.1051/apido:2005013] Baudry, E, Solignac, M, Garnery, L, Gries, M, Cornuet, J & Koeniger, N (1998) Relatedness among honeybees (Apis mellifera) of a drone congregation. Proc Biol Sci, 265, 2009-14.

Mating and Reproduction

Polyandrous Queen Honey Bee 243

[http://dx.doi.org/10.1098/rspb.1998.0533] Bol'Shakova, M.D. and BOL'SHAKOVA, M.D., The flight of honey bee drones, Apis mellifera L.(Hymenoptera, Apidae), to the queen concerning various ecological factors. Brandstaetter, AS, Bastin, F & Sandoz, JC (2014) Honeybee drones are attracted by groups of consexuals in a walking simulator. J Exp Biol, 217, jeb.094292. [http://dx.doi.org/10.1242/jeb.094292] [PMID: 24436379] Conte, YL & Hefetz, A (2008) Primer pheromones in social hymenoptera. Annu Rev Entomol, 53, 523-42. [http://dx.doi.org/10.1146/annurev.ento.52.110405.091434] [PMID: 17877458] Danforth, BN, Sipes, S, Fang, J & Brady, SG (2006) The history of early bee diversification based on five genes plus morphology. Proc Natl Acad Sci USA, 103, 15118-23. [http://dx.doi.org/10.1073/pnas.0604033103] [PMID: 17015826] Demaree, GW (1881) Fertilization in confinement. Am Bee J, 17 Drescher, W (1969) Die flugaktivität von drohnen der rasse apis mellifica carnica l. Und a. Mell. Ligustica l. In abhängigkeit von lebensalter und witterung. Z Bienenforsch, 9, 390-409. Estoup, A, Solignac, M & Cornuet, JM (1994) Precise assessment of the number of patrilines and of genetic relatedness in honeybee colonies. Proc Biol Sci, 258, 1-7. [http://dx.doi.org/10.1098/rspb.1994.0133] Fletcher, DJC & Tribe, GD (1977) b.—Natural emergency queen rearing by the African bee Apis mellifera adansonii and its relevance for successful queen production by beekeepers, II. In: Pretoria, South Africa, (Ed.), Proc Apimondia Inter Symp, 161-8. Franck, P, Solignac, M, Vautrin, D, Cornuet, JM, Koeniger, G & Koeniger, N (2002) Sperm competition and last-male precedence in the honeybee. Anim Behav, 64, 503-9. [http://dx.doi.org/10.1006/anbe.2002.3078] Free, JB (1987) Pheromones of social beesChapman and Hall. Gary, NE (1962) Chemical mating attractants in the queen honey bee. Science, 136, 773-4. [http://dx.doi.org/10.1126/science.136.3518.773] [PMID: 17752107] Gerig, L (1972) Ein weiterer Duftstoff zur Anlockung der Drohnen von Apis mellifica (L.)1. Z Angew Entomol, 70, 286-9. [http://dx.doi.org/10.1111/j.1439-0418.1972.tb02183.x] Gries, M & Koeniger, N (1996) Straight forward to the queen: pursuing honeybee drones (Apis mellifera L.) adjust their body axis to the direction of the queen. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 179, 539-44. [http://dx.doi.org/10.1007/BF00192319] Heidinger, I, Meixner, M, Berg, S & Büchler, R (2014) Observation of the mating behaviour of honey bee (Apis mellifera L.) queens using radio-frequency identification (RFID): factors influencing the duration and frequency of nuptial flights. Insects, 5, 513-27. [http://dx.doi.org/10.3390/insects5030513] [PMID: 26462822] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Janscha, A (1775) Vollständige Lehre von der Bienenzucht; G, Münzberg, Wien, Austria 236. Jean-Prost, P (1957) Observations sur le vol nuptial des reines d’abeilles. Acad Sci, 245, 2107-10. Jean-Prost, PL (1960) Jean-Prost, P.L. 1960 Apiculture Méridionale: ses Bases, ses Techniques, en 20 Leçons (Hyeres, Koeniger, G (1986) Reproduction and mating behaviour. In: Rinderer, T.E., (Ed.), Bee Genetics and Breeding, Academic Press Inc., London, UK 255-80.

244 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1016/B978-0-12-588920-9.50015-7] Koeniger, G (1990) The role of the mating sign in honey bees, Apis mellifera L.: does it hinder or promote multiple mating? Anim Behav, 39, 444-9. [http://dx.doi.org/10.1016/S0003-3472(05)80407-5] Koeniger, G, Koeniger, N & Fabritius, M (1979) Some detailed observations of mating in the honeybee. Bee World, 60, 53-7. [http://dx.doi.org/10.1080/0005772X.1979.11097736] Koeniger, G, Koeniger, N, Pechhacker, H, Ruttner, F & Berg, S (1989) Assortative mating in a mixed population of European Honeybees, Apis mellifera ligustica and Apis mellifera carnica. Insectes Soc, 36, 12938. [http://dx.doi.org/10.1007/BF02225908] Koeniger, G, Koeniger, N & Tiesler, FK (2014) Paarungsbiologie und Paarungskontrolle bei der HonigbieneDruck H. Buschhausen. Koeniger, N & Koeniger, G (2000) Reproductive isolation among species of the genus Apis. Apidologie (Celle), 31, 313-39. [http://dx.doi.org/10.1051/apido:2000125] Koeniger, N & Koeniger, G (2010) Mating behavior in honey bees (Genus Apis). Trop Agric Res Ext, 7, 13-28. [http://dx.doi.org/10.4038/tare.v7i0.5415] Koeniger, N & Koeniger, G (2007) Mating flight duration of Apis mellifera queens: As short as possible, as long as necessary. Apidologie (Celle), 38, 606-11. [http://dx.doi.org/10.1051/apido:2007060] Koeniger, N, Koeniger, G, Gries, M & Tingek, S (2005) Drone competition at drone congregation areas in four Apis species. Apidologie (Celle), 36, 211-21. [http://dx.doi.org/10.1051/apido:2005011] Koeniger, N, Koeniger, G & Pechhacker, H (2005) The nearer the better? Drones (Apis mellifera) prefer nearer drone congregation areas. Insectes Soc, 52, 31-5. [http://dx.doi.org/10.1007/s00040-004-0763-z] Laidlaw, HH, Jr & Page, RE, Jr (1984) Polyandry in honey bees (Apis mellifera L.): sperm utilization and intracolonic genetic relationships. Genetics, 108, 985-97. [http://dx.doi.org/10.1093/genetics/108.4.985] [PMID: 17246245] Langstroth, LL (1861) Copulation of the queen bee. Am Bee J, 1, 65-6. Lensky, Y, Cassier, P, Notkin, M, Delorme-Joulie, C & Levinsohn, M (1985) Pheromonal activity and fine structure of the mandibular glands of honeybee drones (Apis mellifera L.) (Insecta, Hymenoptera, Apidae). J Insect Physiol, 31, 265-76. [http://dx.doi.org/10.1016/0022-1910(85)90002-2] Lensky, Y & Demter, M (1985) Mating flights of the queen honeybee (Apis mellifera) in a subtropical climate. Comp Biochem Physiol A Comp Physiol, 81, 229-41. [http://dx.doi.org/10.1016/0300-9629(85)90127-6] Loper, GM (1985) Influence of age on the fluctuation of iron in the œnocytes of honey bee (Apis mellifera) drones. Apidologie (Celle), 16, 181-4. [http://dx.doi.org/10.1051/apido:19850208] Loper, GM, Wolf, WW & Taylor, OR, Jr (1992) Honey bee drone flyways and congregation Journal of Insect Science: Vol. 12areas radar observations. J Kans Entomol Soc, 65, 223-30. Loper, GM, Wolf, WW & Taylor, OR, Jr (1987) Detection and monitoring of honeybee drone congregation areas by radar. Apidologie (Celle), 18, 163-72. [http://dx.doi.org/10.1051/apido:19870206]

Mating and Reproduction

Polyandrous Queen Honey Bee 245

Michener, CD (2000) The Bees of the WorldJohns Hopkins University Press. Oertel, E (1956) Observations on the flight of drone honey bees. Ann Entomol Soc Am, 49, 497-500. [http://dx.doi.org/10.1093/aesa/49.5.497] Palmer, KA & Oldroyd, BP (2000) Evolution of multiple mating in the genus Apis. Apidologie (Celle), 31, 235-48. [http://dx.doi.org/10.1051/apido:2000119] Pankiw, T, Winston, ML, Plettner, E, Slessor, KN, Pettis, JS & Taylor, OR (1996) Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.). J Chem Ecol, 22, 605-15. [http://dx.doi.org/10.1007/BF02033573] [PMID: 24227572] Pechhacker, H (1994) Physiography influences honeybee queen’s choice of mating place (Apis mellifera carnica Pollmann). Apidologie (Celle), 25, 239-48. [http://dx.doi.org/10.1051/apido:19940210] Plettner, E, Otis, GW, Wimalaratne, PDC, Winston, ML, Slessor, KN, Pankiw, T & Punchihewa, PWK (1997) Species-and caste-determined mandibular gland signals in honeybees (Apis). J Chem Ecol, 23, 36377. [http://dx.doi.org/10.1023/B:JOEC.0000006365.20996.a2] Roberts, WC (1944) Multiple mating of queen bees proved by progeny and flight tests. Gleanings in Bee Culture, 72, 225-59. Ruttner, F (1954) Mehrfache begattung der bienenkönigin. Zool Anz, 153, 99-105. Ruttner, F (1956) The mating of the honeybee. Bee World, 37, 3-15. [http://dx.doi.org/10.1080/0005772X.1956.11094913] Rutttner, F (1957) Die sexualfunktionen der honigbienen im dienste ihrer sozialen gemeinschaft. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 39, 577-600. [http://dx.doi.org/10.1007/BF00348457] Ruttner, F (1962) Drohnen-sammelplätze. Bienenvater, 83, 1-2. Ruttner, F & Ruttner, H (1965) Untersuchungen über die Flugaktivität und das Paarungsverhalten der Drohnen. II. Beobachtungen an Drohnensammelplätzen. Z Bienenforsch, 8, 1-9. Ruttner, F & Ruttner, H (1966) Untersuchungen über die Flugaktivität und das Paarungsverhalten der Drohnen. III. Flugweite und Flugrichtung der Drohnen. Zeitschrift für Bienenforschung, 8, 332-54. Ruttner, F & Ruttner, H (1968) Untersuchungen über die Flugaktivität und das Paarungsverhalten der Drohnen. IV. Zur Fernorientierung und Ortsstetigkeit der Drohnen auf ihren Paarungsflügen. Z Bienenforsch, 9, 259-68. Ruttner, H (1976) Investigations on the flight activity and the mating behaviour of the drones, 6. The flight on and over mountain ridges. Apidologie (Celle), 7, 331-41. [http://dx.doi.org/10.1051/apido:19760404] Ruttner, H & Ruttner, F (1972) Untersuchungen über die Flugaktivität und das Paarungsverhalten der Drohnen. V.-Drohnensammelplätze und Paarungsdistanz. Apidologie (Celle), 3, 203-32. [http://dx.doi.org/10.1051/apido:19720301] Ruttner, F (1988) Biogeography and Taxonomy of HoneybeesSpringer. [http://dx.doi.org/10.1007/978-3-642-72649-1] Ruttner, F (1985) Reproductive Behaviour in honeybees. Fortschr Zool, 31, 225-36. Schlüns, H, Moritz, RFA, Neumann, P, Kryger, P & Koeniger, G (2005) Multiple nuptial flights, sperm transfer and the evolution of extreme polyandry in honeybee queens. Anim Behav, 70, 125-31. [http://dx.doi.org/10.1016/j.anbehav.2004.11.005] Shuck, SA (1882) Mating of a queen bee. Am Bee J, 18, 789.

246 Polyandrous Queen Honey Bee

Lovleen Marwaha

Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438] Soucy, SL & Giray, T (2003) Solitary and group nesting in the orchid bee Euglossa hyacinthina (Hymenoptera, Apidae). Insectes Soc, 50, 248-55. [http://dx.doi.org/10.1007/s00040-003-0670-8] Strang, GE (1970) A study of honey bee drone attraction in the mating response. J Econ Entomol, 63, 641-5. [http://dx.doi.org/10.1093/jee/63.2.641] Taber, S, III (1954) The frequency of multiple mating of queen honey bees. J Econ Entomol, 47, 995-8. [http://dx.doi.org/10.1093/jee/47.6.995] Taber, S, III & Wendel, J (1958) Concerning the number of times queen bees mate. J Econ Entomol, 51, 7869. [http://dx.doi.org/10.1093/jee/51.6.786] Taber, S, III (1964) Factors influencing the circadian flight rhythm of drone honey bees. Ann Entomol Soc Am, 57, 769-75. [http://dx.doi.org/10.1093/aesa/57.6.769] Tarpy, DR, Nielsen, R & Nielsen, DI (2004) A scientific note on the revised estimates of effective paternity frequency in Apis. Insectes Soc, 51, 203-4. [http://dx.doi.org/10.1007/s00040-004-0734-4] Tarpy, DR & Page, RE, Jr (2000) No behavioural control over the mating frequency in queen honey bees (Apis mellifera L.): implications for the evolution of extreme polyandry. Am Nat, 155, 820-7. [http://dx.doi.org/10.1086/303358] [PMID: 10805647] Tryasko, VV (1956) Repeated and multiple mating of queens. Pchelovodstvo, 33, 43-50. Verbeek, B (1976) Investigation of the flight activity of young honeybee queens under continental and insular conditions by means of photoelectronic control. Apidologie (Celle), 7, 151-68. [http://dx.doi.org/10.1051/apido:19760205] Winston, ML (1987) The biology of the honey beeHarvard University Press. Witherelli, PC (1972) Flight activity and natural mortality of normal and mutant drone honeybees. J Apic Res, 11, 65-75. [http://dx.doi.org/10.1080/00218839.1972.11099702] Woyke, J (1955) Multiple mating of the honeybee queen (Apis mellifica L.) in one nuptial flight. Bull Acad Polon Sci Cl, 3, 175-80. Woyke, J (1956) Anatomy-physiological changes in queen bees were returning from mating flights and the process of multiple mating. Bull Acad Polon Sci, 4, 81-7. Woyke, J (1958) The process of mating in the honeybee. Pszczel Zesz Nauk, 2, 1-42. Woyke, I (1960) Natural and artificial insemination of queen honey bees. Pszczel Zesz Nauk, 4, 183-275. [Polish, English summary.]. Woyke, J (1964) Causes of repeated mating flights by queen honeybees. J Apic Res, 3, 17-23. [http://dx.doi.org/10.1080/00218839.1964.11100077] Woyke, J (1966) Wovon hängt die Zahl der Spermien in der Samenblase der auf natürlichem Wege begatteten Königinnen ab. Z Bienenforsch, 8, 236-47. Zmarlicki, C & Morse, RA (1963) Drone congregation areas. J Apic Res, 2, 64-6. [http://dx.doi.org/10.1080/00218839.1963.11100059]

Polyandrous Queen Honey Bee, 2022, 247-273

247

CHAPTER 13

Swarming and Queen Honey Bee Abstract: The reproductive swarms usually include queens, young worker bees and drones, leaving the native hive to explore the pre-selected site and construct a hive there. Various factors which accelerate swarming events include congestion in the colony, reduced queen pheromones, limited available food resource, different ecological conditions, genetic possession of the colony, etc. Swarming is a significant event for a honey bee colony but drastically affects beekeeping. Therefore apiarists generally take specific measures to control packing events, including proper management of the colony, clipping of queen honey bee's wings, destruction of a queen cell, maintenance of adequate strength of the colony, re-queening of the colony and use of swarm resistance honey bees.

Keywords: Controls, Queen's role, Swarming. 13.1. INTRODUCTION Swarming is a fascinating event in which thousands of bees exhibit collective behaviour of specific movements out from the colony sequentially and coordinatedly (Seeley 2010 and Grozinger et al. 2014). At swarming, about ¼ to ¾ of colonial strength, including the older queen, workers and drones, forms the temporary cluster known as a bivouac. Afterwards, the swarm group migrates toward the selected nest site(Rangel and Seeley, 2012; Rangel et al., 2013). In addition, several reports witness the behavioural signal the queen and workers produced for regulating swarming events (Seeley 2010 and Grozinger et al. 2014). Before swarming, the older queen communicates to give pheromone consent and coordination signals for raising a new queen in the colony (Martin 1963; Butler et al. 1964; Butler and Simpson 1967; Avitabile et al. 1975; Getz et al. 1982; Winston et al. 1982; Fefferman and Starks 2006; Rangel and Seeley 2012; Grozinger et al. 2014; Chauzat et al., 2016). In other words, the presence of a queen honey bee is exquisitely mandatory in the swarm. Otherwise, worker honey bees usually abandon the swarm. Furthermore, reproduction swarming requires simultaneous coordination between queen and worker honey bees. For the execution of a specific queen event, workLovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

248 Polyandrous Queen Honey Bee

Lovleen Marwaha

ers coherently perform nest execution, house hunting and relocation to a new selected site in a synchronized manner. In the original colony, the remaining workers raise new queens from available workers' larvae (Winston 1987; Seeley 2010). In other words, swarming is an event of colony fission involving the migration of queen honey bees with colonial workers and drones. Swarming is helpful for growth and dispersion near food sources (Ratti et al., 2016). Nonreproductive Swarming possesses collective behaviour adaptation for survival and healthy flourishment. When the pathogen number reaches up to threshold, there is an increase in nonreproductive swarms (Fell et al., 1977; Antunez et al., 2015; Goulson et al., 2015; Loftus et al., 2016; Kurze et al., 2016). Loftus et al. 2016 concluded that colonies in a small hive swarm more frequently than colonies in a large hive. Insects can avoid infection by migrating or swarming (Fuchs, 1990; de Roode and Lefèvre, 2012; Rangel and Seeley, 2012). Swarming increases genetic diversity, as a newly hatched Queen can mate with multiple drones (Mattila and Seeley, 2017;) 13.2. FACTORS AFFECTING SWARMING Swarming is a complicated, sequentially stepped process mediated by multiple environmental, social, physiological and molecular factors. Swarming occurs when resources are plentiful and other conditions permit rapid population growth (Simpson 1959). In addition, certain conditions promote swarming, which include an increase in colony size, congestion in the hive, and reduced queen substance/ pheromones (Simpson 1958: Winston 1980, 1987). Honey bee colony, with a density of more than 2.3 workers/mm workers, starts constructing queen cell buildings (LenskyandSlabezki, 1981). In a colony with a strength of about 20,000 bees, about 1600 bee swarm on average (Winston 1979; Winston et al. 1981). The colony also starts building a new Queen when the brood comb is 90% occupied, indicating a correlation between congestion and queen rearing (Winston et al. 1981). Few reports witness that generally, honey bee colonies with congestion swarm earlier than non-congested colonies, whereas some of the reports confirmed swarming in non-congested colonies (Simpson 1957a; Simpson 1957b; Winston et al. 1991). The swarming process could not be triggered by manipulating congestion in the brood nest (Simpson1957a).

