Nanoparticles and the Immune System: Volume 2 Immune System of Animals 9783110655872, 9783110654080

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Shyamasree Ghosh Nanoparticles and the Immune System

Also of Interest Nanoparticles and the Immune System. Volume : Human Immune System Shyamasree Ghosh,  ISBN ----, e-ISBN ----

Nanomaterials Safety. Toxicity And Health Hazards Shyamasree Ghosh,  ISBN ----, e-ISBN ----

Nanotoxicology and Nanosafety. Fundamentals on Nanomaterials Toxicological Assessment Eliana Maria Barbosa Souto and Maria C. Teixeira (Eds.), planned  ISBN ----, e-ISBN ---- Nanoscience and Nanotechnology. Advances and Developments in Nano-sized Materials Marcel Van de Voorde (Ed.),  ISBN ----, e-ISBN ----

Nanoanalytics. Nanoobjects and Nanotechnologies in Analytical Chemistry Sergei Shtykov (Ed.),  ISBN ----, e-ISBN ----

Shyamasree Ghosh

Nanoparticles and the Immune System Volume 2: Immune System of Animals

Author Dr. Shyamasree Ghosh School of Biological Sciences National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute Odisha 752050 India [email protected]

ISBN 978-3-11-065408-0 e-ISBN (PDF) 978-3-11-065587-2 e-ISBN (EPUB) 978-3-11-065417-2 Library of Congress Control Number: 2022933744 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the internet at http://dnb.dnb.de. © 2022 Walter de Gruyter GmbH, Berlin/Boston Cover image: unoL/iStock/Getty Images Plus Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

Contents 1 1.1 1.2 1.3 1.4 1.5

Nanoparticles and porifera 1 Shyamasree Ghosh Introduction 1 Immune molecules and Porifera 3 Nanoparticles, Porifera, and applications Nanotoxicity and sponges 7 Discussion 8 References 8

6

2

Cnidaria immune system and nanoparticles 13 Garima Hore, Shyamasree Ghosh, Dhriti Banerjee 2.1 Introduction: general biology 14 2.2 Cnidaria classes in brief 15 2.2.1 Class Anthozoa 15 2.2.2 Class Hydrozoa 16 2.2.3 Class Cubozoa 17 2.2.4 Class Scyphozoa 17 2.2.5 Class Staurozoa 18 2.3 Origin of phylum Cnidaria 19 2.4 Early diversification 19 2.5 Zoogeographical distribution and ecological significance 2.5.1 Class Anthozoa 20 2.5.1.1 Subclass Hexacorallia 20 2.5.1.2 Subclass Octocorallia 21 2.5.2 Class Hydrozoa 22 2.5.3 Class Cubozoa 23 2.5.4 Class Scyphozoa 24 2.5.5 Class Staurozoa 24 2.6 Regenerative capacity of cnidarians 24 2.7 Cnidarian immunity: an overview 25 2.7.1 Recognition of pathogen by immune system 26 2.7.2 Toll-like receptors (TLRs) 26 2.7.3 Lectins 27 2.7.4 Nucleotide oligomerization domain (NOD)-like receptor (NLRs) 27 2.7.5 Intracellular signaling cascade 27 2.7.6 TLR pathways and TIR domains 28

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Contents

2.7.7

2.7.8 2.7.9 2.7.10 2.7.10.1 2.7.10.2 2.7.11 2.7.12 2.7.13 2.8 2.9 2.10 2.11 2.12 2.13 2.13.1 2.13.2 2.13.3 2.13.4 2.14

3 3.1 3.2 3.3 3.4 3.5 3.5.1 3.5.2 3.5.3 3.5.4

GTPase immune-related protein mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) pathway 28 Prophenoloxidase (PPO) signaling pathways 28 Complement pathway 29 Effector responses 29 Surface mucus layer 29 Antimicrobial activity 30 Cellular responses in cnidarians 30 Mobile cytotoxic cells: ROS and reactive nitrogen species (RNS) 30 Encapsulation 31 Repair mechanisms 31 Antioxidants 31 Apoptosis 31 Wound healing 32 Overlap between immunity and symbiosis: anthozoan and symbiotic dinoflagellates 32 Nanoparticles and Cnidaria 33 Cnidarians in nanowaste decontamination 33 Nanoparticle-mediated environmental toxicity and Cnidarians 33 Nanoparticles in augmenting our understanding of the anatomy and physiology of cnidarians 34 Anti-inflammatory potential and applications 34 Discussion 35 References 35

Caenorhabditis elegans and nanoparticles Shyamasree Ghosh Introduction 50 Phylum Nematoda: a brief study 51 C. elegans 52 C. elegans as animal model 52 Immune system in C. elegans 53 Antiviral immune defense 53 Antimicrobial immune responses 54 Immunity and stress pathways 56 Immunity and neuronal control 56

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3.6 3.7

4 4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.2 4.3 4.3.1 4.3.1.1 4.3.1.2 4.3.2 4.3.3 4.3.4 4.3.5 4.3.5.1 4.3.5.2 4.3.5.3 4.3.5.4 4.3.6 4.3.6.1 4.3.6.2 4.3.6.3 4.3.6.4 4.3.6.5 4.3.7 4.3.8 4.4 4.5