Swarming and Queen

Polyandrous Queen Honey Bee 249

Moreover, colonies before swarming generally carry a predominant population of younger bees (Butler 1940; Winston and Taylor 1980; Winston et al. 1981; Gilley1998). Recent reports also indicate that various factors, including colony congestion, colony strength, and the presence of younger bees, promote swarming in honey bees (Fefferman and Starks 2006). According to Christina et al., 2014, various factors promoting swarming act as cues to convey the message about replacing the colony's stability, which means the strength of workers is more in the colony than the brood production capacity of the queen. Therefore colony has reached upto the level of swarming. Therefore, honey bee workers can consider the level of queen pheromone perception to measure colony congestion and strength of the colony (Fefferman and Starks 2006). Swarming changes colony size, worker age distribution and crowding at the nest (Simpson 1958, 1973; Simpson and Riedel 1963; Simpson and Moxley 1971; Winston 1979; Winston and Taylor 1980). In Apis mellifera, as the queen takes multiple nuptial flights and is polyandrous, therefore genetic variation is added to progenies in the same colony, workers inherit different paternal genomic inheritance, consequently behavioural polymorph in swarming tendency also (Roberts 1944; Taber 1954; Woyke 1955; Ruttner and Ruttner 1972; Getz et al., 1982; Christina et al., 2014). Various factors which promote swarming are described in the following sections. 13.2.1. Queen Age As Promoting Factor for Swarming Forster,1969, reported that in honey bee colonies with first-year spring-reared queens, there is no tendency to swarm compared to colonies with two-year-old queens. Honey bee colonies, which swarm, produce less honey in comparison to colonies that do not swarm. A re-queening colony can increase the colony's productivity for honey with spring queen by clipping queens or destroying queen cells. Honey bees swarm to maintain their number. Therefore, Swarming can be considered an intrinsic component of bee behaviour, although Swarming reduces apicultural productivity. Apiarists apply different methods to increase colony production and for prevention of swarming by using manipulating combs, broods, the addition of a queen excluder at the entrance, etc. The addition of empty combs adjacent to the brood provides the Queen ample space for egg laying, which eventually discourages swarming. A single-storey brood nest gives much space to the queen to lay 1800 eggs daily. Additionally, the absence of honey, and stored food

250 Polyandrous Queen Honey Bee

Lovleen Marwaha

provides the queen with enough room to lay eggs. According to Simpson (1958b), colonies that raise new queens but cannot swarm usually produce more honey (Forster,1969). 13.2.2. Reduced Queen Pheromones Queen honey bee secretes certain volatile substances, which regulate the physiology, development, and behaviour of workers and all of these witness the queen's presence in the colony (Kocher and Grozinger 2011). Queen pheromones are circulated in the colony by air current, wax, or contact-to-contact transmission (Seeley1979; LenskyandSlabezki1981; Gilley et al., 2006). It had been considered that reduced concentration of queen pheromones triggers queen-rearing events in the colony. Before swarming, the colony has a massive queen-rearing event, and workers usually construct multiple 10-20 new queen cups (Allen 1956). About two weeks earlier, workers reduced the quantity of food provided to the older Queen (Allen 1955, 1956). A queen, which is about to swarm, possesses less weight than a non-swarming Queen as an adaptation to fly at a specific distance (Seeley and Fell 1981; Allen 1955, 1956; Pierce et al. 2007). Queen pheromones regulate the formation of a new Queen, as worker honey bees consider it a triggering factor to raise a new Queen (Simpson 1958; Winston1980, 1987). Therefore, Queen pheromones’ concentration can decrease due to less production of pheromones or an increase in colony strength, or workers respond less to queen pheromones. Eventually, reduced concentration of queen pheromones toward edges promotes the rearing of queen cells (Seeley 1979). Whole body extract of swarming and non-swarming queens contains equal concentrations of 9-ODA; further, both extracts possess the similar potential to inhibit the production of the queen in the queenless colony (Seeley and Fell 1981; Butler 1960). However, it has been reported that on the addition of 9-ODA in lure in overcrowding colony, the specific chemical does not inhibit queen rearing during swarming season, whereas spraying of specific pheromone does delay the process (Boch and Lensky 1976; Winston et al. 1991). Additionally, applying tarsal and mandibular gland extract on comb edges synergizes the production of queen cells (Lensky and Slebezki 1981). In strong colonies, the queen usually spends less time on edges, which reduces the concentration of footprint pheromones and other queen pheromones (Lensky and Slebezki 1981). Christina et al., 2014, described the chemical and non-chemical factors affecting the Swarming of honey.

Swarming and Queen

Polyandrous Queen Honey Bee 251

13.2.3. Infection as Cause of Swarming Various pathogens and parasites pose significant threats to the fitness and survival of the queen and other castes of honey bees in a colony (Goulson et al., 2015). With innate immunity, some additional behavioural alterations for protection from specific pathogens and parasites (de Roode and Lefèvre, 2012) include complete colony migration or swarming, also termed reproductive Swarming. Polyandry condition helps better resist infection against the pathogen (Lee et al., 2013). It has been reported that diverse genetic colonies possess resistance against multiple pathogens (Diao and Hou, 2018). 13.3. ROLE OF QUEEN MANDIBULAR PHEROMONES IN SWARMING The details of the queen's role in the swarm coordination are unknown. Still, the swarm does not exhibit bivouac formation and relocation in the absence of the queen, which reflects that the queen might signal a specific event's coordination (Avitabile et al. 1975; Pierce et al. 2007). Richards et al., 2011, studied the pheromonal bouquet composition of the queen before swarming and compared that to the non-swarming queen to characterize the dynamic change in the design of volatile chemicals which act as the responsible agent for changing the behaviour of swarming bees. Queen honey bees provide the pheromonal signal to initiate the first step of swarming, which is the rearing of a new Queen. Furthermore, the queen's presence or pheromones increase the swarm cohesion after swarms leave the parental nest in the clustered or airborne swarm. If the queen is not with the swarm, then the swarm group of worker honey bees return to the hive (Morse1963; Simpson, 1963; Avitabile et al., 1975). However, in queen less swarm, if exposed to 9-ODA, the swarm does not return to the hive, which suggests that the queen indicates her presence by secreting Queen Mandibular Pheromone (QMP), predominantly by the 9-ODA component (Avitabile et al. 1975).QMP is composed of 9-oxo-(E)-2-decanoic acid (9-ODA), (R)- and (S)-9-hydroxy-(E)-2-decanoic acid (9-HDA), methyl p-hydroxybenzoate (HOB), and 4hydroxy-3-methyoxyphenylethanol (HVA) (Slessor et al. 1988; Grozinger et al. 2003; Hoover et al. 2003). Additionally, about 20 other chemicals act as essential ingredients of mandibular gland secretion, which collectively regulate reproductive, development, physiology and Behaviour of workers (Richard et al. 2007, 2011; Kocher et al. 2009; Nino et al.2012, 2013; Peso et al.2013). Further, the swarm exhibits a positive correlation between cluster stability and exposure to 9-ODA, 9-HDA, and other QMP components (Butler et al. 1964; Butler and Simpson 1967; Winston et al. 1982). It has been detected that whole

252 Polyandrous Queen Honey Bee

Lovleen Marwaha

Queen extract, or live queen, can stabilize a swarm more efficiently than only pheromones (Boch et al. 1975; Winston et al. 1989), which suggests the involvement of additional pheromones in cluster cohesion. However, it is not completely clear if the queen conveys her presence by regular pheromonal blend or if there is a change in the specific pheromone blend, especially during Swarming. According to Seeley and Fell, 1981, queens in swarming and non-swarming groups secrete equal concentrations of 9-ODA. The secretion of QMP changes with reproductive state and mating quality. Similarly, a change in the pheromone bouquet changes the Behaviour of workers in the swarm. Further, the queen plays an essential role in organizing the swarming process, as swarming workers exhibit more attraction toward volatile chemicals secreted by the swarming queen than the non-swarming queen. The specific volatile chemical signals were given by swarming workers about the queen's presence within the swarm. Honey bee workers can distinguish their queens from other queens (Breed 1981; Wyatt 2010). Few reports witnessed that, during Swarming, there is a change in queen worker interaction. Older workers, especially foragers and scouts, are not attracted to queen pheromones. Richards et al., 2011, used solid-phase microextraction (SPME)coupled with gas chromatography /mass spectroscopy (GC/MS) to determine whether the queen pheromone blend changed before swarming the clustering of bivouacs or bivouac lift-off. Further, they have compared the volatile chemical profile of Swarming and non-swarming queens, in terms of the change in the quantity and types of various volatile blends during different phases of swarming. Similarly, they have recorded the response of worker honey bees to multiple pheromones to different chemical blends produced by other queens during or after swarming. The volatile emission changes during swarm lift-off as the queen emit more volatile chemicals when bivouacs lift off than the queen in the natal nest before swarming. Furthermore, the queen secretes more pheromones during swarm lift-off than in hive or bivouacking queens. Therefore, the specific chemicals are a good messenger for controlling the Behaviour of swarming workers. Furthermore, Pentadecane also had been secreted relatively high proportionality, specifically in lift-off queen. However, details of volatile chemicals emitted for chemical communication in insects could not be detected due to the inefficiency of currently used methods, as a single type of SPME fibre fails to absorb all volatile chemicals with 100% efficiency.

Swarming and Queen

Polyandrous Queen Honey Bee 253

Further, the process of new queen rearing is also inhibited by the older queen's mandibular gland pheromones (QMP) (Slessor et al. 1988, 1990; Winston et al. 1991). 13.4. OTHER QUEEN GLANDS PHEROMONES IN SWARMING Chemical signals from queens and swarming workers are essential in coordinating various swarming processes. Dufour's glands and the Nasanov gland secrete other influential pheromones for swarming. Queen Dufour's glands secrete trace amounts of specific volatile chemicals, including Pentadecane and heptadecane, and particular chemicals are detectable on the surface of laid eggs. Pentadecane is also traceable from worker-laid-down eggs (Katzav-Gozansky et al., 2003; Richard et al., 2011). Concerned chemicals have not been identified from worker honey bees' Nasanov gland, a gland reasonable for communication among workers at the time of swarming (Pickett et al. 1980). The specific compounds had been detected in the sting apparatus of honey bee workers(McDaniel et al. 1984; Breed et al. 2004). Two specific chemicals, including heptadecane and Pentadecane, had been detected to be secreted in higher concentrations by the queen at lift-off, whereas at the non-swarming time, the same queen secreted different chemicals and even at the time of clustering (Seeley 1982; Grozinger and Robinson 2007; Pierce et al. 2007). Similarly, Butyl butanoate had been detected to be produced in a significant amount by lift-off Queen. (E/Z)-Queen of all stages produces βocimene, but the lift-off queen secretes the concerned chemical in a higher concentration than usual. Egg-laying queen secretes volatile chemical (E)--ocimene at a higher concentration than the unmated Queen (Gilley et al., 2006; Huang et al., 2009). Swarm workers produce Nasonov pheromones, mainly composed of seven terpenoids, (Z)- and (E)citral, nerol, geraniol, nerolic acid, geranic acid and (E, E)-farnesol (Free 1987; McIndoo 1915; Pickett et al. 1980; Blum 1992). It had been reported that nesting boxes marked with Nasanov pheromones are more attractive to swarm (Free et al. 1981b; Schmidt et al. 1993; Schmidt 2001). Scout bees release Nasanov pheromones at the entrance of the new hive and use visual signals to attract swarm clusters toward a specific site (Beekman et al., 2006). Although, a little more time is required for the swarm to enter the new hive. Queen also produce different pheromones, including mandibular, Dufour's, tergal, and tarsal glands, which influence the Behaviour of worker honey bees (Lensky and Slabezki 1981; Slessor et al. 1988; Page et al. 1988; Breed et al.

254 Polyandrous Queen Honey Bee

Lovleen Marwaha

1992; Wossler and Crewe 1999a, 1999b; Katzav-Gozansky et al. 2003; Keeling et al. 2003; Slessor et al. 2005). Lift off queen secretes a higher concentration of Benzyl alcohol, geranyl acetone, and benzyl benzoate than inhive queen. In worker honey bees Nasanov gland also secretes Geranyl acetone, a biosynthetic derivative of farnesol (Pickett et al. 1980; Schulz et al. 2011). Nasanov pheromone blend attracts honey bee swarm (Schmidt 1999). Workers can also secrete pheromones to regulate other workers in bivouacs and swarm lift-off. 9-ODA and HVA had been detected as necessary chemicals for stabilizing swarm (Butler et al. 1964; Avitabile et al. 1975; Winston et al. 1982; Richards et al. 2015). Queen Retinue Pheromone (QRP) helps attract bees over a short distance (Keeling et al. 2003). In addition, the tarsal gland secretes footprint pheromones” (Lensky and Slabezki 1981). Queen honey bee, at the time of Swarming, emits a bouquet of unique pheromones compared to non-swarming queens. The concerned pheromones attract the specific lift-off during the peculiar event. The major proportionality of particular chemicals is composed of Pentadecane and heptadecane. It has been reported that the composition of queen pheromones regulates the Behaviour of worker honey bees. In non-swarming colonies, queen honey bees deposit pheromonal secretion from their tarsal gland on the hive, as footprint pheromones inhibit new queen rearing, even in the congested colony (Lensky and Slabezki 1981). 13.5. MAJOR EVENTS IN SWARMING 13.5.1. Pre-Swarming Phase A detailed study on Swarming was carried out by Huber (1792) and Taranov & Ivanova (1946). Similarly, Allen, 2015, had carried out a detailed analysis of the swarming process from the time of queen cup formation. In the pre-swarming period, there is a progressive reduction in worker honey bees, feeding the queen till the swarming day. The number of worker honey bees feeding to the queen was reduced to the minimum, to almost zero, a fortnight before the swarming day. But the queen lays a small number of eggs continuously upto a swarming day. They had conducted a detailed analysis of the specific process and concluded that workers raise new queens before swarming. Fifteen days before raising the queen cup, workers reduced the feeding queen.

Swarming and Queen

Polyandrous Queen Honey Bee 255

According to Taranov & Ivanova (1946), workers repeatedly forced the queen to accept food in pre-swarming, but Allen, 2015 did not notice this. Taranov & Ivanova (1946) observed that worker honey bees shake queen cells, which is quite common in colonies preparing for swarming. Older Queen is treated roughly before a week of the swarm. Worker honey bee tries to keep the queen moving, but not in a specific direction. Huber, 1792, reported that a mated queen destroyed the sealed queen cell in the hive but induced no harm to the queen cell, which the colony raised before swarming. The Virgin Queen makes a sound commonly known as piping, challenging other unhatched queens. However, according to Woods (1950), the newly mated queen can produce a croaking sound, which he concluded, can occur due to the development of ovaries (ALLEN, 2015). They were swarming represents colony reproduction, involving splitting a parental colony into one or more subunits, each with a reproductively active queen and 1/3 of colonial workers (Winston, 1987; van Veen and Sommeijer, 2000). In the honey bees' swarming process, two critical phases occur queen rearing and queen elimination. Queen Rearing: Worker honey bees rear about 15-20 queens in queen cells, whereas old queens depart with about 1/3 of colonial strength during Swarming. Queen Elimination: Daughter queens get eliminated by multiple processes, which include fight/death with the rival queen before or after the emergence or departure of the new queen with the secondary swarm. After that, survived queen inherits the natal hive. The queen elimination process takes less than seven days, but the specific phase influences the colonial strength. The Queen elimination process involves multilevel selection, group decision outcome, potential nepotism, and complex signalling, which affect the power of the colony (Tarpy and Fletcher, 1998; Gilley, 2001; Schneider et al., 2001; Gilley, 2003; Schneider and DeGrandiHoffman, 2003; Gilley et al., 2003; Tarpy et al., 2004; Tarpy and Gilley, 2004; Schneider and Lewis, 2004; Schneider et al., 2004). Queen elimination can occur through queen duels, which include lethal combat, killing one queen in the fight. For example, Butz and Dietz (1994) explained the combat between mated queens introduced in the same colony (Huber, 1814; Weaver and Weaver, 1980; Ohtani, 1994; Gilley, 2001). During the duel phase, queens usually spend most of their time moving and finding queen cells, concomitantly emitting audible piping sounds.

256 Polyandrous Queen Honey Bee

Lovleen Marwaha

Dueling Queens do not come in contact frequently and sometimes hide within worker cells for several hours. Queens, on coming in contact with each other, they can leave after antennal contact or be positioned using their legs and mandible to sting their rival. The specific process can take about 4-15 minutes, usually resulting in the death or wounding of one queen. Rival queens sometimes do spraying. Which comprise instead of fighting, one queen spray ejects faecal material over her rival by raising her abdomen (Page and Erickson, 1986; Post et al., 1987; Page et al., 1988; Bernasconi et al., 1999, 2000; Gilley, 2001; Tarpy and Fletcher, 2003). Worker honey bees interact extensively with queens during duelling (Huber, 1814; Weaver and Weaver, 1980; Ohtani, 1994; Gilley, 2001). Available reports indicate non-random outcomes of queen rivals and correlation with queen characteristics, usually due to workers' participation. Queens survive more in cases where queens are related to workers or are older than their opponents or when queen cells are shaken by workers frequently (Tarpy and Fletcher, 1998; Tarpy et al., 2000; Schneider et al., 2001; Gilley, 2003). Queen elimination can occur by the pre-emergence destruction method, which includes the pre-hatching elimination of queens by their hatched rival queens. Emerged queen usually takes several minutes to an hour to make about a 3-5 mm diameter hole on the side of the queen cell and subsequently push its abdomen into that hole, to sting the developing rival queen (Huber, 1814; Boch et al., 1979). Emerged queens destroy queen cells near to emergence than queen cells with immature queens. Therefore, emerged queen removed their most dangerous rival first (Caron and Greve, 1979; Harano and Obara, 2004). Near the specific action, the worker honey bee either ignores the event or makes a hole that had been chewed by Queen (Huber, 1814). Further, workers can destroy queen cells that have damaged pupae and discard queen pupae present there. Workers who had searched for new nest sites acted as scouting for new homes and advertised new sites to their nest mates. Scout bees advocate their searched nest site through waggle dance before swarming (Lindauer 1955; Seeley and Visscher 2004; Seeley et al. 2006; Michelsen et al. 1986; Nieh 1993; Seeley et al. 2012). Scout bees select multiple sites for swarming. After that, the informed bee decides on one quorum for one nest site. After debate, that scout bee instructs the nest bee to be a swarm to form the cluster by producing piping signals about 1-2 hours before lift-off (Seeley 2010; Seeley and Tautz 2001; Visscher and Seeley 2007). Piping stimulates other workers, who usually maintain low body temperature, to raise their flight muscle temperature of appendages and thorax to 33–35 °C (Heinrich 1981; Seeley and Tautz 2001; Esch 1976; Heinrich 1979). As the thoracic temperature of worker bees increases up to 35 °C, the swarm lifts off into the air to move toward the new nest home (Seeley et al. 2003). Informed bees

Swarming and Queen

Polyandrous Queen Honey Bee 257

signal the remaining bees about the selected site through specific signals. Vibration signals are essential in bivouacking swarm for lift-off preparation (Schneider et al. 1998; Visscher et al. 1999). Only 5% of worker honey bees in bivouac produce this vibrational signal. About an hour before lift-off, the signal frequency of the vibration signal increases (Lewis and Schneider 2000; Visscher et al. 1999; Schneider and Lewis 2004). Few members who produce vibration also waggle dance, and removing vibrating bees from the swarm delay swarm liftoff (Visscher et al. 1999; Lewis and Schneider 2000; Donahoe et al. 2003). Therefore, vibrational signals are used for swarm lift off, but not in locating, debating or selecting a new nest site. Queen and worker interaction in swarming events differ from the interaction of two specific castes in the colony before Swarming and departure. Queen frequently vibrates before swarming in the colony, but in bivouacs, they rarely show vibration movement (Pierce et al. 2007). Queen is piped intensively by workers about 1 to 2 hours before lift-off (Pierce et al. 2007). 13.5.2. Exodus of Swarm It includes the colonial exit of the queen and their workers, which takes place suddenly, furious and in a few seconds. To coordinate departure during Swarming, numerous physical, visual and auditory communications are given at the time of swarming. For Swarming, a signal known as vibration or shaking signal is exchanged between workers, which comprising rapid movement of the body in dorsoventrally, for a fraction of 1-2 seconds, while holding receiver bees forelegs (Gahl1975; Seeley et al. 1998; Visscher et al. 1999; Schneider and Lewis 2004). Various researchers considered that the specific vibrations are used to convey messages like reassess your current activity, reallocate to different activities, prepare for greater action, increase your training, and stimulate the movement of workers toward the hive (Schneider et al. 1986; Nieh 1998; Seeley et al. 1998; Biesmeijer 2003; Schneider and Lewis 2004; Cao et al. 2007), an increase in task performance (Schneider et al. 1986; Schneider 1987; Schneider and McNally 1991; Cao et al. 2009). According to Schneider et al. 1986 and Rangel and Seeley 2008, worker honey bees receive vibration signals throughout the year. There is no increase in the number of the movement before swarming workers, but the queen honey bee is vibrating only for swarming, with maximum frequency a week before swarming (Schneider 1990a, 1991; Pierce et al. 2007). These vibrating signals stimulate the queen to lay eggs at full to ensure egg production and for queen weight loss (Schneider 1991). Vibration signals are performed by worker bees only, although

258 Polyandrous Queen Honey Bee

Lovleen Marwaha

of the enormous population of young worker bees (Allen 1955; Allen 1959; Painter-Kurt and Schneider 1998; Schneider et al. 1998). Worker activities, including piping” and “buzz running, at the swarm exodus are executed to organize the specific event properly. The concerned signals are initiated by workers who had initiated the house hunting process before leaving the parental nest (Rangel et al. 2010). Worker honey bees produce the piping sound of frequency between 100-250 Hz while pressing their abdomen to the surface and vibrating their wings (Seeley and Tautz 2001; Pierce et al. 2007; Rangel and Seeley 2008). Swarming workers also produce buzz runs, in which workers run quickly in a bee crowd while vibrating their wings every second, with a characteristic buzzy sound of frequency180-250 Hz (Lindauer 1955; Esch 1967). The number of buzz runs increases tremendously before 15 minutes of swarm departure, as both scout and other bees start producing buzz runs (Martin 1963; Rangel and Seeley 2008; Rangel et al. 2010). The specific signal just increases the run of worker bees on the comb (Rangel and Seeley 2008). Queen does not act as leader of the swarm but is pushed by hive workers out from the colony, or sometimes she does not exit the parent hive, forcing workers to return to the hive and attempt to exit some later on (Simpson 1958, 1963; Pierce et al. 2007). After exiting the parental hive, the swarm initially forms a bivouac near the parental pack, which is just a temporary stay. 13.5.3.Swarming On the swarming day, worker honey bees coordinate with each other through chemical and non-chemical signals for relocation to a temporary site to form a bivouac. Before swarming, scout bees search for new nest sites and communicate specific information with other scout bees. Additionally, non-chemical and chemical signals are used for the break-up and lift-off of the bivouac for further site migration. Before Swarming occurs in honey bees, about 5% of worker honey bees have already visited the site. Therefore, they are already familiar with the specific path to be followed during Swarming. This small group of scouts guides a large group of swarms unfamiliar with the new nest site. Two possible mechanisms are known for this specific activity: informed bees fly in a particular direction without elevated speed. Other mechanisms include the familiar path, which passes for a specific approach at a comparatively fast pace. Honey bees flying at the top in the swarm group possess maximum velocity as they move toward their new home. Swarming generally occurs in spring when stronger colonies divide for reproduction.