5 5.1 5.2

Nanoparticles and the immune system in C. elegans Discussions 57 References 58 Insects and nanoparticles 61 Rashmi Bhattacherje, Shyamasree Ghosh, Dhriti Banerjee Introduction 62 Apterygotan assemblage 63 Palaeopteran assemblage 63 Paraneopteran assemblage 63 Polyneopteran assemblage 65 Endopterygotan assemblage 66 Contribution of insects to ecosystem functioning 66 Insect immunity 67 Pathogen recognition 67 TOLL and IMD signaling in Drosophila 68 Other pathways 68 Immune responses 71 Humoral response 71 Antimicrobial peptides (AMP) 72 Receptors 73 Peptidoglycan recognition proteins (PGRPs) 73 β‐1,3‐Glucan recognition proteins (βGRPs) 74 Hemolins 74 C‐type lectins (CTLs) 74 Cellular immune response 74 Hemocytes 74 Nodule formation 75 Encapsulation 75 Phagocytosis 75 Melanization 76 Proteasome 76 Apoptosis 76 Nanoparticles and insect immune system 76 Discussion 81 References 81 Nanoparticles and the immune system in Mollusca Shyamasree Ghosh Introduction 91 Phylum Mollusca: an overview 91

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VII

VIII

5.3 5.4 5.5 5.6

6 6.1 6.2 6.3 6.4

7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 7.10 7.11 7.12 7.13 7.14 7.15 7.16 7.17 7.18 7.19 7.20

Contents

Phylum Mollusca: a brief study 93 Immune molecules in Mollusca 94 Nanoparticles and the immune system in Mollusca Discussion 101 References 102 Echinodermata and the immune system 105 Shyamasree Ghosh Introduction 105 Echinodermata: immune system 106 Echinodermata immune system and nanoparticles Discussion 110 References 110 Fish and nanoparticles 113 Rashmi Bhattacherjee, Dhriti Banerjee, Shyamasree Ghosh Introduction 114 Core immune components in fish 115 Thymus 115 Kidney 116 Spleen 116 Cells involved in immune response 116 Nonspecific immunity 117 Physical barriers 117 Nonspecific cellular cytotoxicity 117 Antimicrobial peptides 117 Phagocytosis 118 Tumor necrosis factor 118 Interferon 118 Interleukins 118 Protease inhibitors 119 Lysozyme 119 Natural antibodies 119 Pentraxins 119 Transferrin 120 Specific immune response 120

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7.21 7.22

8 8.1 8.2 8.3

Index

Benefits and hazards of nanoparticles in fish Discussion 123 References 124 Nanoparticles and bird’s immune system Shyamasree Ghosh Introduction 132 Nanoparticles and birds 132 Discussion 136 References 137 141

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IX

1 Nanoparticles and porifera Shyamasree Ghosh Abstract: Sponges are primitive organisms without organs but with cells to perform vital and different functions. Inhabiting the marine habitat, they are often exposed to harmful stress factors of the environment. In this chapter, we highlight the immune molecules in sponges and reveal the impact of exposure of sponges to nanoparticles and the negative impact of nanoparticles on sponge health, physiology, and immune responses. Keywords: Sponge, porifera, nanoparticles, oxidative stress

Abbreviations Suberites domuncula Geodia cydonium Cysteine Toll-like receptors Pattern recognition receptors Lipopolysaccharide 2′,5′-Oligoadenylate synthetase Mesencephalic astrocyte-derived neurotrophic factor Silver nanoparticle

S. domuncula G. cydonium C TLRs PRRs LPS OAS MANF AgNP

1.1 Introduction Sponge or phylum Porifera (Fig. 1.1) includes the most primitive group of multicellular animals, lacking organs, but has a well-developed connective tissue and cells with different functions. Sessile forms are with well-developed water canal systems. With a few freshwater species, most of the 5,000 described species reveal marine existence. Rocks, shells, coral, soft sand, and mud act as their substratum. They may live in shallow waters or in deep waters. Sponges are of different sizes ranging from the size of a grain to more than a meter in height ad diameter. They exhibit radial symmetry. Most species are brightly colored like green, orange, red, and purple. The sponge architecture reveals a water canal system, the simplest being in the asconoid sponges, for example, Leucosolenia. Here the body is perforated by several

Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India https://doi.org/10.1515/9783110655872-001

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pores called ostia, which are the incurrent pores that open into the interior cavity called spongocoel, which then opens out through a large opening called as osculum. The body wall is simple with the outer face covered by pinacocytes and together comprises the pinacoderm which secretes the material that enables the sponge to attach to the substratum. The skeleton is rather complex comprising calcareous or siliceous spicules, protein spongin fibers, or combination of all of the three components. The skeleton is located in the mesohyl with spicules projecting through the pinacoderm which may remain in interlocked or fused form. Mesohyl contains collagen fibrils, and some sponges contain skeleton comprising interconnected fibers. Sponges bearing excessive spongin make them tough and rubbery and with the siliceous spicules embedded partially in the fibers that stiffen them.

Fig. 1.1: Some common sponges from India. Picture courtesy Director, Zoological Survey of India, Kolkata 700053, India.

Ameboid cells in the mesohyl include archeocytes with large sized nucleus, which are phagocytic with roles in digestion. They bear the property of totepotency that gives rise to new and different cells. The other types of cells include the collencytes which are fixed by long cytoplasmic strands and play a role in secretion of collagen fibers. Sclerocytes secrete single spicules in calcareous sponges while spongocytes secrete the spongin skeleton. In the interior side of the mesohyl, the spongocoel is lined by the ovoid-shaped choanocytes, bearing similarity to protozoan choanoflagellate. On the one side, the choanocyte projects into the spongocoel, bearing a flagellum surrounded by microvilli. Sponges are devoid of gut. The Syconoid sponges exhibit radial symmetry and folded body, for example, genus Grantia and Sycon. Choanocytes here line the invaginations called the radial

1.2 Immune molecules and Porifera

3

canals. The invaginations extend outward from the spongocoel. In more complex forms, the choanocytes line the radial canals. The invaginations from the pinacoderm end are lined by pinacocytes and termed as incurrent canals and the two canals are interconnected by prosopyles. Water enters through the incurrent canals, passes through the prosopyles, flagellated canals, spongocoel, and moves out of the osculum. In further complex forms, pinacocytes and mesohyl block the open ends of the incurrent canals, and only water enters through ostia into incurrent canals. Leuconoid sponges reveal the most complex form, where the flagellated canals form small flagellated chambers. Water flows in through ostia, passes into spaces flowing into branched incurrent canals that open into flagellated chambers through prosopyles, and water leaves through the apopyle and flows out through excurrent canals, and all canals open out through osculum. Pinacocytes line the inner side of all canals. Due to the efficient water canal system, they are large sized with greater mass. They reveal diversity in body forms of erect, flattened, vase shaped, or tubular forms, that is, body with many ostia and one osculum [1–4]. The World Porifera Database has been designed with updated global biodiversity of Porifera with members of known classes [2]. Over more than 3,500 compounds, identified till 1998, isolated from Porifera have been applied to sponge systematics, and classification and identification of 475 species of marine sponges. However, this mode of classification faces the challenges of lack of identification of source of chemicals from the endosymbionts of poriferan host [4], and thus reliability of classification based on these chemicals is difficult.

1.2 Immune molecules and Porifera Porifera or sponges are a primitive metazoan phylum with common ancestor with other metazoan phyla. Most of our knowledge on the immune molecules of sponge is based on studies from marine demosponges including Geodia cydonium (G. cydonium) and Suberites domuncula (S. domuncula). Sponges have immune molecules bearing similarity in structure to mammalian innate immune system molecules including molecules with scavenger receptor cysteine (C)-rich domains, cytokine-like molecules released by macrophages, 2′,5′ oligoadenylate synthetase (OAS) as revealed from grafting experiments in marine demosponges including G. cydonium and S. domuncula. Molecules with properties of the adaptive immune system have been recorded in sponges, including cytokines from lymphocytes that enable self- versus non-self-recognition in S. domuncula and in G. cydonium, and receptors with Ig-like domains including receptor tyrosine kinases and sponge adhesion molecules with polymorphic Ig-like domains that reveal to be overexpressed during grafting, thereby playing a role in adaptive immune recognition [5]. Molecules recognizing cell–cell and cell–matrix have been reported in the immune system of sponge. They reveal the evolution of antimicrobial and antiparasitic

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defense mechanisms by engulfment and killing of bacteria by lipopolysaccharide (LPS)-mediated pathway, role played by kinase, and presence of interferons. Cytokines like allograft inflammatory factor 1 play a role in controlling allograft rejection, and transcription factors with Tcf-like factor have been reported from Porifera indicative of adaptive immune system like features that came into light during graft rejection experiments [6]. Toll-like receptors (TLRs) with a role in microbial recognition bind to microbial parts, thereby activating the signaling cascades leading to synthesis of antimicrobial molecules. TLR, IL-1 receptor-associated kinase-4-like protein, has been reported to be constitutively expressed, and effector caspases inducibly expressed in S. domuncula bear homology to higher metazoan molecules [7]. Marine sponges bear a close relationship to the commensal bacteria they harbor. LPS of commensal bacteria Endozoicomonas sp. reveal differences with that of LPS of opportunistic Pseudoalteromonas sp. sponge Suberites. Although there are observed differences in the structure of LPS in sponges, it is not clear how sponges can differentiate the different types of bacteria, namely, the ones they harbor and the other invading opportunistic form and respond accordingly [8]. Pattern recognition receptors (PRRs) have been known to identify the microbial structures and on binding activate the downstream signaling cascades inducing innate immunity observed by experimental studies with LPS in Halichondria panicea inducing immune response, altered gene regulation of signaling and recognitionrelated genes, GTPases, and processes including phosphorylation and ubiquitination which are posttranslational modification, revealing different expressions including constitutive expression, individual expression, and inducible expressions [9]. S. domuncula sponges reveal the expression of few members of antiviral OAS and 2′-phosphodiesterase, aiding in recognition of self versus non-self, and reveal phagocytosis against pathogens [10]. SDMANF, a homolog of mesencephalic astrocyte-derived neurotrophic factor (MANF) member of the neurotrophic factors, has been reported from S. domuncula, although they lack a proper nervous system. Its protein binding domain has been reported to bear similarities to Bcl-2 family of apoptotic regulators, and the protein is upregulated in response to bacterial LPS, decreased apoptotic activity by reduced caspase-3 activity, increase in cell viability, and no expression of Bax indicating the role of MANF in conferring cytoprotection in sponges [11]. Studies from Amphimedon queenslandica have revealed that PRRs including nucleotide-binding domain and leucine-rich repeat are known to play a role in antibacterial defenses and in interactions with symbionts [12]. Scalarane-type sesterterpenoids from marine sponge Lendenfeldia sp. reveal that anti-inflammatory properties are also reported of therapeutic effects in cancer [13]. The marine sponges Hyrtios and Haliclona species, producing compounds 1304KO-327 and 1304KO-328, revealed antibacterial, antiviral, anti-inflammatory, antitumoral, antifungal, and antiplasmodial properties [14]. Marine sponges G. cydonium and S. domuncula revealed molecules