Swarming and Queen

Polyandrous Queen Honey Bee 259

During swarming older queens, about 2/3 of the strength comprising daughter workers and drones leave the parental hive to form new colonies. In contrast, the daughter queen stayed with her sisters to perpetuate the older colony in the original hive (Winston, 1987). During primary Swarming, the mother queen leaves the swarm with workers (Huber, 1814; Allen, 1956; Lindauer, 1961; Martin, 1963; Caron, 1970), whereas secondary swarms contain fewer workers in comparison to primary swarm, with separate young queens (Huber, 1814; Hepburn and Radloff, 1998), although the basic mechanism is same, with that of immediate swarming process. During the swarming process, worker honey bees run near to hive entrance. The specific moment spread within 3-4 minutes within the hive, resulting in a massive moment of bees near to hive entrance (Caron, 1970). Secondary swarming time is related to events of queen elimination, although several factors influence the time of secondary swarm departure. It has been observed that environmental factors, including temperature and daylight, influence swarm formation (Huber, 1814; Allen, 1956; Winston, 1987; Schneider and DeGrandiHoffman, 2002; Tarpy et al., 2000; David C. Gilley, David R. Tarpy, 2005). Colonies with swarming events generally have adult worker bees forming crowds in brood areas (Simpson 1958; Winston and Taylor 1980). The older queen can either join the swarm voluntarily, or workers can force her. Subsequently, she can try to enter the hive again (Allen, 1956; Caron, 1970). During Swarming, honey bees fly near the hive's entrance, and most depart, though few of them could return within an hour. After exiting the natal hive, bees cluster on a tree branch or other nearby objects. Sometimes, the queen does not leave the hive with the swarm. In that case, the departed swarm returns to the hive within about 30 minutes and swarms the next day (Allen, 1956; Caron, 1970). Approximately ten days before, workers started feeding on honey as a primary energy producer for nest building, migration and starting a new colony (Combs 1972). In swarming worker bees, stomach quantity and quality of food are significantly higher than in non-swarming colonies (Combs 1972). Scout bees activate the nest bees to form swarm clusters and assess flight take-off activities of nestmates (Seeley and Tautz 2001; Rittschof and Seeley 2008). The swarm's movement toward the new nest site has been explained by multiple hypotheses, including the olfactory gradient hypothesis, which states that worker honey bees in a hive follow a specific odour released by the scent glands of informers (Avitabile et al. 1975; Beekman et al. 2006). Further, few researchers considered that informed workers guide un-information about the selected site by visual guidance (Couzin et al. 2005).

260 Polyandrous Queen Honey Bee

Lovleen Marwaha

During Swarming, about 11,000 bees travel 8–12 m long, 6–8 m wide, and 3–4 m high (Beekman et al. 2006). Queen presence increases cohesion in honey bees, as a swarm without a queen is more dispersed, spanning a diameter of about 60 m (Morse 1963). Swarm without a queen usually returns to the last place, where they have clustered with her or moved to her if she is nearby. In other words, the swarm moves with the queen only (Avitabile et al. 1975; Simpson 1963; Morse 1963). It has been reported that, in the absence of a queen, if certain workers are marked with 9-ODA, the swarm does not return to the hive but moves to the new location (Avitabile et al. 1975). Swarm get clustered around lure impregnated with queen pheromones 9-ODA and/or 9-HDA. However, the live queen gets a more effectively swarm response than the pheromonal lure (Butler et al. 1964; Butler and Simpson 1967; Boch et al. 1975; Winston et al. 1982; Winston et al. 1989). It has been reported that a queen during lift off of a swarm produces more volatile pheromones than the queen in an average colony in bivouacs (Seeley and Buhrman 1999), which indicates that a queen can change pheromonal blend composition under different circumstances. Queen is not a passive member but an active participant who organizes a swarm. Further on reaching the new site, scouts settle at the entrance, attracting remaining bees by forming visual clusters and secretion of specific pheromones (Ambrose 1976; Seeley et al. 1979; Beekman et al. 2006). 13.5.4. Migration During Swarming About 11,000 flying bees leave the hive but initially do not cover a considerable distance. At the beginning of swarming, they cluster near the hive at about 50m from the parental nest (Ambrose, 1976). Subsequently, on the next day, clustered swarm bees decide sophisticatedly to migrate to their future home site (Seeley et al., 2006; Passino et al., 2008). After decision-making, swarm bees fly toward a new location, a tree cavity or other site (Seeley and Morse, 1977; Villa, 2004). About 3-5% of bees are involved in selecting the home site, commonly known as scout bees (Seeley et al., 1979; Seeley and Visscher, 2007). Avitabile and colleagues, 1975 considered that informed bees guide the follower bees about another place by releasing pheromones, which create a particular odour gradient, indicating the direction of flight. Two specific hypotheses explain pathways for guidance information to other bees visually. One theory includes a subtle guide, and another streaker bee hypothesis. • The subtle guide hypothesis explains that the informed bees tend to steer the swarm by moving toward the new home. In a moving swarm, individual bees avoid collision by turning away from an individual neighbour within a specific distance, aligning with neighbours within a critical distance and flying in a

Swarming and Queen

Polyandrous Queen Honey Bee 261

particular direction. Other swarm steered toward a specific order (Couzin et al., 2005). • Another hypothesis had been proposed by Lindauer (Lindauer, 1955), commonly known as the streaker bee hypothesis, explains that an informed bee provides a signal about the correct travel direction by repeatedly making highspeed flights through an airborne swarm in the direction of the new home, followed by low-speed flights, toward the edge of the swarm to return to the back of swarm. It had been reported through photographic analysis that streaker bees in a swarm fly in the upper half of a cloud of flying bees (Beekman et al., 2006). Schultz et al., 2008, recorded that streaker bees fly toward the direction of the swarm's new home. The subtle guide hypothesis does not agree with the presence of highspeed informed bee movement toward new hives. In contrast, the streaker bee hypothesis witnesses the presence of highspeed fly movement toward the new home site. High-speed streaker bees are not only informed bees but could be uninformed bees reacting to the streaker bees. It is difficult to track the movement of individual bees in a swarm, as a swarm is composed of large clouds of small bees. Generally, Swarm measurements include 8-12 m from front to rear, 6-8 m from side to side, and 3-4 m from top to bottom, whereas an individual fly is about 14 mm long. Therefore, due to the large dimension of the swarm, it is difficult to capture the entire swarm and track the record of individual bees within it. Additionally, swarm movement cannot be captured from a specific site due to the large distance cover, which usually ranges from hundreds or thousands of meters. Schultz et al., 2008, reported that within the swarm, bees in the top and middle portion of hives showed a maximum tendency to move faster toward the nest box. The top of the swarm usually includes fast-flying bees, the middle portion comprises high alignment, and the lower amount includes slower bees. The bottom amount of Swarming includes less aligned bees than fast-flying bees in all directions. The swarm is chaotic, with bees flying in all directions at variable speeds. Schultz et al., 2008, studied flight speed data, which had been found per the streaker bee hypothesis. According to the subtle guide hypothesis, informed bees will differ from uninformed bees only in showing a preference to move in a particular direction, but the streaker bee hypothesis suggests that informed bees move toward the preferred approach with a higher speed of flight. Further, they concluded that fast-flying bees travel faster than the remaining swarm bees must have implemented some method of repeatedly streaking movement or through the swarm. Finally, Schultz et al., 2008, reported that the

262 Polyandrous Queen Honey Bee

Lovleen Marwaha

streaker bee hypothesis is more robust than the subtle guide hypothesis concerning flight guidance in honey bee swarming(Schultz et al., 2008). 13.6.TYPES OF SWARMING WORKERS In a swarming colony, three types of workers include scout, non-scout workers and workers who remain in the parental colony. Scout bees are older bees in a swarm (Gilley 1998). Nest-site scouts also search for various types of food sources at the new site, therefore, are considered “novelty seekers” (Liang et al., 2012). In scouts and non-scouts bees, several genes have differential expressions involved in biogenic amine signalling (Liang et al. 2012). It has been reported that treating worker bees with glutamate and octopamine increases scouting activity, which indicates that the behaviour aspect is controlled by various neuro signalling pathways (Liang et al. 2012). During Swarming, about 1/4th of worker honey bees leave the nest with the swarm, whereas the remaining workers remain in the parental colony (Martin 1963; Getz et al.1982). Even newly introduced queens could join the swarm (SimpsonandRiedel1964). Generally, younger bees are included in a swarm than non-swarming bees (Butler 1940; Winston and Taylor 1980; Winston etal.1981; Gilley1998). Swarming bees' age corresponds to nurse bees/ brood care taking non-swarming bees (Winston 1987; Seeley 1995). 13.7.AGE AND PHYSIOLOGY OF SWARMING WORKERS Swarming workers are also physiologically younger than non-swarming bees. It has been reported that the Juvenile hormone of swarming bees is comparatively lower than non-swarming bees. Juvenile hormone is positively co-related with behavioural maturity in worker honey bees (Sullivan et al. 2000; Zeng et al. 2005). Swarm generally carries workers of low age for different reasons, which include that in a new hive generally, workers perform various tasks, which are performed by younger bees only. Additionally, initially foraging task is not executed by worker bees, as bees take energy from the stored reserve. Further, younger bees carry a greater quality of lipids, high storage of vitellogenin, trehalose, glucose, and fructose, and body glycogen reserves than non-swarming bees (Butler 1940; Combs 1972; Fluri et al. 1982; Winston 1987; Leta et al.1996; Toth and Robinson 2005;). The workers of specific patrilines swarm more frequently than other patrilines(Getz et al. 1982; Kryger and Moritz 1997). Data from the workers' brains indicated that about 140 genes express differently in swarming and non-swarming worker honey bees. Further, nearly 1200 other genes are differentially expressed in scouts and non-scouts workers(Liang et al. 2012). Additionally, about 900 genes are expressed differentially among honey bees producing vibration signals more than in nonswarming bees (Alaux et al., 2009; Christina et al., 2014).

Swarming and Queen

Polyandrous Queen Honey Bee 263

13.8. NATAL COLONY AFTER SWARMING New queen takes over the colony after the departure of the original queen. In the strong colony, after the prime swarm, swarms also depart the colony (Winston 1987). After emergence, new virgin queens take over the colony's charge within a few days after eliminating other rival developing queens or emerging queens (Allen 1956; Fletcher 1978a; Gilley 2001; Gilley and Tarpy 2005; Schneider and DeGrandi-Hoffman 2008). In the more robust colony, with prime and after swarms, the colony takeover process by the queen is delayed (Allen 1956; Fletcher1978a; Gilley 2001; Gilley and Tarpy 2005; Schneider and DeGrandiHoffman 2008). The rival queens emerged attract each other by a piping sound, known as tooting and quacking, and after that they fight (Michelsen et al. 1986; Page et al. 1988; Bernasconi et al. 2000; Gilley2001; Schneider et al. 2001; Tarpy and Fletcher 2003; Gilley and Tarpy 2005). Workers support queen replacement by stimulating the queen to increase her activity and to produce more queendirected pipes or by vibrating queen cells to slow their emergence from queen cells (Fletcher 1978a, b; Bruinsma et al. 1981; Grooters 1987 Schneider et al. 2001; Gilley 2001). The specific signal interaction coordinates the emergence of the queen with the departure of prime and after swarms. It ensures the exit of one queen only with each swarm and a single queen to take over the charge of the parental colony. Subsequently, of migration to the new site, worker honey bees start building new comb, whereas, in the native hive, older workers raise a new queen, which on hatching, becomes the new queen of the original colony. As multiple queen cells are grown, the queen, which will emerge earliest, kills other queen cells enclosing developing queen pupae to take over the colony. After hatching, the queen mates with the drone in the congregation area and starts laying eggs post-mating. CONCLUSION Overgrowth of the colony results in congestion in the colony and reduced pheromone distribution per honey bee. Therefore, honey bees colony prefer to swarm to expand the colony. Swarm usually consist of old queen honey bee, young worker bees and drones, which leave the colony. Swarms migrate to the new select site with profound food availability and establish a settlement there. REFERENCES Alaux, C, Duong, N, Schneider, SS, Southey, BR, Rodriguez-Zas, S & Robinson, GE (2009) Modulatory communication signal performance is associated with a distinct neurogenomic state in honey bees. PLoS One, 4, e6694. [http://dx.doi.org/10.1371/journal.pone.0006694] [PMID: 19693278] Allen, MD (1955) Observations on honeybees attending their queen. Br J Anim Behav, 3, 66-9.

264 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1016/S0950-5601(55)80015-9] Delia Allen, M (1956) The behaviour of honeybees preparing to swarm. Br J Anim Behav, 4, 14-22. [http://dx.doi.org/10.1016/S0950-5601(56)80011-7] Ambrose, JT (1976) Swarms in Transit. Bee World, 57, 101-9. [http://dx.doi.org/10.1080/0005772X.1976.11097603] Antúnez, K, Anido, M, Branchiccela, B, Harriet, J, Campa, J, Invernizzi, C, Santos, E, Higes, M, MartínHernández, R & Zunino, P (2015) Seasonal variation of honeybee pathogens and its association with pollen diversity in Uruguay. Microb Ecol, 70, 522-33. [http://dx.doi.org/10.1007/s00248-015-0594-7] [PMID: 25794593] Avitabile, A, Morse, RA & Boch, R (1975) Swarming honey bees are guided by pheromones. Ann Entomol Soc Am, 68, 1079-82. [http://dx.doi.org/10.1093/aesa/68.6.1079] Beekman, M, Fathke, RL & Seeley, TD (2006) How does an informed minority of scouts guide a honeybee swarm as it flies to its new home? Anim Behav, 71, 161-71. [http://dx.doi.org/10.1016/j.anbehav.2005.04.009] Bernasconi, G, Bigler, L, Hesse, M & Ratnieks, FLW (1999) Characterization of queen-specific components of the fluid released by fighting honey bee queens. Chemoecology, 9, 161-7. [http://dx.doi.org/10.1007/s000490050049] Biesmeijer, JC (2003) The occurrence and context of the shaking signal in honey bees (Apis mellifera) exploiting natural food sources. Ethology, 109, 1009-20. [http://dx.doi.org/10.1046/j.0179-1613.2003.00939.x] Blum, MS (1992) Honey bee pheromones in the hive and the honey bee, Dadant and Sons 385-9. Boch, R & Lensky, Y (1976) Pheromonal control of Queen rearing in honeybee colonies. J Apic Res, 15, 5962. [http://dx.doi.org/10.1080/00218839.1976.11099835] Boch, R, Shearer, DA & Shuel, RW (1979) Octanoic and other volatile acids in the mandibular glands of the honeybee and in royal jelly. J Apic Res, 18, 250-2. [http://dx.doi.org/10.1080/00218839.1979.11099977] Breed, MD (1981) Individual recognition and learning of queen odors by worker honeybees. Proc Natl Acad Sci USA, 78, 2635-7. [http://dx.doi.org/10.1073/pnas.78.4.2635] [PMID: 16593008] Breed, MD, Stiller, TM, Blum, MS & Page, RE, Jr (1992) Honeybee nestmate recognition: Effects of queen fecal pheromones. J Chem Ecol, 18, 1633-40. [http://dx.doi.org/10.1007/BF00993235] [PMID: 24254293] Bruinsma, O (1981) Delay of the emergence of honey bee queens in response to tooting sounds. Proc K Ned Akad Wet C, 84, 381-7. Butler, CG (1940) The ages of bees in a swarm Butler, CG & Simpson, J (1967) Pheromones of the queen honeybee (Apis mellifera L.) which enable her workers to follow her when swarming. Proc R Entomol Soc Lond, Ser A Gen Entomol, 42, 149-54. [). Oxford, UK: Blackwell Publishing Ltd.]. [http://dx.doi.org/10.1111/j.1365-3032.1967.tb00806.x] Butler, CG (1960) The significance of queen substance in Swarming and supersedure in honey bee (Apis mellifera L.) colonies. Proc R Entomol Soc Lond, Ser A Gen Entomol, 35, 129-32. [). Oxford, UK: Blackwell Publishing Ltd.]. [http://dx.doi.org/10.1111/j.1365-3032.1960.tb00681.x] Butler, CG, Callow, RK & Chapman, JR (1964) 9-Hydroxydec-trans-2-enoic acid, a pheromone stabilizing honeybee swarms. Nature, 201, 733-3.