1.2 Immune molecules and Porifera

5

with cysteine-rich domains, cytokine-like molecules released by macrophages, and OAS system [15]. Porifera has been believed to be branched from common progenitor of metazoans namely Urmetazoa with the evolution of cell-to-cell and cell-to-matrix adhesion molecules leading to colonial life followed by establishment of an immune system, together with apoptosis, chemokines, endothelial-monocyte-activating polypeptide glutathione peroxidase, pre-B-cell colony-enhancing factor, and myotrophin [2–5], a synthetase as revealed from studies in S. domuncula and G. cydonium [16]. Invariant natural killer T are T cells with properties of innate NK cells and adaptive memory T cells. Such cells have been reported in sponges that play a role in antibacterial defenses [17]. Diverse cell surface receptors and adhesion molecules including collagen, fibronectin, galectin, integrin, receptor tyrosine kinase, scavenger receptor cysteine rich (SRCR), and short consensus repeats have been reported to play a role in processes of allogeneic graft rejection in transplantation experiment-based studies and have also been reported to involve in an upregulation of phenylalanine hydroxylase, leading to melanin synthesis [18]. The galectins with high affinity to N-acetyllactosamine disaccharides have been identified from marine ball sponge (Cinachyrella sp.) (Fig. 1.2) with property to modulate function of mammalian ionotropic glutamate receptor [19]. A vinculin ortholog (Fig. 1.2) has been reported from Oscarella pearsei (O. pearsei) sponge with cell adhesion like functions [20]. Disulfide-containing peptides, barrettides, have been isolated from marine sponge Geodia barretti (G. barretti) (Fig. 1.2) that had biological properties of inhibiting the growth of gram-negative Escherichia coli and gram-positive Staphylococcus aureus [21]. A

B

C

Fig. 1.2: 4AGR: tetrameric galectin from Cinachyrella sp. [19], 6BFI vinculin homolog in sponge [20], 6CFB, structure of barrettides, a disulfide-containing peptide from G. barretti with an antifungal property (21N3) from PDB [22].

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1.3 Nanoparticles, Porifera, and applications Bioactive compound, avarol or avarone, has been reported to be produced by the sponge Dysidea avara [23]. Molecules derived from sponges have been used to prevent type I diabetes [24]. Silver nanoparticles (AgNP) synthesized from marine sponge (Amphimedon sp.) were used to synthesize hepatitis C virus (HCV) NS3 helicase and protease activities and revealed their anti-HCV mode of action by in silico studies [25]. Chondrosia reniformis sponge collagen nanoparticles have been reported to function in enhancing penetration for transdermal drug 17beta-estradiol-hemihydrate and have applications in hormone replacement therapy [26]. Three-dimensional chitinous scaffolds obtained from marine sponges covered with AgNPs and Ag-bromide with antibacterial properties are being applied as water filtration system [27]. Biosilica, SiO2, synthesized by siliceous marine demosponges is catalyzed by the enzyme silicatein and involves other enzymes including silicatein and silintaphin-1 in biomineralization process and finds application in nanobiotechnology and biomedicine in tooth and bone regeneration [28]. Biosilica finds biomedical application in 3D bioprinting of implants for biomedical use [29]. Micro- or nanodispersion chitin isolated from marine demosponge Lanthella basta (L. basta) silicified by Stöber silica forming micro- and nanoaggregates have been designed [30]. Biosilica in siliceous sponge spicules, like Petrosia ficiformis, contains silicatein beta [31]. Biosynthesis of silica nanocrystals could be done by using recombinant silicatein from marine sponges Latrunculia oparinae [32]. OAS [2–5] (a synthetase) has been cloned and isolated from freshwater sponge Lubomirskia baicalensis [33]. Silicateinα has been reported to cause biomineralization of silicates in sponges [34]. Gelatin-Bletilla striata gum/Salvia miltiorrhiza nano-Ag (GBS-Ag) from sponges has found application in dressing and healing of refractory orthopedic wound [35]. Chitinous scaffolds from marine demosponge L. basta is known to contain copper and copper oxide mineral deposits [36]. The formation of silica nanostructures involves biomineralization through complex molecular pathways [37, 38]. The process involved during fabrication of siliceous spicule has been studied using fluorescent and magnetic nanoparticles [39]. Atomic force microscopy and scanning electron microscopy were used to study sponges and revealed the uniqueness of formation of nanoparticulate deposits in annular substructure of demosponge biosilica spicules [40]. Silica, calcium phosphate, or carbonate from glass sponges or Hexactinellida, Hyalonema sieboldi, reveal properties of durability, flexibility, and optics [41]. Demospongiae and Hexactinellida reveal siliceous spicules produced by biosintering process in Monorhaphis chuni [42]. In sponges, biosilicification occurs in the presence of silicateins, when silintaphin-1 enables filament formation and enables gamma-Fe2O3 nanoparticles assembly to form spicules and forms the base for biosilica material formation that finds application in

1.4 Nanotoxicity and sponges

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clinical and biomedical applications [43]. Siliceous skeletal system in Euplectella aspergillum reveals structural complexities [44]. The bacteria observed and isolated from Hymeniacidon heliophila sponge cells revealed its role in bioleaching, wherein the bacteria has been reported to secrete substances that could leach copper metallic nanoparticles finding application in copper leaching from electronic waste [45]. Marine sponges Haliclona exigua are used to synthesize silver nanoparticles revealing antibacterial and antiproliferative activities [46]. Marine sponge Acanthella elongata has been used to synthesize gold nanoparticles [47].

1.4 Nanotoxicity and sponges Due to the immense application of nanoparticles in human lives, their synthesis and usage find their exposure to the environment and pose a potential threat to all forms of life. They enter the human and animal bodies by routes of inhalation, oral intake, entry, and passage through subcutaneous, transdermal, and intravenous routes. Their physicochemical properties of size, charge, morphology, engineering through coating and surface modification, and diversity in chemical composition affect the biological systems and can increase their toxicity [48] (Fig. 1.3). Altered Physiology Stress Lysosomal membrane stability

Nanoparticles

Phagocytic activity Lysozymes

Marine environment

Sponge

Acid phosphatases

Impact on Health, Physiology and immune molecules

Free radicals Superoxides ROS NO SOD Catalase GST

Fig. 1.3: Impact of nanotoxicity of sponges.