Swarming and Queen

Polyandrous Queen Honey Bee 265

[http://dx.doi.org/10.1038/201733b0] Butz, VM & Dietz, A (1994) The mechanism of queen elimination in two-queen honey bee ( Apis mellifera L.) colonies. J Apic Res, 33, 87-94. [http://dx.doi.org/10.1080/00218839.1994.11100855] Cao, TT, Hyland, KM, Malechuk, A, Lewis, LA & Schneider, SS (2007) The influence of the vibration signal on worker interactions with the nest and nest mates in established and newly founded colonies of the honey bee, Apis mellifera. Insectes Soc, 54, 144-9. [http://dx.doi.org/10.1007/s00040-007-0921-1] Cao, TT, Hyland, KM, Malechuk, A, Lewis, LA & Schneider, SS (2009) The effect of repeated vibration signals on worker behavior in established and newly founded colonies of the honey bee, Apis mellifera. Behav Ecol Sociobiol, 63, 521-9. [http://dx.doi.org/10.1007/s00265-008-0686-1] Caron DM (1970) A study of swarming and the Behaviour of swarming in honey bees, Apis mellifera L, PhD. Thesis, Cornell University. Caron, DM & Greve, CW (1979) Destruction of queen cells placed in queenright Apis mellifera colonies. Ann Entomol Soc Am, 72, 405-7. [http://dx.doi.org/10.1093/aesa/72.3.405] Chauzat, MP, Jacques, A, Laurent, M, Bougeard, S, Hendrikx, P & Ribière-Chabert, M (2016) Risk indicators affecting honeybee colony survival in Europe: one year of surveillance. Apidologie (Celle), 47, 348-78. [http://dx.doi.org/10.1007/s13592-016-0440-z] Combs, GF, Jr (1972) The engorgement of swarming worker honeybees. J Apic Res, 11, 121-8. [http://dx.doi.org/10.1080/00218839.1972.11099711] Couzin, ID, Krause, J, Franks, NR & Levin, SA (2005) Effective leadership and decision-making in animal groups on the move. Nature, 433, 513-6. [http://dx.doi.org/10.1038/nature03236] [PMID: 15690039] de Roode, JC & Lefèvre, T (2012) Behavioural immunity in insects. Insects, 3, 789-820. [http://dx.doi.org/10.3390/insects3030789] [PMID: 26466629] Diao, Q & Hou, C (2018) Does nonreproductive swarming adapt to pathogens? PLoS Pathog, 14, e1006742. [http://dx.doi.org/10.1371/journal.ppat.1006742] [PMID: 29370276] Esch, H (1967) The sounds produced by swarming honey bees. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 56, 408-11. [http://dx.doi.org/10.1007/BF00298058] Fefferman, NH & Starks, PT (2006) A modeling approach to swarming in honey bees (Apis mellifera). Insectes Soc, 53, 37-45. [http://dx.doi.org/10.1007/s00040-005-0833-x] Fell, RD, Ambrose, JT, Burgett, DM, De Jong, D, Morse, RA & Seeley, TD (1977) The seasonal cycle of swarming in honeybees. J Apic Res, 16, 170-3. [http://dx.doi.org/10.1080/00218839.1977.11099883] Fletcher, DJC (1978) The influence of vibratory dances by worker honeybees on the activity of virgin queens. J Apic Res, 17, 3-13. a [http://dx.doi.org/10.1080/00218839.1978.11099893] Fletcher, DJC (1978) The vibration of queen cells by worker honeybees and its relation to the issue of swarms with virgin queens. J Apic Res, 17, 14-26. b [http://dx.doi.org/10.1080/00218839.1978.11099894] Forster, IW (1969) Swarm control in honey bee colonies. N Z J Agric Res, 12, 605-10. [http://dx.doi.org/10.1080/00288233.1969.10421245]

266 Polyandrous Queen Honey Bee

Lovleen Marwaha

Free, JB (1987) Pheromones of social beesCornell University Press, Ithaca. Free, JB, Pickett, JA, Ferguson, AW & Smith, MC (1981) Synthetic pheromones to attract honeybee ( Apis mellifera ) swarms. J Agric Sci, 97, 427-31. [http://dx.doi.org/10.1017/S0021859600040879] Fuchs, S (1990) Preference for drone brood cells by Varroa jacobsoni Oud in colonies of Apis mellifera carnica. Apidologie (Celle), 21, 193-9. [http://dx.doi.org/10.1051/apido:19900304] Gahl, RA (1975) The shaking dance of honey bee workers: Evidence for age discrimination. Anim Behav, 23, 230-2. [http://dx.doi.org/10.1016/0003-3472(75)90068-8] Getz, WM, Brückner, D & Parisian, TR (1982) Kin structure and the swarming Behaviour of the honey bee Apis mellifera. Behav Ecol Sociobiol, 10, 265-70. [http://dx.doi.org/10.1007/BF00302815] Gilley, DC & Tarpy, DR (2005) Three mechanisms of queen elimination in swarming honey bee colonies. Apidologie (Celle), 36, 461-74. [http://dx.doi.org/10.1051/apido:2005033] Gilley, DC (1998) The identity of nest-site scouts in honey bee swarms. Apidologie (Celle), 29, 229-40. [http://dx.doi.org/10.1051/apido:19980303] Gilley, DC (2001) The Behaviour of honey bees (Apis mellifera ligustica) during queen duels. Ethology, 107, 601-22. [http://dx.doi.org/10.1046/j.1439-0310.2001.00692.x] Gilley, DC (2003) Absence of nepotism in the aggressive interactions between workers and duelling queen honey bees. Proc Biol Sci, 270, 2045-9. [http://dx.doi.org/10.1098/rspb.2003.2461] [PMID: 14561293] Gilley, DC, DeGrandi-Hoffman, G & Hooper, JE (2006) Volatile compounds emitted by live European honey bee (Apis mellifera L.) queens. J Insect Physiol, 52, 520-7. [http://dx.doi.org/10.1016/j.jinsphys.2006.01.014] [PMID: 16530783] Gilley, DC, Tarpy, DR & Land, BB (2003) Effect of queen quality on interactions between workers and dueling queens in honeybee ( Apis mellifera L.) colonies. Behav Ecol Sociobiol, 55, 190-6. [http://dx.doi.org/10.1007/s00265-003-0708-y] Goulson, D, Nicholls, E, Botías, C & Rotheray, EL (2015) Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science, 347, 1255957. [http://dx.doi.org/10.1126/science.1255957] [PMID: 25721506] Grooters, HJ (1987) Influences of queen piping and worker behaviour on the timing of emergence of honey bee queens. Insectes Soc, 34, 181-93. [http://dx.doi.org/10.1007/BF02224083] Grozinger, CM & Robinson, GE (2007) Endocrine modulation of a pheromone-responsive gene in the honey bee brain. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 193, 461-70. [http://dx.doi.org/10.1007/s00359-006-0202-x] [PMID: 17192826] Grozinger, CM, Fan, Y, Hoover, SR & Winston, ML (2007) Genome-wide analysis reveals differences in brain gene expression patterns associated with caste and reproductive status in honey bees (Apis mellifera). Mol Ecol, 16, 4837-48. [http://dx.doi.org/10.1111/j.1365-294X.2007.03545.x] [PMID: 17927707] Grozinger, CM, Richards, J & Mattila, HR (2014) From molecules to societies: mechanisms regulating swarming behavior in honey bees (Apis spp.). Apidologie (Celle), 45, 327-46. [http://dx.doi.org/10.1007/s13592-013-0253-2] Harano, K & Obara, Y (2004) Virgin queens selectively destroy fully matured queen cells in the honeybee

Swarming and Queen

Polyandrous Queen Honey Bee 267

Apis mellifera L. Insectes Soc, 51, 253-8. [http://dx.doi.org/10.1007/s00040-004-0735-3] Hoover, SER, Keeling, CI, Winston, ML & Slessor, KN (2003) The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477-80. [http://dx.doi.org/10.1007/s00114-003-0462-z] [PMID: 14564409] Huang, MH, DeGrandi-Hoffman, G & LeBlanc, B (2009) Comparisons of the queen volatile compounds of instrumentally inseminated versus naturally mated honey bee ( Apis mellifera ) queens. Apidologie (Celle), 40, 464-71. [http://dx.doi.org/10.1051/apido/2009008] Huber, F (1926) 1814 New Observations Upon BeesAm Bee J., Hamilton, Illinois. Katzav-Gozansky, T, Soroker, V, Francke, W & Hefetz, A (2003) Honeybee egg-laying workers mimic a queen signal. Insectes Soc, 50, 20-3. [http://dx.doi.org/10.1007/s000400300003] Katzav-Gozansky, T, Soroker, V, Kamer, J, Schulz, CM, Francke, W & Hefetz, A (2003) Ultrastructural and chemical characterization of egg surface of honeybee worker and queen-laid eggs. Chemoecology, 13, 12934. [http://dx.doi.org/10.1007/s00049-003-0238-0] Keeling, CI, Slessor, KN, Higo, HA & Winston, ML (2003) New components of the honey bee ( Apis mellifera L.) queen retinue pheromone. Proc Natl Acad Sci USA, 100, 4486-91. [http://dx.doi.org/10.1073/pnas.0836984100] [PMID: 12676987] Kocher, SD & Grozinger, CM (2011) Cooperation, conflict, and the evolution of queen pheromones. J Chem Ecol, 37, 1263-75. [http://dx.doi.org/10.1007/s10886-011-0036-z] [PMID: 22083225] Kocher, SD, Richard, FJ, Tarpy, DR & Grozinger, CM (2009) Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees. Behav Ecol, 20, 1007-14. [http://dx.doi.org/10.1093/beheco/arp090] [PMID: 22476212] Kryger, P & Moritz, RFA (1997) Lack of kin recognition in swarming honeybees ( Apis mellifera ). Behav Ecol Sociobiol, 40, 271-6. [http://dx.doi.org/10.1007/s002650050342] Kurze, C, Routtu, J & Moritz, RFA (2016) Parasite resistance and tolerance in honeybees at the individual and social level. Zoology, 119, 290-7. [http://dx.doi.org/10.1016/j.zool.2016.03.007] [PMID: 27106014] Lee, GM, McGee, PA & Oldroyd, BP (2013) Variable virulence among isolates of Ascosphaera apis: testing the parasite–pathogen hypothesis for the evolution of polyandry in social insects. Naturwissenschaften, 100, 229-34. [http://dx.doi.org/10.1007/s00114-013-1016-7] [PMID: 23340580] Lensky, Y & Slabezki, Y (1981) The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups. J Insect Physiol, 27, 313-23. [http://dx.doi.org/10.1016/0022-1910(81)90077-9] Leta, MA, Gilbert, C & Morse, RA (1996) Levels of hemolymph sugars and body glycogen of honeybees (Apis mellifera L.) from colonies preparing to swarm. J Insect Physiol, 42, 239-45. [http://dx.doi.org/10.1016/0022-1910(95)00106-9] Liang, ZS, Nguyen, T, Mattila, HR, Rodriguez-Zas, SL, Seeley, TD & Robinson, GE (2012) Molecular determinants of scouting behavior in honey bees. Science, 335, 1225-8. [http://dx.doi.org/10.1126/science.1213962] [PMID: 22403390] Lindauer, M (1955) Schwarmbienen auf Wohnungssuche. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 37, 263-324.

268 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1007/BF00303153] Loftus, JC, Smith, ML & Seeley, TD (2016) How honey bee colonies survive in the wild: testing the importance of small nests and frequent Swarming. PLoS One, 11, e0150362. [http://dx.doi.org/10.1371/journal.pone.0150362] [PMID: 26968000] Martin, P (1963) Die Steuerung der Volksteilung beim Schwärmen der bienen. Zugleich ein Beitrag zum Problem der Wanderschwärme. Insectes Soc, 10, 13-42. [http://dx.doi.org/10.1007/BF02223520] Mattila, HR & Seeley, TD (2007) Genetic diversity in honey bee colonies enhances productivity and fitness. Science, 317, 362-4. [http://dx.doi.org/10.1126/science.1143046] [PMID: 17641199] McDaniel, CA, Howard, RW & Blomquist, GJ (1984) Hydrocarbons of the Cuticle, Sting Apparatus, and Sting Shaft of Apis mellifera L. Identification and Preliminary Evaluation as. Sociobiology, 8 McIndoo, NE (1914) The scent-producing organ of the honey bee. Proceedings of the Academy of Natural Sciences of Philadelphia, 542-55. [http://dx.doi.org/10.5962/bhl.title.57035] Morse, RA (1963) Swarm orientation in honeybees. Science, 141, 357-8. [http://dx.doi.org/10.1126/science.141.3578.357] [PMID: 17815993] Nieh, JC (1993) The stop signal of honey bees: reconsidering its message. Behav Ecol Sociobiol, 33, 51-6. [http://dx.doi.org/10.1007/BF00164346] Nieh, JC (1998) The honey bee shaking signal: function and design of a modulatory communication signal. Behav Ecol Sociobiol, 42, 23-36. [http://dx.doi.org/10.1007/s002650050408] Niño, EL, Malka, O, Hefetz, A, Tarpy, DR & Grozinger, CM (2013) Chemical profiles of two pheromone glands are differentially regulated by distinct mating factors in honey bee queens (Apis mellifera L.). PLoS One, 8, e78637. [http://dx.doi.org/10.1371/journal.pone.0078637] [PMID: 24236028] Niño, EL, Malka, O, Hefetz, A, Teal, P, Hayes, J & Grozinger, CM (2012) Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology. J Insect Physiol, 58, 1082-9. [http://dx.doi.org/10.1016/j.jinsphys.2012.04.015] [PMID: 22579504] Ohtani, T (1994) Behaviors of adult queen honeybees within observation hives. I. behavior patterns. Hum Nat, 3, 37-77. Page, RE, Jr & Erickson, EH, Jr (1986) Kin recognition and virgin queen acceptance by worker honey bees (Apis mellifera L.). Anim Behav, 34, 1061-9. [http://dx.doi.org/10.1016/S0003-3472(86)80165-8] Page, RE, Blum, MS & Fales, HM (1988) o-Aminoacetophenone, a pheromone that repels honeybees (Apis mellifera L.). Experientia, 44, 270-1. [http://dx.doi.org/10.1007/BF01941735] [PMID: 3350140] Painter-Kurt, S & Schneider, SS (1998) Age and behavior of honey bees, Apis mellifera (Hymenoptera: Apidae), that perform vibration signals on queens and queen cells. Ethology, 104, 475-85. [http://dx.doi.org/10.1111/j.1439-0310.1998.tb00084.x] Passino, KM, Seeley, TD & Visscher, PK (2008) Swarm cognition in honey bees. Behav Ecol Sociobiol, 62, 401-14. [http://dx.doi.org/10.1007/s00265-007-0468-1] Peso, M, Niño, EL, Grozinger, CM & Barron, AB (2013) Effect of honey bee queen mating condition on worker ovary activation. Insectes Soc, 60, 123-33. [http://dx.doi.org/10.1007/s00040-012-0275-1] Pickett, JA, Williams, IH, Martin, AP & Smith, MC (1980) Nasonov pheromone of the honey bee,Apis

Swarming and Queen

Polyandrous Queen Honey Bee 269

mellifera L. (Hymenoptera: Apidae). J Chem Ecol, 6, 425-34. [http://dx.doi.org/10.1007/BF01402919] [PMID: 24420593] Pierce, AL, Lewis, LA & Schneider, SS (2007) Theuseof the vibration signal and working piping behavior during swarming in honey bees, Apis mellifera. Ethology, 113, 267-75. [http://dx.doi.org/10.1111/j.1439-0310.2006.01314.x] Post, DC, Page, RE, Jr & Erickson, EH, Jr (1987) Honeybee (Apis mellifera L.) queen feces: Source of a pheromone that repels worker bees. J Chem Ecol, 13, 583-91. [http://dx.doi.org/10.1007/BF01880101] [PMID: 24301896] Rangel, J & Seeley, TD (2008) The signals initiating the mass exodus of a honeybee swarm from its nest. Anim Behav, 76, 1943-52. [http://dx.doi.org/10.1016/j.anbehav.2008.09.004] Rangel, J & Seeley, TD (2012) Colony fissioning in honey bees: size and significance of the swarm fraction. Insectes Soc, 59, 453-62. [http://dx.doi.org/10.1007/s00040-012-0239-5] Rangel, J, Griffin, SR & Seeley, TD (2010) An oligarchy of nest-site scouts triggers a honeybee swarm’s departure from the hive. Behav Ecol Sociobiol, 64, 979-87. [http://dx.doi.org/10.1007/s00265-010-0913-4] Rangel, J, Reeve, HK & Seeley, TD (2013) Optimal colony fissioning in social insects: testing an inclusive fitness model with honey bees. Insectes Soc, 60, 445-52. [http://dx.doi.org/10.1007/s00040-013-0309-3] Ratti, V, Kevan, PG & Eberl, HJ (2016) A discrete-continuous modeling framework to study the role of swarming in a honeybee-Varroa destrutor-virus system. Mathematical and computational approaches in advancing modern science and engineering, Springer, Cham 299-308. [http://dx.doi.org/10.1007/978-3-319-30379-6_28] Richard, FJ, Schal, C, Tarpy, DR & Grozinger, CM (2011) Effects of instrumental insemination and insemination quantity on Dufour’s gland chemical profiles and vitellogenin expression in honey bee queens (Apis mellifera). J Chem Ecol, 37, 1027-36. [http://dx.doi.org/10.1007/s10886-011-9999-z] [PMID: 21786084] Richard, FJ, Tarpy, DR & Grozinger, CM (2007) Effects of insemination quantity on honey bee queen physiology. PLoS One, 2, e980. [http://dx.doi.org/10.1371/journal.pone.0000980] [PMID: 17912357] Richards, J, Carr-Markell, M, Hefetz, A, Grozinger, CM & Mattila, HR (2015) Queen-produced volatiles change dynamically during reproductive swarming and are associated with changes in honey bee (Apis mellifera) worker behavior. Apidologie (Celle), 46, 679-90. [http://dx.doi.org/10.1007/s13592-015-0358-x] Rittschof, CC & Seeley, TD (2008) The buzz-run: how honeybees signal ‘Time to go!’. Anim Behav, 75, 189-97. [http://dx.doi.org/10.1016/j.anbehav.2007.04.026] Roberts, WC (1944) Multiple mating of queen bees proved by progeny and flight tests. Gleanings in Bee Culture, 72, 225-59. Ruttner, H & Ruttner, F (1972) Untersuchungen über die Flugaktivität und das Paarungsverhalten der Drohnen. V.-Drohnensammelplätze und Paarungsdistanz. Apidologie (Celle), 3, 203-32. [http://dx.doi.org/10.1051/apido:19720301] Schmidt, JO (1999) Attractant or pheromone: The case of Nasonov secretion and honeybee swarms. J Chem Ecol, 25, 2051-6. [http://dx.doi.org/10.1023/A:1021084706241] Schmidt, JO (2001) Hierarchy of attractants for honey bee swarms. J Insect Behav, 14, 469-77.