Impact on population ?

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1 Nanoparticles and porifera

When the freshwater sponge Eunapius carteri was exposed to copper oxide nanoparticles and copper sulfate from industrial toxic effluents by feeding/activity by flagella on contaminated waters, it has been reported to affect sponge physiology, revealing symptoms of stress and a compromised immune system, causing inhibited phagocytosis, affecting adversely lysosomal membrane stability, generating oxidative stress as understood by the release of nitric oxide, ROS, and superoxide anion, and altering the enzymatic activity by catalase, glutathione-S-transferase, lysozyme, phosphatases, phenoloxidase, and superoxide dismutase [49].

1.5 Discussion Nanoparticles are an environmental hazard due to their toxicity based on their physicochemical properties [48]. Although toxicity of nanoparticles and their impact on the marine invertebrates have been less studied in this chapter, we have highlighted the impact of toxicity of nanoparticles even on the primitive sponges revealing different alterations in physiology and causing features of stress, oxidative stress, altered phagocytosis and enzymatic profiles, and generation of free radicals [49]. Microplastics released in seawater is proven to be a health hazard for the marine sponges [50]. This domain of biology and its findings are alarming due to its immense impact of wildlife, diversity, and conservation and ecological balance. Thus, more research in this domain needs to be focused on understanding the potential threats to the life and health of sponges and their impact on sponge physiology and immune molecules.

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Wiens M, Korzhev M, Perovic-Ottstadt S, Luthringer B, Brandt D, Klein S, Müller WE. Toll-like receptors are part of the innate immune defense system of sponges (demospongiae: Porifera). Mol Biol Evol. 2007 Mar 24(3):792–804. Gardères J, Bedoux G, Koutsouveli V, Crequer S, Desriac F, Pennec GL. Lipopolysaccharides from Commensal and Opportunistic Bacteria: Characterization and Response of the Immune System of the Host Sponge Suberites domuncula. Mar Drugs. 2015 Aug 7 13(8):4985–5006. Schmittmann L, Franzenburg S, Pita L. Individuality in the Immune Repertoire and Induced Response of the Sponge Halichondria panicea. Front Immunol. 2021 Jun 16 12:689051. Saby E, Poulsen JB, Justesen J, Kelve M, Uriz MJ. 2ʹ-Phosphodiesterase and 2ʹ,5ʹoligoadenylate synthetase activities in the lowest metazoans, sponge [porifera]. Biochimie. 2009 Nov-Dec 91(11–12):1531–34. Sereno D, Müller WEG, Bausen M, Elkhooly TA, Markl JS, Wiens M. An evolutionary perspective on the role of mesencephalic astrocyte-derived neurotrophic factor (MANF): At the crossroads of poriferan innate immune and apoptotic pathways. Biochem Biophys Rep. 2017 Mar 18(11):161–73. Yuen B, Bayes JM, Degnan SM. The characterization of sponge NLRs provides insight into the origin and evolution of this innate immune gene family in animals. Mol Biol Evol. 2014 Jan 31 (1):106–20. Peng BR, Lai KH, Chang YC, Chen YY, Su JH, Huang YM, Chen PJ, Yu SS, Duh CY, Sung PJ. Sponge-derived 24-homoscalaranes as potent anti-inflammatory agents. Mar Drugs. 2020 Aug 19 18(9):434. Koh SI, Shin HS. The anti-rotaviral and anti-inflammatory effects of Hyrtios and Haliclona species. J Microbiol Biotechnol. 2016 Nov 28 26(11):2006–11. Müller WE, Blumbach B, Müller IM. Evolution of the innate and adaptive immune systems: Relationships between potential immune molecules in the lowest metazoan phylum (Porifera) and those in vertebrates. Transplantation. 1999 Nov 15 68(9):1215–27. Müller WE, Wiens M, Müller IM, Schröder HC. The chemokine networks in sponges: Potential roles in morphogenesis, immunity and stem cell formation. Prog Mol Subcell Biol. 2004 34, 103–43. Wingender G. From the Deep Sea to Everywhere: Environmental Antigens for iNKT Cells. Arch Immunol Ther Exp (Warsz). 2016 Aug 64(4):291–98. Müller WE, Koziol C, Müller IM, Wiens M. Towards an understanding of the molecular basis of immune responses in sponges: The marine demosponge Geodia cydonium as a model. Microsc Res Tech. 1999 Feb 15 44(4):219–36. Freymann DM, Nakamura Y, Focia PJ, Sakai R, Swanson GT. Structure of a tetrameric galectin from Cinachyrella sp. (ball sponge). Acta Crystallogr D Biol Crystallogr. 2012, Sep 68(Pt 9): 1163–74. Miller PW, Pokutta S, Mitchell JM, Chodaparambil JV, Clarke DN, Nelson WJ, Weis WI, Nichols SA. Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution. J Biol Chem. 2018 Jul 27 293(30):11674–86. Carstens BB, Rosengren KJ, Gunasekera S, Schempp S, Bohlin L, Dahlström M, Clark RJ, Göransson U. Isolation, characterization, and synthesis of the barrettides: disulfidecontaining peptides from the marine sponge Geodia barretti. J Nat Prod. 2015 Aug 28 78(8): 1886–93. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The Protein Data Bank. Nucleic Acids Res. 2000 28:235–42. Müller WE, Grebenjuk VA, Le Pennec G, Schröder H, Brümmer F, Hentschel U, Müller IM, Breter H. Sustainable production of bioactive compounds by sponges–cell culture and gene cluster approach: A review. Mar Biotechnol (NY). 2004 Mar–Apr 6(2):105–17.