270 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1023/A:1011120021964] Schmidt, JO, Slessor, KN & Winston, ML (1993) Roles of Nasonov and queen pheromones in attraction of honeybee swarms. Naturwissenschaften, 80, 573-5. [http://dx.doi.org/10.1007/BF01149276] Schneider, SS & Lewis, LA (2004) The vibration signal, modulatory communication and the organization of labor in honey bees, Apis mellifera. Apidologie (Celle), 35, 117-31. [http://dx.doi.org/10.1051/apido:2004006] Schneider, SS & McNally, LC (1991) The vibration dance behavior of queenless workers of the honey bee,Apis mellifera (Hymenoptera: Apidae). J Insect Behav, 4, 319-32. [http://dx.doi.org/10.1007/BF01048281] Schneider, SS (1987) The modulation of worker activity by the vibration dance of the honeybee, Apis mellifera. Ethology, 74, 211-8. [http://dx.doi.org/10.1111/j.1439-0310.1987.tb00934.x] Schneider, SS (1990) Queen Behavior and Worker: Queen Interactions in Absconding and Swarming Colonies of the African Honey Bee, Apis mellifera scutellata (Hymenoptera: Apidae). J Kans Entomol Soc, 179-86. Schneider, SS (1991) Modulation of queen activity by the vibration dance in swarming colonies of the African honey bee, Apis mellifera scutellata (Hymenoptera: Apidae). J Kans Entomol Soc, 269-78. Schneider, SS, Painter-Kurt, S & Degrandi-Hoffman, G (2001) The role of the vibration signal during queen competition in colonies of the honeybee, Apis mellifera. Anim Behav, 61, 1173-80. [http://dx.doi.org/10.1006/anbe.2000.1689] Schneider, SS, Stamps, JA & Gary, NE (1986) The vibration dance of the honey bee. I. Communication regulating foraging on two time scales. Anim Behav, 34, 377-85. [http://dx.doi.org/10.1016/S0003-3472(86)80105-1] Schultz, KM, Passino, KM & Seeley, TD (2008) The mechanism of flight guidance in honeybee swarms: subtle guides or streaker bees? J Exp Biol, 211, 3287-95. [http://dx.doi.org/10.1242/jeb.018994] [PMID: 18840663] Schulz, S, Yildizhan, S & van Loon, JJA (2011) The biosynthesis of hexahydrofarnesylacetone in the butterfly Pieris brassicae. J Chem Ecol, 37, 360-3. [http://dx.doi.org/10.1007/s10886-011-9939-y] [PMID: 21465267] Scott Schneider, S, DeGrandi-Hoffman, G & Smith, DR (2004) The African honey bee: factors contributing to a successful biological invasion. Annu Rev Entomol, 49, 351-76. [http://dx.doi.org/10.1146/annurev.ento.49.061802.123359] [PMID: 14651468] Seeley, TD (1995) The wisdom of the hive: the social physiology of honey bee coloniesHarvard University Press, Cambridge. [http://dx.doi.org/10.4159/9780674043404] Seeley, TD (2010) Honeybee DemocracyPrinceton University Press, Princeton, New Jersey. Seeley, TD (1982) Adaptive significance of the age polyethism schedule in honeybee colonies. Behav Ecol Sociobiol, 11, 287-93. [http://dx.doi.org/10.1007/BF00299306] Seeley, TD & Buhrman, SC (1999) Group decision making in swarms of honey bees. Behav Ecol Sociobiol, 45, 19-31. [http://dx.doi.org/10.1007/s002650050536] Seeley, TD & Morse, RA (1977) Dispersal behavior of honey bee swarms. Psyche (Camb, Mass), 84, 199209. [http://dx.doi.org/10.1155/1977/37918] Seeley, TD & Morse, RA (1978) Nest site selection by the honey bee, Apis mellifera. Insectes Soc, 25, 323-

Swarming and Queen

Polyandrous Queen Honey Bee 271

37. [http://dx.doi.org/10.1007/BF02224297] Seeley, TD, Morse, RA & Visscher, PK (1979) The natural history of the flight of honey bee swarms. Psyche (Camb, Mass), 86, 103-13. [http://dx.doi.org/10.1155/1979/80869] Seeley, T & Tautz, J (2001) Worker piping in honey bee swarms and its role in preparing for liftoff. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 187, 667-76. [http://dx.doi.org/10.1007/s00359-001-0243-0] [PMID: 11763965] Visscher, PK & Seeley, TD (2007) Coordinating a group departure: who produces the piping signals on honeybee swarms? Behav Ecol Sociobiol, 61, 1615-21. [http://dx.doi.org/10.1007/s00265-007-0393-3] Seeley, TD, Weidenmüller, A & Kühnholz, S (1998) The shaking signal of the honey bee informs workers to prepare for greater activity. Ethology, 104, 10-26. [http://dx.doi.org/10.1111/j.1439-0310.1998.tb00026.x] Seeley, T, Passino, K & Visscher, K (2006) \. Group decision making in honey bee swarms. Am Sci, 94, 2209. [http://dx.doi.org/10.1511/2006.59.220] Seeley, TD & Fell, RD (1981) Queen substance production in honey bee (Apis mellifera) colonies preparing to swarm (Hymenoptera: Apidae). J Kans Entomol Soc, 54, 192-6. Seely, TD (1979) Queen substance dispersal by messenger workers in honeybee colonies. Behav Ecol Sociobiol, 5, 391-415. [http://dx.doi.org/10.1007/BF00292527] Simpson, J (1963) Queen perception by honeybee swarms. Nature, 199, 94-5. [http://dx.doi.org/10.1038/199094a0] Simpson, J & Riedel, IBM (1963) The factor that causes swarming by honeybee colonies in small hives. J Apic Res, 2, 50-4. [http://dx.doi.org/10.1080/00218839.1963.11100056] Simpson, J (1957) OBSERVATIONS ON COLONIES OF HONEYBEES SUBJECTED TO TREATMENTS DESIGNED TO INDUCE SWARMING. Proc R Entomol Soc Lond, Ser A Gen Entomol, 32, 185-92. a [). Oxford, UK: Blackwell Publishing Ltd.]. Simpson, J (1957) The incidence of swarming among colonies of honey-bees in England. J Agric Sci, 49, 387-93. b [http://dx.doi.org/10.1017/S0021859600038375] Simpson, J (1958) The factors which cause colonies of Apis mellifera to swarm. Insectes Soc, 5, 77-95. [http://dx.doi.org/10.1007/BF02222430] Simpson, J (1959) Variation in the incidence of swarming among colonies of Apis mellifera throughout the summer. Insectes Soc, 6, 85-99. [http://dx.doi.org/10.1007/BF02223793] Simpson, J (1973) Influence of hive-space restriction on the tendency of honeybee colonies to rear queens. J Apic Res, 12, 183-6. [http://dx.doi.org/10.1080/00218839.1973.11099747] Slessor, KN, Kaminski, LA, King, GGS, Borden, JH & Winston, ML (1988) Semiochemical basis of the retinue response to queen honey bees. Nature, 332, 354-6. [http://dx.doi.org/10.1038/332354a0] Slessor, KN, Winston, ML & Le Conte, Y (2005) Pheromone communication in the honeybee (Apis mellifera L.). J Chem Ecol, 31, 2731-45. [http://dx.doi.org/10.1007/s10886-005-7623-9] [PMID: 16273438]

272 Polyandrous Queen Honey Bee

Lovleen Marwaha

Sullivan, JP, Jassim, O, Fahrbach, SE & Robinson, GE (2000) Juvenile hormone paces behavioral development in the adult worker honey bee. Horm Behav, 37, 1-14. [http://dx.doi.org/10.1006/hbeh.1999.1552] [PMID: 10712853] Taber, S, III (1954) The frequency of multiple mating of queen honey bees. J Econ Entomol, 47, 995-8. [http://dx.doi.org/10.1093/jee/47.6.995] Taranov, GF & Ivanova, LV (1946) Observations on the Behaviour of the Queen in the colony. Pchelovodstvo, 2, 35-9. Tarpy, DR & Fletcher, DJC (1998) Effects of relatedness on queen competition within honey bee colonies. Anim Behav, 55, 537-43. [http://dx.doi.org/10.1006/anbe.1997.0617] [PMID: 9514667] Tarpy, DR & Gilley, DC (2004) Group decision making during queen production in colonies of highly eusocial bees. Apidologie (Celle), 35, 207-16. [http://dx.doi.org/10.1051/apido:2004008] Tarpy, DR, Gilley, DC & Seeley, TD (2004) Levels of selection in a social insect: a review of conflict and cooperation during honey bee ( Apis mellifera ) queen replacement. Behav Ecol Sociobiol, 55, 513-23. [http://dx.doi.org/10.1007/s00265-003-0738-5] Tarpy, DR, Hatch, S & Fletcher, DJC (2000) The influence of queen age and quality during queen replacement in honeybee colonies. Anim Behav, 59, 97-101. [http://dx.doi.org/10.1006/anbe.1999.1311] [PMID: 10640371] Toth, AL & Robinson, GE (2005) Worker nutrition and division of labour in honeybees. Anim Behav, 69, 427-35. [http://dx.doi.org/10.1016/j.anbehav.2004.03.017] Villa, JD (2004) Swarming behavior of honey bees (Hymenoptera: Apidae) in southeastern Louisiana. Ann Entomol Soc Am, 97, 111-6. [http://dx.doi.org/10.1603/0013-8746(2004)097[0111:SBOHBH]2.0.CO;2] Visscher, PK, Shepardson, J, McCart, L & Camazine, S (1999) Vibration signal modulates the behavior of house-hunting honey bees (Apis mellifera). Ethology, 105, 759-69. [http://dx.doi.org/10.1046/j.1439-0310.1999.00462.x] Weaver, EC & Weaver, N (1980) Physical domination of workers by young queen honeybees (Apis mellifera L.; Hymenoptera: Apidae). J Kans Entomol Soc, 752-62. Winston, ML & Taylor, OR (1980) Factors preceding queen rearing in the Africanized honeybee (Apis mellifera) in South America. Insectes Soc, 27, 289-304. [http://dx.doi.org/10.1007/BF02223722] Winston, ML (1979) Intra-colony demography and reproductive rate of the Africanized honeybee in South America. Behav Ecol Sociobiol, 4, 279-92. [http://dx.doi.org/10.1007/BF00297648] Winston, ML The biology of the honey beeharvard university press. (1991) Winston, ML, Dropkin, JA & Taylor, OR (1981) Demography and life history characteristics of two honey bee races (Apis mellifera). Oecologia, 48, 407-13. [http://dx.doi.org/10.1007/BF00346502] [PMID: 28309760] Winston, ML, Higo, HA, Colley, SJ, Pankiw, T & Slessor, KN (1991) The role of queen mandibular pheromone and colony congestion in honey bee (Apis mellifera L.) reproductive swarming (Hymenoptera: Apidae). J Insect Behav, 4, 649-60. [http://dx.doi.org/10.1007/BF01048076] Winston, ML, Slessor, KN, Smirle, MJ & Kandil, AA (1982) The influence of a queen-produced substance, 9HDA, on swarm clustering behavior in the honeybee Apis mellifera L. J Chem Ecol, 8, 1283-8.

Swarming and Queen

Polyandrous Queen Honey Bee 273

[http://dx.doi.org/10.1007/BF00987761] [PMID: 24414734] Winston, ML, Slessor, KN, Willis, LG, Naumann, K, Higo, HA, Wyborn, MH & Kaminski, LA (1989) The influence of queen mandibular pheromones on worker attraction to swarm clusters and inhibition of queen rearing in the honey bee (Apis mellifera L.). Insectes Soc, 36, 15-27. [http://dx.doi.org/10.1007/BF02225877] Wossler, TC & Crewe, RM (1999) The releaser effects of the tergal gland secretion of queen honeybees (Apis mellifera). J Insect Behav, 12, 343-51. [http://dx.doi.org/10.1023/A:1020839505622] Woyke, J (1955) Multiple mating of the honeybee queen (Apis mellifica L.) in one nuptial flight. Bull Acad Polon Sci Cl, 3, 175-80. Wyatt, TD (2010) Pheromones and signature mixtures: defining species-wide signals and variable cues for identity in both invertebrates and vertebrates. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 196, 685-700. [http://dx.doi.org/10.1007/s00359-010-0564-y] [PMID: 20680632] Zeng, Z, Huang, ZY, Qin, Y & Pang, H (2005) Hemolymph juvenile hormone titers in worker honey bees under normal and preswarming conditions. J Econ Entomol, 98, 274-8. [http://dx.doi.org/10.1093/jee/98.2.274] [PMID: 15889713]

274

Polyandrous Queen Honey Bee, 2022, 274-288

CHAPTER 14

Requeen Process and Importance Abstract: Polyandrous queen honey bee plays a crucial role in regulating colony strength, sex ratio, colony productivity, social communication, pheromonal regulation of colonial events and developmental controls. On the other hand, a honey bee colony without a queen fails to perpetuate. Therefore for profitable beekeeping, apiarists try to inoculate a colony with a queen with considerable fertility and strong pheromonal profiling. The present chapter highlights the importance of requeening and its method .

Keywords: Re-queening and general method, Role of the queen,. 14.1. INTRODUCTION The Queen honey bee serves as an essential part of the honey bee colony, as she can regulate the strength and sex ratio of the colony. In case of unsatisfactory performance of the queen honey bee, an apiarist can replace the older queen with the reproductively active queen. The queen can be raised in specially constructed queen cells Laidlaw, 1997; Donald, 2014. A Healthy and active queen can lay about 2000 eggs/day (Root and Root, 1980). Queens can lay two types of eggs, fertilized or unfertilized, depending on the cell's width (Mattila and Seeley, 2007). Differential development occurs from queen larvae, as it is fed on royal jelly, a protein-rich secretion from the gland of young workers. If larvae, which develop from fertilized eggs, are provided on worker jelly, they will grow into worker bees (Gensen, 2000). Queen larvae develop inside of the queen cell. On pupation, the head is projected toward the downward direction, which workers seal. Later on, the virgin queen emerges out of that cell. Artificial queen bee rearing is a unique process involving a peculiar application of bee biology. In the specific process, nurse worker bees can produce queens from young female grafted larvae. The healthy queen bee is an essential part of the colony, as she regulates the population of available worker bees versus broods in the colony. The queen replacement strategy is generally implemented for thriving colonies and apicultural production flourishment. Worker honey bees raise a queen in a peanut-shaped wax cell (Laidlaw and Page, 1997;kumar, 2018). Queen honey bees, after successful mating and with a proper diet, can oviposit Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

Process and Importance

Polyandrous Queen Honey Bee 275

about 2000 eggs/day (Root and Root, 1980). Queens can lay down two types of eggs; fertilized eggs and unfertilized eggs, depending upon the width of wax cells on the comb(Mattila and Seeley, 2007). A queen that develops from a larva is formed from the fertilized egg and continuously feeds on royal jelly until complete development. The product will include a worker honey bee if a specific larva is not provided on royal jelly (Jensen, 2000). Subsequently, upon completion of the larval feeding stage, pupation occurs, and the worker honey bee seals the queen cell until the queen honey bee emerges. During swarming, older queens leave the hive before the emergence of the first, virgin queen honey bees (Laidlaw and Page, 1997). Artificial queen rearing had been practised in ancient Greece, and apiarists usually put worker honey bee comb with younger larvae comb in the queen-less colony. Subsequently, it has been noticed that worker honey bees could raise emergency queen cells. Jacob Nickel, 1565 described the protocol for raising honey bee queens from worker eggs or younger larvae. Queen production for sale was practised in 1861, by Alley, Carey and Pratt, from Massachusetts, U.S.A. Earlier, producers used a narrow strip of comb containing eggs and larvae, which they fascinate on top of the comb. Modern queen rearing technique was initiated in the 19th Century, and Gilbert Doolittle (1889) in the U.S.A. developed a comprehensive system for rearing queen bees. He carried out the queen rearing method by transferring worker bee larvae to queen cells. Doolittle, 1915, had carried queen rearing using a queen excluder that is still plasticized (Doolittle, 1915). Apiarists rear many queens for multi-purposes, including requeening of the colony, swarming reduction, brood and honey production, for initiation of the new colony (Laidlaw and Page, 1997; Ruttner, 1983). For queen rearing workers, larvae younger than 12-24 hours are selected and grafted to the queen cell cup. After that, larvae are fed royal jelly. Different artificial queen production methods are available, which can be implemented to replace old reproductively less active queens. Honey bee colonies respond differently to various bee rearing techniques, as the specific process is dependent upon ecology, climatic factors, the genetic integrity of species, pollen source, and behavioural and biological factors (Zhadanova, 1967; Wen and Chong, 1985; Morse, 1994; Nuru and Dereje, 1999; Koç and Karacaoğlu, 2004; Dodologlu et al., 2004; Cengiz et al., 2009; Nuru, 2012; Crailsheim et al., 2013). Proper control of the quality of queen, progeny production, apicultural productivity, strength of the colony, swarming and colony behaviour can be regulated. Various environmental factors influence the quantity, quality and queen honey bee production (Mahbobi et al., 2012; Kumar, 2018). Further, artificial queen rearing helps in reducing swarming tendency, enhancing apicultural productivity of colonies, modulation of colonial strength, increasing colony

276 Polyandrous Queen Honey Bee

Lovleen Marwaha

number and genetic improvement of the specific colony (Morse, 1979, 1994; Crane, 1990; Laidlaw and Page, 1997). Queen honey bees influence the colony's productivity and behavioural and immunological aspects (Laidlaw, 1979; Morse, 1979; Ruttner, 1983). In past decades, different technologies have been implemented to rear queens from single colonies (Johansson and Johansson, 1973; Morse, 1979; Harry and Laidlaw, 1981; Ruttner, 1983). Wilkinson and Brown (2002) concluded that there is significant variation in the length of queen wax cells in proper queen colonies and queen fewer colonies. According to Büchler et al. (2013), acceptance or rejection of queen larvae is influenced by the presence or absence of a queen in a colony and the protocol of queen rearing. Skowronek and Skubida (1988) suggested that larval grafting was successful in queen cups with a diameter of 7.8 - 9.0 mm in Apis mellifera. Therefore it could be concluded that the acceptance rate of grafted larvae depends upon queen cup size (Ratnieks and Nowogrodzski, 1988; El-Din, 1999; Buchler et al., 2013). Additionally, the number and quality of the reared queen are influenced by technique, the strength of the colony and food resources (Morse, 1979; Laidlaw, 1979; Wilkinson and Brown, 2002; Buchler et al., 2013). Adgaba et al., 2018, studied the acceptance, sealed and emerging rate of grafted larvae concomitantly with the comparative quality of queens developed from different cells (Adgaba et al., 2018). The method of queen rearing facilitates the apiarist to replace old and reproductively inactive queens in honey bee colonies (Joseph Latshaw, 2011). In addition, the artificial rearing of the queen method allows apiarists to understand honey bee behaviour and genetics (Hamdan, K. 2010). By specific process, apiarists can get queens with high honey production, high reproduction potential, good temperament, resistance to disease, swarming and colour(Jonestone, 2008). Queen production requires the grafting of larvae into the queen cell. Queen-less condition and cell shape are stimulants for producing new queens (Abbasi et al., 2015). 14.2. QUEEN'S ROLE AND GENERAL DEVELOPMENT In a honey bee colony, queen production is an essential task, as colony flourishment depends upon the queen's quality. However, the preference of honey bee workers for selecting particular larvae for queen rearing is poorly understood. Female caste development depends on a larval diet, as worker honey bees provide a special diet to developing queen larvae. In other words, developmental determination is controlled by the diet given to developing larvae (Rhetn, 1933; Haydak, 1970; Mao et al., 2015). Larvae develop from the fertilized egg. It is fed on royal jelly, it develops into the queen, whereas larvae fed on worker jelly

Process and Importance

Polyandrous Queen Honey Bee 277

develop into workers (Rhetn, 1933; Mao et al., 2015; Haydak, 1970; Bruwers et al.,1987; Shi,2011; Wang et al., 2016). Sagili et al. 2018 reported that worker honey bees select well-fed larvae for queen rearing than deprived larvae. They further concluded that worker honey bees perceive the nutritional status of growing larvae and can rear them as queens, depending upon the colony requirement. The colonial organization depends upon a reproductively active queen, as only she can lay fertilized eggs. Additionally, she possesses the longest life span of about 1-8 years than other castes (Bozina, 1961; Page et al., 2001). After hatching, the queen can mate with about 5-21 drones in a few days (Estoup et al., 1994; Robinson et al.,1994; Arnold et al., 1996; Oldroyd et al., 1997; Tarpy and Nielsen, 2002; Seeley et al., 2007; Heidinger et al., 2014). Therefore, this polyandrous specification results in the distribution of multiple patrilines among fertilized eggs (Estoup et al., 1994; Oldroyd et al., 1997). Fertilized eggs exhibit developmental plasticity upto the third instar larval stage, as they can develop into queen or worker honey bees (Weaver, 1957; Rembold et al., 1980; Dedej et al., 1998). A colony rears a new queen to replace the queen in case if older queen becomes reproductively inactive or dead or in case of a missing queen, diseased queen or dead queen (Butler, 1957; Lensky and Slabezki, 1981; Graham et al., 1992; Tofilski and Czekonska, 2004). If a colony fails to requeen, eventually colony will be lost in case of the sudden loss of a queen or in case of an inactive reproductive queen, the colony rear queen in an emergency. In other words, the colony has only six days to begin the process of queen rearing, as three days are required for the hatching of the egg and afterwards, only for next three days for larvae to remain totipotent (Woyke, 1971; Fell and Morse,1984; Dedej et al., 1998; Shi et al., 2011). Therefore, emergency queen rearing is a great urgent process. Workers select totipotent larvae for queen rearing. The specific method is signified by modifying the worker wax cell into a queen cell, which is a peanut-shaped queen cell (Punnett and Winston, 1983; Hatch et al., 1999; Shi et al., 2011). The queen cell size, shape and orientation specify the queen bee rearing behaviour (Fell and Morse, 1984; Shi et al., 2011). Honey bees can differentiate between broods, castes, and various developmental stages (Free, 1967; Free et al.,1975; Free and Winder, 1983; Free et al., 1989). It has been reported that worker honey bees recognize surface pheromones secreted from hungry or well-fed larvae (Free and Winter, 1983; Free et al.,1989). In addition, worker honey bees detect volatile pheromone E-β-ocimene produced by hungry larvae (He et al.,2016). However, the mechanism of worker bee preference for emergency rearing is not fully known.