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[24] Van Kaer L. Drugs from the sea: A marine sponge-derived compound prevents Type 1 diabetes. Sci World J. 2001 Nov 6 1:630–32. [25] Shady NH, Khattab AR, Ahmed S, Liu M, Quinn RJ, Fouad MA, Kamel MS, Muhsinah AB, Krischke M, Mueller MJ, Abdelmohsen UR. Hepatitis C Virus NS3 protease and helicase inhibitors from red sea sponge (Amphimedon) species in green synthesized silver nanoparticles assisted by in silico modeling and metabolic profiling. Int J Nanomedicine. 2020 May 12(15):3377–89. [26] Nicklas M, Schatton W, Heinemann S, Hanke T, Kreuter J. Preparation and characterization of marine sponge collagen nanoparticles and employment for the transdermal delivery of 17beta-estradiol-hemihydrate. Drug Dev Ind Pharm. 2009 Sep 35(9):1035–42. [27] Machałowski T, Czajka M, Petrenko I, Meissner H, Schimpf C, Rafaja D, Ziętek J, Dzięgiel B, Adaszek Ł, Voronkina A, Kovalchuk V, Jaroszewicz J, Fursov A, Rahimi-Nasrabadi M, Stawski D, Bechmann N, Jesionowski T, Ehrlich H. Functionalization of 3D chitinous skeletal scaffolds of sponge origin using silver nanoparticles and their antibacterial properties. Mar Drugs. 2020 Jun 10 18(6):304. [28] Wang X, Schröder HC, Wiens M, Schloßmacher U, Müller WE. Biosilica: molecular biology, biochemistry and function in demosponges as well as its applied aspects for tissue engineering. Adv Mar Biol. 2012 62, 231–71. [29] Schröder HC, Grebenjuk VA, Wang X, Müller WE. Hierarchical architecture of sponge spicules: Biocatalytic and structure-directing activity of silicatein proteins as model for bioinspired applications. Bioinspir Biomim. 2016 Jul 25 11(4):041002. [30] Wysokowski M, Behm T, Born R, Bazhenov VV, Meissner H, Richter G, Szwarc-Rzepka K, Makarova A, Vyalikh D, Schupp P, Jesionowski T, Ehrlich H. Preparation of chitin-silica composites by in vitro silicification of two-dimensional Lanthella basta demosponge chitinous scaffolds under modified Stöber conditions. Mater Sci Eng C Mater Biol Appl. 2013 Oct 33(7):3935–41. [31] Armirotti A, Damonte G, Pozzolini M, Mussino F, Cerrano C, Salis A, Benatti U, Giovine M. Primary structure and post-translational modifications of silicatein beta from the marine sponge Petrosia ficiformis (Poiret, 1789). J Proteome Res. 2009 Aug 8(8):3995–4004. [32] Shkryl YN, Bulgakov VP, Veremeichik GN, Kovalchuk SN, Kozhemyako VB, Kamenev DG, Semiletova IV, Timofeeva YO, Shchipunov YA, Kulchin YN. Bioinspired enzymatic synthesis of silica nanocrystals provided by recombinant silicatein from the marine sponge Latrunculia oparinae. Bioprocess Biosyst Eng. 2016 Jan 39(1):53–58. [33] Schröder HC, Natalio F, Wiens M, Tahir MN, Shukoor MI, Tremel W, Belikov SI, Krasko A, Müller WE. The 2ʹ-5ʹ-oligoadenylate synthetase in the lowest metazoa: Isolation, cloning, expression and functional activity in the sponge Lubomirskia baicalensis. Mol Immunol. 2008 Feb 45(4):945–53. [34] Natalio F, Corrales TP, Panthöfer M, Schollmeyer D, Lieberwirth I, Müller WE, Kappl M, Butt HJ, Tremel W. Flexible minerals: Self-assembled calcite spicules with extreme bending strength. Science. 2013 Mar 15 339(6125):1298–302. [35] Jiao C, Deng M, Ma Y, Hu G. Effect and Repair Mechanism of Nano Ag Sponge Dressing Combined with Gelatin-Bletilla Striata Gum/Salvia Miltiorrhiza on Refractory Orthopedic Wounds. Biomed Res Int. 2021 Mar 30(2021):8872235. [36] Petrenko I, Bazhenov VV, Galli R, Wysokowski M, Fromont J, Schupp PJ, Stelling AL, Niederschlag E, Stöker H, Kutsova VZ, Jesionowski T, Ehrlich H. Chitin of poriferan origin and the bioelectrometallurgy of copper/copper oxide. Int J Biol Macromol. 2017 Nov 104(PtB): 1626–32.

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[37] Ehrlich H. Silica Biomineralization, Sponges. In: Reitner J, Thiel V eds Encyclopedia of Geobiology. Encyclopedia of Earth Sciences Series. Springer, Dordrecht, 2011, https://doi. org/10.1007/978-1-4020-9212-1_31. [38] Coradin T, Marchal A, Abdoul-Aribi N, Livage J. Gelatine thin films as biomimetic surfaces for silica particles formation. Colloids Surf B Biointerfaces. 2005 Sep 44(4):191–96. [39] Markl JS, Müller WEG, Sereno D, Elkhooly TA, Kokkinopoulou M, Gardères J, Depoix F, Wiens M. A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic-inorganic hybrid materials in sponge primmorphs. Biotechnol Bioeng. 2020 Jun 117(6):1789–804. [40] Weaver JC, Pietrasanta LI, Hedin N, Chmelka BF, Hansma PK, Morse DE. Nanostructural features of demosponge biosilica. J Struct Biol. 2003 Dec 144(3):271–81. [41] Ehrlich H, Deutzmann R, Brunner E, Cappellini E, Koon H, Solazzo C, Yang Y, Ashford D, Thomas-Oates J, Lubeck M, Baessmann C, Langrock T, Hoffmann R, Wörheide G, Reitner J, Simon P, Tsurkan M, Ereskovsky AV, Kurek D, Bazhenov VV, Hunoldt S, Mertig M, Vyalikh DV, Molodtsov SL, Kummer K, Worch H, Smetacek V, Collins MJ. Mineralization of the metre-long biosilica structures of glass sponges is templated on hydroxylated collagen. Nat Chem. 2010 Dec 2(12):1084–88. [42] Müller WE, Wang X, Burghard Z, Bill J, Krasko A, Boreiko A, Schlossmacher U, Schröder HC, Wiens M. Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni. J Struct Biol. 2009 Dec 168(3):548–61. [43] Wiens M, Bausen M, Natalio F, Link T, Schlossmacher U, Müller WE. The role of the silicateinalpha interactor silintaphin-1 in biomimetic biomineralization. Biomaterials. 2009 Mar 30(8): 1648–56. [44] Weaver JC, Aizenberg J, Fantner GE, Kisailus D, Woesz A, Allen P, Fields K, Porter MJ, Zok FW, Hansma PK, Fratzl P, Morse DE. Hierarchical assembly of the siliceous skeletal lattice of the hexactinellid sponge Euplectella aspergillum. J Struct Biol. 2007 Apr 158(1):93–106. [45] Rozas EE, Mendes MA, Nascimento CA, Espinosa DC, Oliveira R, Oliveira G, Custodio MR. Bioleaching of electronic waste using bacteria isolated from the marine sponge Hymeniacidon heliophila (Porifera). J Hazard Mater. 2017 May 5; 329:120–30. [46] Inbakandan D, Kumar C, Bavanilatha M, Ravindra DN, Kirubagaran R, Khan SA. Ultrasonicassisted green synthesis of flower like silver nanocolloids using marine sponge extract and its effect on oral biofilm bacteria and oral cancer cell lines. Microb Pathog. 2016 Oct 99: 135–41. [47] Inbakandan D, Venkatesan R, Ajmal Khan S. Biosynthesis of gold nanoparticles utilizing marine sponge Acanthella elongata (Dendy, 1905). Colloids Surf B Biointerfaces. 2010 Dec 1 81(2), 634–39. [48] Savage DT, Hilt JZ, Dziubla TD. In Vitro Methods for Assessing Nanoparticle Toxicity. Methods Mol Biol. 2019 1894:1–29. 10.1007/978-1-4939-8916-4_1. [49] Mukherjee S, Gautam A, Pal K, Karmakar P, Ray M, Ray S. Copper oxide nanoparticle and copper sulfate induced impairment of innate immune parameters in a common Indian sponge. J. Hazard Mater Lett. 2021 2:100036. [50] Fallon BR, Freeman CJ. Plastics in Porifera: The occurrence of potential microplastics in marine sponges and seawater from Bocas del Toro, Panamá. Peer J. 2021 9:e11638. 2021 Jul 8 10.7717/peerj.11638.

2 Cnidaria immune system and nanoparticles Garima Hore, Shyamasree Ghosh, Dhriti Banerjee Abstract: The phylum Cnidaria of invertebrates, although primitive in its organization, reveals an innate immune system. Diseases are caused when exposed to pathogens. The body is capable to show reactions while exposing to the environmental stress factors. Different aspects such as Cnidarian immunity, molecules and pathways involved, symbiosis, regeneration, and healing process of wound and cellular immune process are discussed in this chapter. Nanoparticles have been reported to induce toxic effects on the Cnidarians affecting their physiological and cellular processes. They have found their applications in trapping nanoparticle waste and model nanoparticle-mediated environmental toxicity. We discuss in this chapter nanoparticle-mediated toxicity and its impact on Cnidarians. Keywords: Cnidaria, nanoparticles, immune system

Abbreviations A. aureta A. digitifera A. millepora A. pallida AMP C. werneri CASP-3 CTLs ERK G. ventalina H. magnipapillata H. vulgaris MACPF MAPK Myd88 N. vectensis N

Aurelia aurita Acropora digitifera Acropora millepora Aptasia pallida Antimicrobial peptides Corumbella werneri Caspase-3 C-type lectins Extracellular signal-regulated kinases Gorgonia ventalina Hydra magnipapillata Hydra vulgaris Membrane-attack complex/perforin Mitogen-activated protein kinases Myeloid differentiation primary response 88 Nematostella vectensis Nitrogen

Garima Hore, Department of Zoology, Dr. Kanailal Bhattacharyya College, Dharmatala, Ramrajatala, Santragachi, Howrah 711104, West Bengal, India Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India Dhriti Banerjee, Diptera Section, Zoological Survey of India, Ministry of Environment, Forest and Climate Change, M-Block, New Alipore, Kolkata 700053, India https://doi.org/10.1515/9783110655872-002

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NF-κB NLRs NP O. faveolata P. cylindrical P. noctiluca P PAMPs PPO PRRs QDs RNS ROS S. pistillata S SML SOD TIR TLRs TNFRSF1A W. annularis

Nuclear factor kappa light chain enhancer of activated B cells Nucleotide oligomerization domain (NOD)-like receptors Nanoparticles Orbicella faveolata Porites cylindrical Pelagia noctiluca Phosphorus Pathogen-associated molecular pattern Prophenoloxidase Pattern recognition receptors Quantum dots Reactive nitrogen species Reactive oxygen species Stylophora pistillata Sulfur Surface mucus layer Superoxide dismutase Toll/interleukin-1 receptor Toll-like receptors Tumor necrosis factor receptor superfamily member 1A Wutubus annularis