278 Polyandrous Queen Honey Bee

Lovleen Marwaha

Grafting larva into a queen cell for queen rearing is an old technique, implemented since the 1800s (Webster et al., 1987; Doolittle, 1889; Crailsheim et al.,2013). Various explorations indicate the difference between the queen-rearing process, which occurs under natural conditions or artificial queen-rearing conditions (Breed et al., 1994). Nurse bees select larvae for queen making during emergency queen emergence and transfer them to the queen cell. (Sagili et al., 2018) analyzed queen development by considering the correlation of specific processes under provided nutrition under natural or artificial queen rearing conditions. Further, they carried out experiments to understand the behaviour of nurse bees to deprived larvae and the selection of specific larvae for the queen rearing process. It had been reported that healthy feed possesses more chance of developing into a high-quality queen. Workers can assess the reproductive activeness of other caste, therefore, modify their behaviour according to caring specific caste (Long et al., 2017; Slone et al., 2012). Food-deprived larva secretes pheromonally message of a hunger signal to nurse bees for being fed. Although, the same hunger signal worker honey bees can consider a negative signal for the likelihood of being selected for queen rearing. Further, they concluded that worker honey bees like larvae for queen rearing based on chemicals secreted by developing larva, not exclusively based on nutritional status. Furthermore, worker honey bees consider the E-β-Ocimene levels and other chemicals for preferential selection of larva for queen rearing among well-fed and malnourished larvae. Additionally, short deprivation of larvae from food can profoundly affect the final fate of larva development. Sagili et al., 2018 observed that queen rearing by natural emergency and larval grafting methods provided significantly different results. Natural queen rearing methods exhibited differential behaviour for non-deprived and deprived larvae, whereas for the artificial grafting method, worker bees were indifferent to both types of larvae(Sagili et al., 2018). It has been reported that pheromone E--Ocimene regulates inhibition of worker ovarian development, enhance hypopharyngeal gland development and control foraging behaviour in worker (Gilley et al., 2006; DeGrandt-Hoffman et al., 2007; Malsonnasse et al., 2009; Malsonnassae et al., 2010; Carroll and Duehl, 2012; Ma et al., 2016). Wei et al., 2019, noted that queen honey bees laid more large queen cells than worker cells. Further, they reached a queen developed from a queen egg and a queen reared by worker larvae artificially, indicating that the queen possesses more ovarioles in the former case. Additionally, gene expression analysis revealed differential gene expression in the queen, which develops from the queen egg and from worker larvae. The differentially expressed genes in both castes include a gene which regulates hormone signalling and development, which regulates

Process and Importance

Polyandrous Queen Honey Bee 279

immune pathways. It has been reported that larval diet changes methylation patterns in honey bees [Kucharaski et al., 2008; Lyko et al., 2010; Shi et al., 2011:Wei, et al., 2019). 14.3. METHOD OF QUEEN PRODUCTION Queen can be produced by the natural or artificial method. For healthy growth of the colony, requeening is required every year. However, in case of queen injury or disease, artificial queen production is needed. The queen rearing method was modified by Johanson and Johanson (1973), Laidlaw (1979), and Wongsiri et al. (1990). Further, the queen can be reared by using natural or artificial grafting larva in queen cups (Miller, 1912; Doolittle, 1915; Laidlaw, 1979; Wongsiri et al. 1990; Kumar and Kumar, 1999; Kumar and Kumar, 2000), whereas in commercial queen rearing method, usually 12-24 hrs are grafted in artificial prepared wax or plastic cups (Laidlaw, 1979; Ruttner, 1983). Further, nurse worker honey bees can reen grafted larvae, which provide royal jelly and can change the fate of grafted larvae development. Wongsiri et al. (1990) reported queen cell production by feeding larvae on a sugar diet. Chang and Hsieh (1993) said that tea pollen, natural pollen and sugar syrup give the best results. Furthermore, according to Eckert and Shaw (1960), the presence of larvae with one day old age, more nurse bees, and food availability lead best results. Winston (1987) suggests that the availability of protein food to worker honey bees increases the development of hypopharyngeal glands in honey bees. Brodschneider and Crailsheim (2010) reported that brood production and larval development are influenced by protein availability to worker honey bees. Maurizio (1950) said that the development of the hypopharyngeal gland and fat bodies is controlled by a lack of vitamins in the diet. Haydak and Dietz (1965) and Anderson and Dietz (1976) reported the influence of vitamin pyridoxine and inositol on the development of honey bee larvae. They concluded that specific vitamin is required for normal development. It has been reported that vitamin B1 (thiamine) increases the frequency of queen cell production, whereas vitamin B complex, combined with vitamin C, the blend together induces mortality in control. Herbert et al. (1985) concluded that vitamin C fed to adult workers results in a high brood survival rate. According to Kaftanoglu et al. (2010), including sugar in the diet composite also positively affects ovariole number, larval survival and weight (Haleem et al., 2015). Artificial bee rearing technique helps in requeening the colony to minimize swarming tendency, increase apicultural productivity, enhance brood and honey production, and increase colony number (Morse, 1979, 1994; Crane, 1990;

280 Polyandrous Queen Honey Bee

Lovleen Marwaha

Laidlaw and Page, 1997; Laidlaw,1979; Morse,1979; Ruttner,1983). A queen influences the general behaviour and disease resistance in the colony. Therefore artificial queen rearing is essential for honey bee colony growth and flourishment (Morse,1979; Ratnieks and Nowogrodzki, 1988). Accordingly, different techniques have been developed to raise a queen from a single colony (Johansson and Johansson, 1973; Morse, 1979; Harry and Laidlaw, 1981; Ruttner, 1983). Moreover, the colony's response to different queen-rearing techniques varies from race to race due to other environmental conditions and behavioural and biological factors (Morse, 1994; Nuru and Dereje, 1999; Nuru, 2012; Crailsheimet al., 2013). The quality of queen rearing is affected by factors like temperature, relative humidity and pollen source plants (Zhadanova,1967; KoçandKaracaog˘lu,2004; Cengiz et al., 2009; Wen-Cheng and Chong-Yuan, 1985; Morse, 1994; Dodologlu et al., 2004; Cengizetal.,2009; Crailsheimetal.,2013). Büchler et al. (2013) reported that the acceptance of grafted queens is affected by the presence or absence of the queen-rearing method. Skowronek and Skubida (1988) noticed that the grafting process by more successful in queen cups with the dimension of 7.8–9.0 mm, in the case of Apis mellifera. Few explorations witness that queen grafting methods in queen-less or of queen righted colonies affect the quality of queen and queen acceptance (Laidlaw, 1979; Laidlaw and Page, 1997; Emsen et al., 2003; Cengiz et al., 2009, Ahmad and Dar, 2013; Büchler et al., 2013). Additionally, queen quality is affected by available resources in the colony and the strength of young worker bees (Morse, 1979; Laidlaw, 1979; Wilkinson and Brown, 2002; Büchler et al., 2013). For queen rearing, equipment used includes cell cups, bars and frames. Artificial queen-rearing larvae from worker honey bees are placed into cell cups in bars and bars, in turn, placed on frames. Queen cups are about 8-9 mm in diameter at the rim. Ruttner (1983) and Laidlaw (1979) described the production of queen cell cups that could be prepared from bee wax, which is appropriately cleaned with the soap solution and stored in a sealed box to keep them free from dirt. Most apiarists attach queen cups with cell bars. Additionally, plastic cups can be used for queen rearing. Plastic cups can be recycled after washing with detergent. Additionally, plastic queen cups can be introduced about a day before, so that honey bees can clean and polish the cups. The plastic cups can be fixed on a bar, and 2-4 bars can be fixed on a frame. Furthermore, about 10-20 cells can be attached to each bar. The grafting of larvae can be carried out with the help of grafting needles. Some grafting needles carry magnifying glasses fitted to the stem for magnification. In a grafting needle, usua-

Process and Importance

Polyandrous Queen Honey Bee 281

lly, both ends are designed for grafting, but with different configurations. Even an artist's paintbrush can be used for grating. Usually, grafting is accessible from a dark wax comb than a light wax comb because of the better contrast of dark background with white larvae. An illuminated grafting magnifier can be used for better larvae vision. The grafting process can be carried out in the room or in indirect light, as larvae do not dry out and remain protected from U.V. radiation from direct sunlight. Colonies accept larvae easily when larvae are taken from the same colony. Ripe queen cells can be transferred from the rearing colony to the mating colony, about 1-2 days before for proper acceptance. If queen cells are allowed to emerge in the brood chamber, they must be protected against other queens and workers, which can be carried out by emergence cells. Queen cell protectors can be made from insulation tape, tin foil or plastic tubing, which can be placed on developing queen cells, which helps in queen cell protection from worker honey bees, which can chew queen cells. Additionally, many wooden or plastic emergence cages are available, which help protect all queen cells on the cell bar. Queens can be produced by the Alley method (Ruttner, 1983), which involves grafting the one-day-old larva into a downward-pointing queen cell. Large-scale production of the queen requires grafting procedures and proper rearing. Different ways are available, which help in the production of high-quality queens. Queenrearing colonies need plenty of younger and well-fed bees to supply royal jelly to the colony. The grafting of larvae can be easily carried out from dark combs. For large-scale production, the quality of the queen depends upon the grafting method and colony management.To enhance queen acceptability, different ways are available for the successful production of queen cells and to rear high queen quality. An additional comb filled with pollen cells is supplied to the colony to accelerate the process. For a successful rearing comb of pollen, honey and a sealed brood is also placed, with grafted queen cell. For the grafting procedure, worker larvae are transferred from the original cell to the artificial queen cell. Grafting of larvae can be carried out under suitable environmental conditions, with optimum temperature about 24-26°C and relative humidity > 50%. Grating should be carried out in the laboratory, as larvae are sensitive to temperature, direct sunlight and low humidity. Additionally, grafting in the laboratory helps in protection against robbing bees. The grafting laboratory should be near to breeder and nurse colonies—furthermore, cool light helps in avoiding additional heat, which could harm the colony. For grafting larvae, larvae must be selected from well-fed as nurse bees because nurse bees do not accept food-deprived larvae. Brood comb should not expose to direct sunlight, as it can cause drying of larvae. If the

282 Polyandrous Queen Honey Bee

Lovleen Marwaha

weather is dry, wet cloth can be spread over eggs, preventing them from drying and desiccation. Additionally, the damp cloth also protects from light and dust. For experienced beekeepers, larvae grafting can be done on three bars within 8-10 minutes or less. After grating one bar, it should be covered with a damp cloth. To carry grafted larvae, special carrying boxes are used, which help to protect larvae from drying. After grafting frame should be placed in a rearing colony soon, and Queen larvae can be placed in a mixture of royal jelly and half-warm water. Queen rearing depends on factors like quality, strength, age of workers, developmental stage of nurse colonies, grafted larval age, queen-less or queenrighted condition, number of drafted cells, and rearing method. Furthermore, additional factors like humidity regulation, temperature, the vitality of queen cells, nectar supply, and supplemental feeding affect the queen rearing process. With proper management, even bad weather conditions do not influence the acceptability of grafted larvae. After hatching, the queen's mating can be achieved by the instrumental insemination technique (Cobey et al., 2013). Additional natural mating can be used through drones of other colonies. Drone colonies can be produced in sufficient numbers, to ensure reproduction. Drone colony building should start well in advance. Drone colonies are developed from healthy and strong colonies. Additionally, a continuous supply of pollen and honey is provided to them. Further, regular monitoring is required to achieve a high-quality control level. Generally, Varroa and other more vital pathogens tend to affect drones. Additionally, careful varroa management in the case of drone colonies helps regulate the concerned problem regulation (Büchler et al. (2010). Drone colony production should start about two months earlier. As for development, about 40 days are required, and the life span of drones is about two months. Queen can be shipped in plastic cages of variable sizes and shapes. There are two different types of cells, including small-sized and large-sized, for shipment of the queen. A larger compartment can house about the queen and 6-12 attendant worker bees, whereas a small shipment cage includes a cage filled with queen and queen candy, which provides food during shipping. For the introduction of the queen into the colony, a small hole can be created, which workers eat slowly to reach upto queen(Büchler et al., 2012) CONCLUSION Complete colony growth is dependent upon the quality of the queen honey bee. Therefore, beekeepers prefer to replace the queen annually. Artificial queen rearing methods better replace queen honey bees with a comparatively good

Process and Importance

Polyandrous Queen Honey Bee 283

quality queen. Above mentioned discussion highlights the protocol for artificial queen rearing REFERENCES Adgaba, N, Al-Ghamdi, A, Tadesse, Y, Alsarhan, R, Single, A, Mohammed, SE & Ali Khan, K (2019) The responses of Apis mellifera jemenitica to different artificial queen rearing techniques. Saudi J Biol Sci, 26, 1649-54. [http://dx.doi.org/10.1016/j.sjbs.2018.08.028] [PMID: 31762639] Ahmad, SB & Dar, SA (2013) Mass rearing of queen bees, Apis mellifera l.(hym: apidae) for bee colony development raised under the temperate conditions of Kashmir. Bioscan, 8, 945-8. Anderson, LM & Dietz, A (1976) Pyridoxine requirement of the honey bee (Apis mellifera) for brood rearing. Apidologie (Celle), 7, 67-84. [http://dx.doi.org/10.1051/apido:19760105] Arnold, G, Quenet, B, Cornuet, J-M, Masson, C, De Schepper, B, Estoup, A & Gasqui, P (1996) Kin recognition in honeybees. Nature, 379, 498-8. [http://dx.doi.org/10.1038/379498a0] Bozina, KD (1961) How long does the queen live? Pchelovodstvo, 38, 13. Breed, MD, Welch, CK & Cruz, R (1994) Kin discrimination within honey bee (Apis mellifera) colonies: An analysis of the evidence. Behav Processes, 33, 25-39. [http://dx.doi.org/10.1016/0376-6357(94)90058-2] [PMID: 24925238] Brodschneider, R & Crailsheim, K (2010) Nutrition and health in honey bees. Apidologie (Celle), 41, 278-94. [http://dx.doi.org/10.1051/apido/2010012] Brouwers, EVM, Ebert, R & Beetsma, J (1987) Behavioural and physiological aspects of nurse bees about the composition of larval food during caste differentiation in the honeybee. J Apic Res, 26, 11-23. [http://dx.doi.org/10.1080/00218839.1987.11100729] Büchler, R, Andonov, S, Bienefeld, K, Costa, C, Hatjina, F, Kezic, N, Kryger, P, Spivak, M, Uzunov, A & Wilde, J (2013) Standard methods for rearing and selection of Apis mellifera queens. J Apic Res, 52, 1-30. [http://dx.doi.org/10.3896/IBRA.1.52.1.07] Butler, CG (1957) The process of queen supersedure in colonies of honeybees (Apis mellifera Linn.). Insectes Soc, 4, 211-23. [http://dx.doi.org/10.1007/BF02222154] Carroll, MJ & Duehl, AJ (2012) Collection of volatiles from honeybee larvae and adults enclosed on brood frames. Apidologie (Celle), 43, 715-30. [http://dx.doi.org/10.1007/s13592-012-0153-x] Cengiz, M, Emsen, B & Dodologlu, A (2009) Some characteristics of queen bees (Apis mellifera L.) rearing in queenright and queenless colonies. J Anim Vet Adv, 8, 1083-5. Chang, CP & Hsieh, FK (1993) Factors affecting royal jelly production. In: Connor, L.J., Rinderer, T.E., Sylvester, H.A., Wongsiri, S., (Eds.), Asian Apiculture, Wicwas Press U.S.A. 316-26. Crailsheim, K, Brodschneider, R, Aupinel, P, Behrens, D, Genersch, E, Vollmann, J & Riessberger-Gallé, U (2013) Standard methods for artificial rearing of Apis mellifera larvae. J Apic Res, 52, 1-16. [http://dx.doi.org/10.3896/IBRA.1.52.1.05] Crane, E (1990) Bees and beekeeping: science, practice and world resourcesHeinemann Newnes. Dedej, S, Hartfelder, K, Aumeier, P, Rosenkranz, P & Engels, W (1998) Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee ( Apis mellifera carnica ). J Apic Res, 37, 183-90. [http://dx.doi.org/10.1080/00218839.1998.11100970]

284 Polyandrous Queen Honey Bee

Lovleen Marwaha

DeGrandi-Hoffman, G, Gilley, D & Hooper, J (2007) The influence of season and volatile compounds on the acceptance of introduced European honey bee ( Apis mellifera ) Queens into European and Africanized colonies. Apidologie (Celle), 38, 230-7. [http://dx.doi.org/10.1051/apido:2007003] Dodologlu, A, Emsen, B & Gene, F (2004) Comparing some characteristics of queen honey bees (Apis mellifera L.) reared using the Doolittle method and natural queen cells. J Appl Anim Res, 26, 113-5. [http://dx.doi.org/10.1080/09712119.2004.9706518] Donald, E (2014) http:/ www.fairmontstate.edu.com Doolittle, GM (1915) Scientific queen-rearing is practically applied, being a method by which the best of the queen -bees are reared in perfect accord with nature's ways. American Bee Journal; 6th edition, Published hamilton, III USA. 126. Doolittle, GM (1889) Doolittle, G.M., 1889. Scientific queen-rearing is practically applied.: being a method by which the best of queen bees are reared in perfect accord with nature's ways (Thomas G Newman & Son, Eckert, JE & Shaw, FR (1960) Beekeeping, The Macmillan Company, New York 536. El-Din, HAES (1999) Biological and ecological studies on rearing honeybee (Apis mellifera L.) for commercial queens production. Mitsubachi Kagaku, 20, 127-30. Emsen, B, Dodologlu, A & Gene, F (2003) Effect of larvae age and grafting method on the larvae accepted rate and height of sealed queen cell (Apis mellifera L.). J Appl Anim Res, 24, 201-6. [http://dx.doi.org/10.1080/09712119.2003.9706457] Estoup, A, Solignac, M & Cornuet, JM (1994) Precise assessment of the number of patrilines and of genetic relatedness in honeybee colonies. Proc Biol Sci, 258, 1-7. [http://dx.doi.org/10.1098/rspb.1994.0133] Fell, RD & Morse, RA (1984) Emergency queen cell production in the honey bee colony. Insectes Soc, 31, 221-37. [http://dx.doi.org/10.1007/BF02223608] Free, JB & Williams, IH (1976) The effect on the foraging behaviour of honeybees of the relative locations of the hive entrance and brood combs. Appl Anim Ethol, 2, 141-54. [http://dx.doi.org/10.1016/0304-3762(76)90042-0] Free, JB & Winder, ME (1983) Brood recognition by honeybee (Apis mellifera) workers. Anim Behav, 31, 539-45. [http://dx.doi.org/10.1016/S0003-3472(83)80077-3] Free, JB (1967) Factors determining the collection of pollen by honeybee foragers. Anim Behav, 15, 134-44. [http://dx.doi.org/10.1016/S0003-3472(67)80024-1] [PMID: 6031100] Free, JB, Ferguson, AW & Simpkins, JR (1989) The effect of different periods of brood isolation on subsequent brood-cell visits by worker honeybees (Apis mellifera L.). J Apic Res, 28, 22-5. [http://dx.doi.org/10.1080/00218839.1989.11100815] Gensen, M (2000) Queen rearing with Apiscerana - the Jensen method. Bees for Development Journal, l, 55. Gilley, DC, DeGrandi-Hoffman, G & Hooper, JE (2006) Volatile compounds emitted by live European honey bee (Apis mellifera L.) queens. J Insect Physiol, 52, 520-7. [http://dx.doi.org/10.1016/j.jinsphys.2006.01.014] [PMID: 16530783] Graham, JM (1992) Haleem, N, Kumar, NR & Kaur, R (2015) Effect of nutritional supplements on queen cell production in honey bee (Apis mellifera). J Appl Nat Sci, 7, 400-3. [http://dx.doi.org/10.31018/jans.v7i1.623] Harry, H & Laidlaw, JR (1981) Contemporary queen rearingDadant and Sons Inc., U.S.A..

Process and Importance

Polyandrous Queen Honey Bee 285

Hatch, S, Tarpy, DR & Fletcher, DJC (1999) Worker regulation of emergency queen rearing in honey bee colonies and the resultant variation in queen quality. Insectes Soc, 46, 372-7. [http://dx.doi.org/10.1007/s000400050159] Haydak, MH & Dietz, A (1965) Influence of the diet on the development and brood rearing of honeybees. Proceedings of the XV Beekeeping Congress, BucharestVol. 20, 158-62. Haydak, MH (1970) Honey bee nutrition. Annu Rev Entomol, 15, 143-56. [http://dx.doi.org/10.1146/annurev.en.15.010170.001043] He, XJ, Zhang, XC, Jiang, WJ, Barron, AB, Zhang, JH & Zeng, ZJ (2016) Starving honey bee (Apis mellifera) larvae signal pheromonally to worker bees. Sci Rep, 6, 22359. [http://dx.doi.org/10.1038/srep22359] [PMID: 26924295] Heidinger, I, Meixner, M, Berg, S & Büchler, R (2014) Observation of the mating behaviour of honey bee (Apis mellifera L.) queens using radio-frequency identification (RFID): factors influencing the duration and frequency of nuptial flights. Insects, 5, 513-27. [http://dx.doi.org/10.3390/insects5030513] [PMID: 26462822] Herbert, EW, Jr, Vanderslice, JT & Higgs, DJ (1985) Vitamin C enhancement of brood rearing by caged honeybees fed a chemically defined diet. Arch Insect Biochem Physiol, 2, 29-37. [http://dx.doi.org/10.1002/arch.940020104] Jensen, M (2000) Queen rearing with Apis cerana-the Jensen method. Bees for Development Journal, l, 55. Johansson, TSK & Johansson, MP (1973) Methods for Rearing Queens. Bee World, 54, 149-75. [http://dx.doi.org/10.1080/0005772X.1973.11097476] Jonstone, M (2008) Prime facts 828, Rearing queen bees, Honey Bees, Richmond. Science, 317, 362-4. Joseph, L (2011) Queen Rearing Basics Kaftanoglu, O, Linksvayer, TA & Page, RE, Jr (2011) Rearing honey bees, Apis mellifera, in vitro 1: effects of sugar concentrations on survival and development. J Insect Sci, 11, 96. [PMID: 22208776] Abbasi, KH, Shafiq, M, Ahmad, KJ, Razzaq, A, Saleem, M & Ullah, MA (2015) Performance of larval grafted queen vs queen produced through the natural method in Apis mellifera. J Entomol Zool Stud, 3, 47-9. Koç, AU & Karacaoglu, M (2004) Effects of rearing season on the quality of queen honey bees (Apis mellifera L.) raised under the conditions of Aegean Region. Mellifera, 4-7, 34-7. Kucharski, R, Maleszka, J, Foret, S & Maleszka, R (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science, 319, 1827-30. [http://dx.doi.org/10.1126/science.1153069] [PMID: 18339900] Kumar, N (2018) Evaluation of larval grafted queen and naturally reared queen of Italian honey bees (Apis mellifera L.). J Pharmacogn Phytochem, SP1, 3181-3. Kumar, R & Kumar, NR (2000) Queen rearing and royal jelly production in Asian honey bee Apis cerana, Asian Bees and Beekeeping. Progress of Research and Development 145-7. Laidlaw, HH & Page, RE (1997) Queen rearing and bee breeding. Wicwas Press. Wicwas, Cheshire, UK, 1997, 73-4. Laidlaw, HH (1979) Contemporary queen rearing Lensky, Y & Slabezki, Y (1981) The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups. J Insect Physiol, 27, 313-23. [http://dx.doi.org/10.1016/0022-1910(81)90077-9] Long, K, Cao, TT, Keller, JJ, Tarpy, DR, Shin, M & Schneider, SS (2017) Levels of selection shaping caste interactions during queen replacement in the honey bee, Apis mellifera. Insectes Soc, 64, 227-40. [http://dx.doi.org/10.1007/s00040-016-0537-4]

286 Polyandrous Queen Honey Bee

Lovleen Marwaha

Lyko, F, Foret, S, Kucharski, R, Wolf, S, Falckenhayn, C & Maleszka, R (2010) The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol, 8, e1000506. [http://dx.doi.org/10.1371/journal.pbio.1000506] [PMID: 21072239] Ma, R, Mueller, UG & Rangel, J (2016) Assessing the role of β-ocimene in regulating foraging behavior of the honey bee, Apis mellifera. Apidologie (Celle), 47, 135-44. [http://dx.doi.org/10.1007/s13592-015-0382-x] Mahbobi, A, Farshineh, AM, Woyke, J & Abbasi, S (2012) Effects of the age of grafted larvae and the effects of supplemental feeding on some morphological characteristics of Iranian queen honey bees (Apis mellifera). J Apic. Res Pp, 56, 93-8. Maisonnasse, A, Lenoir, JC, Beslay, D, Crauser, D & Le Conte, Y (2010) E-β-ocimene, a volatile brood pheromone involved in social regulation in the honey bee colony (Apis mellifera). PLoS One, 5, e13531. [http://dx.doi.org/10.1371/journal.pone.0013531] [PMID: 21042405] Maisonnasse, A, Lenoir, JC, Costagliola, G, Beslay, D, Crauser, D, Plettner, E & Le Conte, Y (2009) E--ocimene is a new volatile primer pheromone that inhibits worker ovary development in honey bees. Apidologie (Celle), 40, 562-4. [http://dx.doi.org/10.1051/apido/2009024] Mao, W., Schuler, M.A. and Berenbaum, M.R., (2015). A dietary phytochemical alters caste-associated gene expression in honey bees: Science Advances, 1(7), p.e1500795. Mattila, HR & Seeley, TD (2007) Genetic diversity in honey bee colonies enhances productivity and fitness. Science, 317, 362-4. [http://dx.doi.org/10.1126/science.1143046] [PMID: 17641199] Miller, CC (1912) How best queens can be secured. Am Bee J, 52, 243. Morse, RA (1979) Rearing Queen Honeybees, Wicwas Press, U.S.A. 18-116. Nuru, A (2012) Suitable honeybee colony multiplication technique using intermediate top-bar hiveshttp://www.apitradeafrica.org/publications/cat apiexpo-Africa-2012.html Nuru, A, Ahmad, AA, Awraris, G, Yilma, T, Anwer, A, Mohammad, JA, Mohammed, A & Deepak, S (2016) Natural nest characteristics of Apis mellifera jemenitica (Hymenoptera; Apidae) and its implications in frame hive adoption. J Anim Plant Sci, 26 Nuru, A & Dereje, W (1999) Responses of Ethiopian honeybees to different queen rearing techniques. Oldroyd, BP, Clifton, MJ, Wongsiri, S, Rinderer, TE, Sylvester, HA & Crozier, RH (1997) Polyandry in the genus Apis, particularly Apis andreniformis. Behav Ecol Sociobiol, 40, 17-26. [http://dx.doi.org/10.1007/s002650050311] Page, RE, Jr & Peng, CYS (2001) Aging and development in social insects with emphasis on the honey bee, Apis mellifera L. Exp Gerontol, 36, 695-711. [http://dx.doi.org/10.1016/S0531-5565(00)00236-9] [PMID: 11295509] Punnett, EN & Winston, ML (1983) Events following queen removal in colonies of european-derived honey bee races (Apis mellifera). Insectes Soc, 30, 376-83. [http://dx.doi.org/10.1007/BF02223969] Ratnieks, FL & Nowogrodzki, R (1988) Small-scale queen rearing by beekeepers in the Northeast Rembold, H, Kremer, JP & Ulrich, GM (1980) Characterizing postembryonic developmental stages of the female castes of the honey bee, Apis mellifera L. Apidologie (Celle), 11, 29-38. [http://dx.doi.org/10.1051/apido:19800104] Von Rhein, W (1933) Über die entstehung des weiblichendimorphismusimbienenstaate. Wilhelm Roux Arch Entwickl Mech Org, 129, 601-65. [PMID: 28353923] Robinson, GE, Page, RE, Jr & Arensen, N (1994) Genotypic differences in brood rearing in honey bee

Process and Importance

Polyandrous Queen Honey Bee 287

colonies: context-specific? Behav Ecol Sociobiol, 34, 125-37. [http://dx.doi.org/10.1007/BF00164183] Root, AI (1910) The ABC and XYZ of bee cultureA.I. Root Company. Ruttner, F (1983) Queen rearing; biological basis and technical instruction, Apimondia Publishing House, Bucharest 358. Sagili, RR, Metz, BN, Lucas, HM, Chakrabarti, P & Breece, CR (2018) Honey bees consider larval nutritional status rather than genetic relatedness when selecting larvae for emergency queen rearing. Sci Rep, 8, 7679. [http://dx.doi.org/10.1038/s41598-018-25976-7] [PMID: 29769574] Seeley, TD & Tarpy, DR (2007) Queen promiscuity lowers disease within honeybee colonies. Proc Biol Sci, 274, 67-72. [http://dx.doi.org/10.1098/rspb.2006.3702] [PMID: 17015336] Shi, YY, Huang, ZY, Zeng, ZJ, Wang, ZL, Wu, XB & Yan, WY (2011) Diet and cell size both affect queenworker differentiation through DNA methylation in honey bees (Apis mellifera, Apidae). PLoS One, 6, e18808. [http://dx.doi.org/10.1371/journal.pone.0018808] [PMID: 21541319] Skowronek, W & Skubida, P (1988) The effect of internal conditions in the honeybee colony on queen rearing and the quality of queens. Pszczelnicze Zeszyty Naukowe, 32, 3-18. Slone, JD, Stout, TL, Huang, ZY & Schneider, SS (2012) The influence of drone physical condition on the likelihood of receiving vibration signals from worker honey bees, Apis mellifera. Insectes Soc, 59, 101-7. [http://dx.doi.org/10.1007/s00040-011-0195-5] Tarpy, DR & Nielsen, DI (2002) Sampling error, effective paternity, and estimating the genetic structure of honey bee colonies (Hymenoptera: Apidae). Ann Entomol Soc Am, 95, 513-28. [http://dx.doi.org/10.1603/0013-8746(2002)095[0513:SEEPAE]2.0.CO;2] Tofilski, A & Czekonska, K (2004) Emergency queen rearing in honeybee colonies with brood of known age. Apidologie (Celle), 35, 275-82. [http://dx.doi.org/10.1051/apido:2004014] Wang, Y, Ma, L, Zhang, W, Cui, X, Wang, H & Xu, B (2016) Comparison of the nutrient composition of royal jelly and worker jelly of honey bees (Apis mellifera). Apidologie (Celle), 47, 48-56. [http://dx.doi.org/10.1007/s13592-015-0374-x] Weaver, N (1957) Effects of larval age on dimorphic differentiation of the female honey bee. Ann Entomol Soc Am, 50, 283-94. [http://dx.doi.org/10.1093/aesa/50.3.283] Webster, TC, Peng, Y-S & Duffey, SS (1987) Conservation of nutrients in larval tissue by cannibalizing honey bees. Physiol Entomol, 12, 225-31. [http://dx.doi.org/10.1111/j.1365-3032.1987.tb00745.x] Wei, H, He, XJ, Liao, CH, Wu, XB, Jiang, WJ, Zhang, B, Zhou, LB, Zhang, LZ, Barron, AB & Zeng, ZJ (2019) A maternal effect on queen production in honeybees. Curr Biol, 29, 2208-2213.e3. [http://dx.doi.org/10.1016/j.cub.2019.05.059] [PMID: 31231048] Wen-Cheng, H & Chong-Yuan, Z (1985) The relationship between the weight of the queen honey bee at various stages and the number of ovarioles, eggs laid and sealed brood produced. Mitsubachi Kagaku, 6, 1136. Wilkinson, D & Brown, MA (2002) Rearing queen honey bees in a queenright colony. Am Bee J, 142, 270-4. Wongsiri, SIRIWAT, Lai, Y & Sylvester, HA (1990) Queen rearing with Apis cerana. Am Bee J, 130, 32-5. Woyke, J (1971) Correlations between the age at which honeybee brood was grafted, characteristics of the resultant queens, and results of insemination. J Apic Res, 10, 45-55.

288 Polyandrous Queen Honey Bee

Lovleen Marwaha

[http://dx.doi.org/10.1080/00218839.1971.11099669] Zhdanova, TS (1967) Influence of nest temperature on quality of queens produced artificially. Internatioinal Apicultural Congress, Romania245-9.

Polyandrous Queen Honey Bee, 2022, 289-299

SUBJECT INDEX A Ability 68, 70, 71, 188 biphasic gene expression 188 Absorbing nutrients 15 Acid(s) 1, 20, 21, 45, 46, 68, 69, 70, 72, 73, 96, 111, 112, 122, 129, 132, 138, 140, 141, 142, 143, 145, 146, 147, 156, 163, 165, 167, 181, 182, 198, 199, 219, 220, 253 amino 69, 72, 73, 199 aspartic 69 decanoic 122, 129, 138, 140, 141, 142, 145, 156, 163, 165, 181, 219, 220 decenoic 20, 21, 46 dodecanoic 72 folic 68 geranic 111, 112, 132, 253 glutamic 69, 72 hexadecanoic 129 linolenic 1, 20, 45, 111, 112, 163, 167, 182 nerolic 111, 112, 132, 253 nonanoic 129 octadecanoic 182 octanoic 72, 96 oleic 122, 143 pantothenic 68, 72 retinoic 198, 199 sebacic 69, 70 stearic 138, 145, 146, 147, 156, 182 Actin 185 agglomerates 185 cytoskeleton 185 Acute bee paralysis virus (ABPV) 40, 86 Adenosine 72, 73, 187 diphosphate 73 monophosphate 72 triphosphate 73, 187 Adhesive triphosphate 186 ADH’s expression 145 Africanized honey bees 131 Aggressive behaviour 129, 130

Alarm pheromones 129, 130, 131, 154 of Africanized honey bees 131 producing 129 Alcohol dehydrogenase 147, 183 Amino acid sequence 198 AMP-binding enzyme gene 201 Antibacterial properties 75 Anticancer drugs 70 Anti-hypercholesterolaemic activity 75 Antioxidant activity 74 Anti-senescence activity 71 Antiviral activity 224 Apicultural productivity 41, 249, 275, 279 Apis 182, 217 florea 217 mellifacapensis 182 Apis mellifera capensis 130, 181 honey bee races 130 Apoptosis 70, 71, 175, 184 Apoptotic cell formation 185 Artificial bee rearing technique 279

B Bee 38, 155, 249, 275 behaviour 249 comb, worker honey 275 hive 38, 155 Bee larvae 1, 71, 184, 186, 201, 202, 275 transferring worker 275 worker honey 1, 184, 186, 201, 202 Behaviour of swarming workers 252 Bifurcated 182 pathway 182 step processes 182 Biochemical 133, 146 pathway 146 synthesis pathways 133 Biogenic amine signalling 262 Biosynthesis 145, 151, 156, 157, 166, 182, 183, 202 bifurcated 157

Lovleen Marwaha All rights reserved-© 2022 Bentham Science Publishers

289

290 Polyandrous Queen Honey Bee

fatty acid 182, 183 insect hormone 202 Black queen cell virus (BQCV) 97 Body glycogen reserves 262 Brood 2, 3, 13, 14, 16, 40, 42, 47, 57, 87, 92, 113, 131, 212, 248, 249, 274, 275, 277, 279, 281 attending worker honey bees 212 capped 16, 42 comb 248, 281 food 3, 92 infected 14 nest 248 production 40, 279 recognition pheromones 131 Brood cells 8, 18, 19, 117, 132, 177 capped 8, 19, 117, 177 uncapped 8, 117 Brood rearing 8, 91, 117 activities 91 process 8, 117 Buzz, producing 258

C Capped drone cells and worker honey bees 216 Caspase activity 184, 187 Caste diversification 97 Cell-cell interaction process 202 Cell death 72, 83, 84, 138, 185, 187, 197 induction Programme 197 massive programmed 84 ovarian programmed 83, 138 queen-induced programmed 187 Chemical 22, 46, 112, 122, 123, 130, 141, 142, 144, 252 communication 252 composition 22, 46, 112, 122, 123, 130, 141, 142, 144 Chromatin 93, 176, 203 condensation 176 modification 93, 203 Chronic bee paralysis virus (CBPV) 96

Lovleen Marwaha

Cloud 24, 218, 239, 240, 261 heavy 218 Cluster 150, 168, 247, 252, 256, 260 cohesion 252 temporary 247 Cognitive processes work 72 Comb 86, 87, 92, 130, 168, 233, 236, 258, 259, 262, 275, 281 colony’s 87 Conditions 2, 7, 24, 116, 247, 278, 282 abiotic 24 artificial queen-rearing 278 ecological 247 honey-filled 7, 116 natural 278 queen-righted 2, 282 Cytochromes 138

D Dark wax comb 281 Daughter queens 255, 259 Defence 57, 224 pathogen 224 Deformed wing virus (DWV) 14, 40, 86, 96 Degeneration 11, 14, 61, 96, 111, 184, 185, 186, 202, 241 oocyte 96 ovarian 14, 184 Dehydrogenase reductase 205 Density, high mitochondrial 202 Developing rival queen 256 Development 15, 47, 60, 83, 121, 180, 181, 184, 187, 197, 198, 278 divergent ovarian 187, 197 embryonic 180 germ cell 60 honey bee 198 inhibition of worker ovarian 47, 278 ovary 121, 181 post-queen egg hatching 15 suppression of worker ovarian 83, 184 Diet 1, 2, 6, 13, 19, 25, 59, 75, 115, 146, 201, 204, 214, 224, 235, 276, 279

Subject Index

artificial 6, 115, 214, 235 coated pollen grain 13 protein-restricted 224 Differential 184, 205 display reverse transcription (DDRT) 205 ovarian development 184 Diseases, neurodegenerative 70 DNA 18, 73, 92, 176, 198, 213 binding domain 198 fragmentation 176 methylation 18, 73, 92 Drone(s) 1, 10, 23, 24, 35, 36, 37, 46, 88, 89, 96, 112, 125, 138, 142, 144, 163, 167, 211, 212, 214, 215, 217, 218, 219, 220, 223, 224, 225, 232, 238, 239, 240, 241, 242 abdomen 10 activity 218 aging 224 attraction 46, 112, 125, 138, 142, 144, 163, 220, 223, 242 comb 214, 215 congregation areas (DCA) 1, 24, 35, 219, 223, 232, 238, 239, 240, 241, 242 drifting 218, 219 flight activity 217 flying 241 haploid 211, 214 honey bee 232 parasite 96 post-mating 239 spermatozoa 88 Drone brood 214, 215 production 214 Drone cells 153, 213, 215, 216 capped 213, 215, 216 Drone colony 282 building 282 production 282 Drone eggs 6, 115, 215, 220 eating 215 Drone larvae 67, 131, 214, 225 feeding diploid 214 Drosophila 180

Polyandrous Queen Honey Bee 291

Dufour’s gland 22, 23, 47, 112, 121, 127, 128, 129, 132, 152, 153, 163, 165, 166, 169, 183 pheromone 132 queen’s 183 secretion 127, 128, 129, 152, 166

E Ecdysis 16, 19 Ecdysone 11, 74, 176, 199, 201, 204 and juvenile hormone 74, 199 hormone influence 199 Ecdysteroid concentration influence 205 Effect 70, 75, 139 anti-inflammatory 70 antioxidant 75 synergistic 139 Egg(s) 3, 11, 14, 15, 16, 24, 25, 48, 59, 60, 128, 132, 182, 212, 220, 221, 225, 232, 236, 242, 249, 275 deposit 15 diploid 59, 212, 225 formation process 15 hatching 15, 221 laying 14, 25, 182, 242 queen’s 14 Egg-laying 11, 12, 94, 253 activity 12 queen 11, 94, 253 Electron microscopy 176, 202 Electrophoretic analysis 17 Environment 19, 39, 41, 60, 83, 90, 147, 184, 186, 187, 199, 211, 214, 217, 280, 281 artificial 60 colonial 90 conditions 41, 147, 184, 211, 214, 217, 280, 281 social 39, 83, 186, 187, 199 Enzyme 71, 74, 96, 138, 145, 183 antioxidant 96 alcohol dehydrogenase 145, 183 citric acid 71

292 Polyandrous Queen Honey Bee

Epidermal growth factor receptor (EGFR) 17, 73, 76 pathway 17 European honey bee 1 Events 185, 203, 250 cytoplasmic dumping 185 ovarian 203 queen-rearing 250 Exponential fertility 83 Expression 12, 67, 111, 121, 156, 184, 197, 199, 200, 201, 202, 203, 205 genetic 12, 121, 197

F Factors 39, 57, 58, 75, 83, 84, 93, 94, 97, 127, 132, 141, 176, 180, 184, 198, 199, 217, 239, 240, 241, 249, 259, 275, 280, 282 biological 275, 280 climatic 275 environmental 93, 97, 141, 176, 184, 259 epigenetic 180 genetic 199 inhibitory 132 transcription 198 Fatty acid(s) 69, 74, 75, 127, 199, 219 binding proteins 199 polyunsaturated 75 Fecundity 40, 83 lower 40 queen’s 83 Feeding 5, 119, 150, 232, 254, 279, 282 brood 232 larvae 279 queen 254 supplemental 282 Fertile 24, 35, 36, 68, 111, 163, 180, 232, 240 honey bee queen 232 Fertile queen 36, 83, 236 poor 236 single 36 Flight activity 214, 217, 218 circadian rhythm influences drone 217 Flourishment, apicultural production 274

Lovleen Marwaha

Fluorescent in situ hybridization 200 Follicle 11, 222 growing 11 testicular 222 Food 9, 17, 41, 248, 262, 276, 278 deprived larva 278 for worker larvae 17 resources 276 sources 9, 248, 262 storage 41 Footprint pheromones 129, 131, 132, 154, 250, 254 Forager 132, 186 bee stage 186 pheromone 132 Forager worker 5, 114 bees of Apis mellifera 5 honey bee 5, 114 Foraging ontogeny 151 influence worker 151 Foraging tendency 154 Formation 10, 11, 15, 16, 72, 76, 146, 147, 153, 175, 180, 185, 241, 242, 259 influence swarm 259 ovarian phenotype 185 Fructose corn 41 Functionality 48, 92, 93, 94, 97, 111, 169 ovarian system 92 Functions 71, 74, 198 catalytic 198 immune 71 nutritional 74

G Gas chromatography 252 Gene expression 11, 18, 44, 47, 73, 93, 111, 169, 176, 199, 200, 201, 204, 205, 278 analysis 278 ecdysteroid-regulated 205 stimulate 73 volatiles change worker brain 47 Genes 14, 44, 60, 156, 157, 198, 199, 200, 201, 202, 204, 205, 262, 278

Subject Index

expressed 156, 198, 201, 278 hypoxia 202 immune 14 killer 202 vitellogenin 60 Genetic 9, 12, 19, 24, 39, 47, 83, 84, 85, 93, 147, 175, 180, 197, 199, 200, 201, 202, 203, 204, 205, 247, 248, 275 diversity 9, 24, 85, 248 elements 19, 83, 84, 175, 180, 197, 199, 200, 201, 202, 203, 204, 205 expression modulation 47 integrity 12, 39, 275 marker analysis 85 possession 247 restrictions 147 variability 197 variance 93 Genomic expression 57, 76, 83 Gentiobiose 69 Geomagnetism 241 Germ cells 15, 180, 184 ovarian 184 Gland(s) 48, 74, 132 thoracic 74 secretion 48, 132 Glandular plasticity in female castes of 111 honey bees 111 Glucoside 69, 72 luteolin 69, 72 Glutamine 69, 72 Glycan chain monomer 68 Glycoprotein 68, 74 Glycosylation pathway 68 Growth 71, 185 oocyte 185 ovarian 185 stimulants 71

H Haplodiploid sex-determination system 212 Harmonious conduct 133

Polyandrous Queen Honey Bee 293

Hatching 15, 26, 37, 74, 175, 212, 233, 263, 277, 282 Health 40, 68, 95, 224 queen’s 40, 95 reproductive 224 Heat shock proteins 205, 224 Helium-filled balloons 240 Histone deacetylases 70 Hive 7, 9, 35, 36, 38, 87, 120, 211, 212, 218, 221, 223, 234, 237, 251, 259, 260 constructed 38 damaged 87 Honey 1, 2, 4, 8, 9, 10, 12, 13, 35, 37, 40, 41, 43, 67, 86, 88, 113, 117, 118, 138, 177, 213, 223, 225, 232, 234, 236, 249, 250, 275, 279, 281, 282 and pollen grains 67, 225 processing 4, 10, 213, 232, 234 production 12, 13, 35, 37, 40, 88, 113, 138, 275, 279 ripe 177 storage 9, 118 unripe 8, 43, 86, 117 Honey bee(s) 1, 2, 3, 5, 10, 11, 12, 13, 16, 23, 35, 36, 37, 40, 41, 45, 48, 67, 68, 84, 85, 87, 88, 97, 111, 129, 144, 153, 169, 186, 247, 248, 249, 263, 274, 276, 278, 280 behaviour 276 brain 144 castes 2, 16, 35, 129 colonies 1, 2, 3, 10, 11, 12, 13, 35, 40, 87, 111, 248, 249, 274, 276 feeding 5 foragers 153 hunger signal worker 278 licking queen 169 old queen 263 post-mating queen 37 queens 2, 11, 23, 36, 41, 45, 48, 68, 84, 88, 97 suppress worker 85 swarm resistance 247 worker Apis mellifera 67Honey bee colony growth 280 influences 186

294 Polyandrous Queen Honey Bee

strength 41 Honey bee workers 41, 148, 168, 181, 182, 187, 199, 211, 249, 252, 253, 276 egg-laying 211 cannibalism 211 Hormone signalling 278 Hydrocarbons 67, 75 Hydrogen peroxide 71

I Immune 68, 86, 97 activity 68 protection measures 86 system 97 Immunity 57, 62, 111, 163, 197, 251 innate 251 Immunofluorescence 202 Indicated queen mandibular pheromones 20 Induction of apoptosis 71 Infected queen honey bees 97 Infection 13, 70, 71, 95, 96, 248, 251 apis 96 virulent bacterial 70 Infertility 96 Influence 20, 40, 83, 92, 125, 149, 169 calming 20, 125, 149 conjugative 92 inhibitory 20 mating frequency 40 queen 83 volatiles 169 Information 2, 35, 45, 57, 67, 176, 184, 213, 258 genetic 57 Informed bees fly 258 Inhibition 71, 21, 111, 127, 151, 152, 169, 183, 278 pseudo-queen formation 111 Inhibitory effects 129, 151 of queen tergal gland secretion 129 Instrumental insemination technique 282 Israeli acute bee paralysis virus 40, 86

Lovleen Marwaha

J Juvenile hormone(s) (JH) 71, 74, 92, 142, 165, 166, 167, 169, 176, 184, 185, 199, 204, 205, 262 acid methyltransferase 205 application 92 formation 142 of swarming bees 262 production 165

K Kaempferol glucosides 69, 72 Kinase, mitogen-activated protein 17, 70 Koschevnikov glands 112, 129, 130 Koschewnikow gland 130, 131, 154

L Large body measurements 84 Larva 16, 17, 41, 141, 220, 275, 278, 281 developing 278 stretched 220 Larvae 25, 166, 203, 278, 281 food-deprived 281 immature 166 malnourished 278 queen-destined 203 young hatched 25 Larval 38, 59, 89, 205, 224, 275 castes 59 developmental programming 38 environment 224 feeding stage 275 queen 205 selection influence 89 Light wax comb 281

M Magnifying glasses 280 Maillard’s reaction 74

Subject Index

Male-attraction bioassays 220 Maltose 69 Mandibles 11, 58, 59 notched 59 Mandibular 145, 156, 182 pheromones synthesis 156, 182 queen gland 145 Mandibular gland 22, 88, 122, 123, 124, 129, 141, 142, 143, 144, 145, 147, 156, 163, 164, 165, 166, 251 blend 166 bouquet 123 components 88, 144, 145, 163, 164 composition 122, 141, 142 compounds 22, 124 pheromonal composition 142 secretion 122, 124, 129, 141, 143, 144, 156, 165, 251 synthesis 147 Mass spectroscopy 252 Mated queens 22, 58, 113, 123, 125, 129, 140, 141, 142, 143, 144, 147, 153, 166, 219, 255 Mating 12, 24, 57, 84, 94, 122, 144, 168, 232, 281, 282 and reproduction in queen honey bee 232 colony 281 frequency 12, 24, 57, 84, 94 natural 168, 282 queen 122, 144 tendency 232 Maturation 21, 48, 132 ovarian 48 Meiotic gametogenesis 213 Metamorphosis 180, 197, 205 Methods 93, 275, 280 artificial queen production 275 queen grafting 280 queen introduction 93 Microsatellite analysis 25 Migration, germ cell 180 Mitogen-activated protein kinase (MAPK) 17, 70, 197, 203, 205 Mitotic 15, 186 activity 186

Polyandrous Queen Honey Bee 295

division, rapid 15 Modern queen rearing technique 275 Modulate oogenesis 184 Monopoly, reproductive 111 mTOR pathways 70, 203 Myosin-based contraction 185

N Nasanov 168, 253 glands 168, 253 pheromones 253 Natural 38, 278 food preference 38 queen rearing methods 278 Nectar 3, 35, 37, 93, 113, 217, 132 fermented 132 workers transport 37 Nest 57, 85, 122, 138, 139, 142, 165, 256, 259 bees 256, 259 mates 57, 85, 122, 138, 139, 142, 165, 256 Neurotransmitters 72 Nosema 14, 224 apis, fungal pathogen 224 infection 14 Notch 202, 203 signalling 203 signalling pathway 202 Nutrition 62, 76, 93, 119, 186, 278 differential larval 62

O Oestrogen receptors 70 Offspring, queen’s 85 Oocytes 11, 15, 185, 187, 200 degenerating 200 protecting maturing 187 Oogenesis 60, 184, 185, 186, 200, 203 inhibiting 186 Ovarian 46, 48, 60, 123, 144, 147, 148, 153, 181, 182, 185, 201, 202 activation 46, 48, 123, 144, 147, 148, 153, 181, 182, 201, 202

296 Polyandrous Queen Honey Bee

phenotype development 185 structural integrity 60 Ovarian developmental 4, 25, 83, 197 plasticity 197 inhibition 83 suppression 4, 25 Ovaries 11, 15, 46, 47, 58, 60, 84, 86, 93, 94, 97, 175, 176, 179, 180, 181, 184, 187, 197, 200, 201 degenerative 175 developed 181 honey bee 201 queen’s 15 worker honey-bees 86 Oxidation 146, 183 Oxidative damage 75 Oxidoreductase 198 Oxygen transport 198

P Path 12, 163, 203, 258 queen developmental 163 queen’s development 12 Pathways 70, 147, 154, 260, 279 biosynthetic 147, 154 immune 279 Pheromonal 35, 90, 121, 127, 139, 147, 151, 163 biosynthetic pathways 147 components of queen honey bee 139 secretion 35, 90, 121, 127, 151, 163 Pheromone(s) 3, 4, 57, 114, 131, 145, 146, 154 thermoregulatory 131, 154 secretion 3, 4, 57, 114, 145 synthesis 146 Photographic analysis 261 Physiological 12, 84 activity 84 influence 12 reproductive division 84 Plasticity 19, 89, 111, 146, 185, 197 organ 146

Lovleen Marwaha

phenotypic 146 Pollen grains 1, 5, 9, 19, 67, 75, 114, 118, 215, 223, 225 Pollen processing 4 Polyandrous 35, 36, 211 honey bee queen 211 queen 35, 36 Process 68, 75, 147, 175, 198, 278 ageing 68 biosynthetic 147 degenerative 175 metabolic 198 moulting 75 queen-rearing 278 Production, progeny 275 Programmed cell death (PCD) 11, 84, 175, 176, 179, 180, 184, 185, 186, 187, 197, 199, 200, 201, 202 Properties 68, 71, 75 anti-inflammatory 75 antimicrobial 71 anti-oxidative 68 antitumor 75 nematicidal 71 Protein 68, 74 glycosylation 68 secretion 74

Q Quality queen 14, 141, 177, 283 Quantity, stomach 259 Queen 21, 22, 25, 48, 62, 95, 122, 126, 145, 147, 148, 163, 164, 165, 166, 167, 183, 187, 215, 241, 249, 263, 277, 280 apis cerana 165 artificial mated 166 clipping 249 colonies 147, 148 commercial produced 95 developing 122, 263 development pathway 122 diseased 277 egg-laying mated 22

Subject Index

honey 62, 126 influences 122, 183, 215, 280 mating status correlation 21 post-mating 25, 95, 167 pseudo 145, 187 sex pheromones 241 signal 48 stationary 163, 164 Queen bee 11, 20, 57, 61, 62, 93, 202 larvae 202 Queen cell(s) 41, 250, 263, 275, 279 emergency 275 production 250, 279 seal wax 41 vibrating 263 Queen development 14, 15, 60, 70, 73, 74, 201 inducing 74 stimulate 73 Queen elimination 44, 255, 256, 259 procedure 44 process 255 Queen honey bee 47, 48, 275 production 275 queen 47, 48 Queen larvae 17, 59, 67, 73, 76, 97, 199, 202, 205, 274, 276, 282 developing 276 infect 97 Queenless colonies 6, 115, 125, 132, 143, 149, 185, 215, 217, 250 Queen mandibular 20, 46, 122, 125, 127, 128, 138, 140, 141, 142, 145, 147, 149, 150, 151, 153, 165, 166, 167, 181, 182, 251 gland pheromones 46, 182 pheromones (QMPs) 20, 122, 125, 127, 128, 138, 140, 141, 142, 145, 147, 149, 150, 151, 153, 165, 166, 167, 181, 251 Queen pheromones 182, 184, 187, 203, 247, 250 influence 182, 187 pseudo 184 reduced 247, 250 suppress 203 Queen production 39, 41, 276, 279

Polyandrous Queen Honey Bee 297

artificial 279 Queen rearing 35, 41, 44, 89, 113, 144, 212, 248, 250, 255, 275, 276, 277, 278, 279, 280, 282, 283 artificial 275, 280, 283 environment 89 inhibition 35 method 275, 276, 279 process 277, 278, 282 Queen replacement 13, 274 process 13 strategy 274 Queen retinue 23, 127, 152, 164, 254 pheromones (QRP) 23, 164, 254 response 127, 152

R Rearing 275, 278, 279 conditions, artificial queen 278 methods, commercial queen 279 queen bees 275 Reduction 12, 23, 71, 74, 143, 151, 183, 218, 240 oxidative enzymatic 183 Regulation 1, 4, 21, 35, 48, 83, 89, 92, 112, 125, 126, 138, 169, 203 colonial 89, 125 developmental 4 epigenetics 92 Reproducible workers 146 Reproduction, sexual 211 Reproductive 57, 59, 60, 241 development 59, 60 organ development 57 queen 241 Reproductive system 36, 111 functional 36 hypertrophied 111 Retinue 46, 112, 123, 126, 144, 150, 165, 224 behaviour of honey bees 126 response 46, 112, 123, 126, 144, 150, 165, 224 Retinue behaviour induction 35, 47, 89, 127

298 Polyandrous Queen Honey Bee

tendency 89 RNA 156, 200 interference (RNAi) 200 sequence technology 156 Royal jelly 11, 71, 76, 203 influences 11 product 76 protein 203 stimulate growth 71 RT-qPCR 205

S Sacbrood virus (SBV) 97 SDR gene 201 Secretion 76, 133, 274 hormonal 133 protein-rich 76, 274 Semen 44, 95, 96, 122, 127, 141, 142, 152, 166, 223, 224 higher-quality 166 Seminal 222, 224 fluid proteins 224 vesicle (SV) 222 Signalling pathway, mediated 76 Signals 23, 125, 128, 129, 149, 167, 184, 247, 251, 253, 256, 257, 258, 261 behavioural 247 drone insemination 125, 129, 149 non-chemical 258 producing piping 256 vibrational 257 visual 253 Social 121, 124, 130, 138, 186, 274 behaviour 121, 138 communications 124, 130, 274 insect colonies 186 Solid-phase microextraction 167, 252 Sperm 25, 89, 225 drone’s 25 production 89, 225 Sperm storage 13, 24, 40, 57, 84, 94, 232 spermatheca influences 40 Spermatogenesis 222

Lovleen Marwaha

Spermatozoa 94, 95, 213 Steroid hormones 199 Stimulate 153, 257 retinal behaviour 153 vibrating signals 257 Subcortical actin network 185 Sugary syrup, honey bees suck 37 Suppression 1, 11, 23, 35, 124, 125, 127, 138, 149, 152, 185 genetic 11 ovarian 1, 35, 124 pseudo-queen formation 1 Suppress worker, insect ecdysis 112 Swarm clusters 126, 151, 253 Swarming 35, 44, 112, 124, 154, 163, 167, 169, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 262, 275 bees 251, 262 behaviour 167 colony 262 honey bee 262 of honey 250 queen pheromones influence 112 reduction 35, 275 regulation 124 reproductive 154, 251 resistance 169 volatiles influence 163 worker bees 259 Swarming tendency 1, 249, 275, 279 reducing 275 Synthesis, enzyme alcohol dehydrogenase 147

T Threonine 69, 72 Tissue, ovarian 179 TOR pathway 176, 203 Transcriptional 84, 93, 202 activity 84 analysis 202 program 93 Transcriptome analyses 201

Subject Index

Transfer, cytoplasmic 185 Transition phenotypes 187 Tumour necrosis factor (TNF) 71 Tyrosine 69, 72

V Variation 37, 39, 84, 88, 89, 111, 122, 131, 142, 212, 214, 249 genetic 249 glandular secretion 111 Vitamin pyridoxine 279 Vitellogenin RNA 168

W Wax 15, 37, 87, 112, 128, 138, 153, 250, 279 artificial prepared 279 capping 15 moths 87 production 37, 112, 128, 153 secretion 128, 138, 153 Wax cell(s) 3, 8, 15, 38, 43, 86, 117, 179, 221, 223, 236, 274, 275 capped brood 86 construction ability 223 honey-filled 43 peanut-shaped 274 Western 1, 71 blotting 71 honey bee 1 Worker(s) 1, 3, 10, 16, 23, 38, 47, 59, 126, 130, 144, 145, 147, 150, 164, 165, 166, 168, 175, 180, 183, 185, 186, 187, 200, 201, 202, 203, 205, 256, 262 capensis parasitic 147 cells, hexagonal wax 38 grafting larvae 59 Koschewnikow gland (WKG) 130 mandibular gland 145 ovarian development 47 ovaries 23, 47, 165, 180, 185, 186, 187, 200, 201, 203, 205 Worker jelly 17, 18, 42, 59, 213, 235, 238

Polyandrous Queen Honey Bee 299

adding 18, 42, 213, 235, 238 compositions 17 influences 59 Worker larvae 17, 39, 40, 42, 59, 73, 75, 84, 86, 89, 144, 186, 187, 198, 200, 201, 202, 213, 238, 278 developing 42, 86, 213 transplanting 202 younger 84, 89, 144

Y Younger larvae 123, 275 comb 275 elicit 123