2.1 Introduction: general biology Cnidarians comprising sea anemones, corals, jellyfishes, and hydroids represent a diversified phylum encompassing near about 9,000 living species, thriving in aquatic, primarily marine, habitats. They reveal radial symmetry and primitive existence. The stinging cells, nematocytes, known as cnidocytes, are unique organelles meant for defense, predation, and adhesion and form a characteristic feature of phylum Cnidaria [1–4]. They reveal a gut cavity lined by endoderm termed as coelenteron or gastrovascular cavity. Body is diploblastic with body wall comprising outer epidermis, inner gastrodermis, and intermediate layer of mesoglea. Phylum Cnidaria is classified into two main groups: members of class Anthozoa including sea pens, sea anemones, and corals, existing as polyps with sessile existence; and free-swimming Medusozoa including sea wasps, jellyfish, and Hydra, in which free-swimming medusoid forms are also prevalent, along with the polyps [4]. In terms of body symmetry, Cnidarians, in both the abovementioned groups, exhibit an external radially symmetrical body form, although bilaterality and internal asymmetries are also evident in several forms [4]. Cnidarians bear a single external aperture, serving as both mouth and anus, and are usually encircled by tentacles containing nematocytes [4]. Cnidaria evolution reveals an early branching with a metazoan descent, and out of the five classes of Cnidaria (Anthozoa, Hydrozoa, Cubozoa, Scyphozoa, and Staurozoa) (Fig. 2.1), four classes have been found to be associated with the Cambrian

2.2 Cnidaria classes in brief

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fossil record [5], suggestive of the notion that substantial cnidarian heterogeneity took place approximately ~500 million years ago [4]. Phylogenetically, Cnidaria represents a sister group to Bilateria [4]. In Bilateria, body form constitutes key features of bilaterally symmetrical body axes, presence of a mesoderm which is the third germ layer, and a central nervous system. These differentiate Bilateria from Cnidaria forms [4]. In contrast to these features, Cnidarians are usually considered as diploblastic animals, that is, they possess only two germ layers including endoderm and ectoderm and radially symmetrical body axes, yet anthozoan polyps are known to exhibit an internal bilaterality in the asymmetric arrangement of pharynx, the siphonoglyph, as well as the retractor muscles present in the mesenteries [6–10, 4].

Fig. 2.1: Classification of phylum Cnidaria, composed of five present cnidarian classes (Anthozoa, Hydrozoa, Cubozoa, Scyphozoa, and Staurozoa). Class Anthozoa is further divided into subclasses, namely, Hexacorallia and Octacorallia. Abbreviations: AR- adradii; IR- interradii; gd- gonad; cl- calyx; and pe- peduncle.

2.2 Cnidaria classes in brief 2.2.1 Class Anthozoa Members of class Anthozoa exhibit either solitary or colonial polypoid forms with complete absence of medusoid forms including a largest group of more than 6,000 species including diverse range of corals, sea fan, sea pansies, and sea anemone.

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Class Anthozoa, at present, is considered monophyletic clade composed of two chief clades recognized with subclasses including Hexacorallia and Octocorallia [11, 12]. Studies on hexacorallian fossils lead to the notion that the group originated during a time ranging between the lower Cambrian period being the first geological period in the Paleozoic that existed around approximately 540 million years ago [13] and the Ediacaran times in the precambrian that existed in the geological timescale of approximately 600 million years ago [12, 14]. Subclass Octocorallia, revealing benthic organisms, is of exceptional worth in the faunal realm, owing to its unique beauty, variety, abundance, as well as noteworthy interspecific associations [15]. Comprising varied forms, ranging from filiform, encrusting and membranous structures to intricate dendriform frameworks, this group is anatomically quite diverse [15]. While considering their distribution patterns, it is to be noted that octocorals are typically marine organisms, displaying noteworthy diversity in shallow reefs in tropical waters and in deep sea or lowest oceanic layer environments (e.g., seamounts), being significant structural units of such communities [15, 16].

2.2.2 Class Hydrozoa Hydrozoans, including more than 2,700 species constituted by medusozoan cnidarian forms prevalent in marine habitats, exhibit varied life cycle patterns [17]. Although freshwater hydrozoans are less widespread than their marine counterparts, the genus Hydra can undoubtedly be regarded as the best studied hydrozoan [18]. They reveal either polypoid or medusoid structures, and some species exhibit both forms in their life cycles. Hydrozoans can be represented on the basis of their way of giving rise to the pelagic stage (hydromedusae), budding off laterally from the polyps, by complete alteration of polyps (characteristic of class Cubozoa), or emerging by strobilation (as seen in class Scyphozoa) [18]. A prime attribute of class Hydrozoa is the formation of polymorphic colonies [18]. Three morphologically distinct forms are prevalent in this taxon, namely, the planula larva that undergoes metamorphosis and develops the polyp form which, subsequently, forms the medusae by the process of budding, a strategy common in Hydroidomedusae [18]. Contradictory to this, however, often it is observed that either the hydroid form or the medusa form are lacking from the biological life cycles of these species [19]. Siphonophores at present comprise 191 living species, in which most of the species possess both forms of polyps and medusa merging into planktonic organisms with polymorphic colonial existence [18, 20].

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2.2.3 Class Cubozoa Cubozoans, commonly known as box jellyfishes, comprising near about 50 described species, form the smallest class of phylum Cnidaria [21]. Yet, Cubozoans are considered as one of the most interesting cnidarians, owing to some of their exceptional attributes [21], such as possession of unique, complex eyes and related visual potentiality [22]; remarkable courtship and mating behavior [23]; and intense toxicity [24]. The presence of a definite cuboidal bell forms the basis of naming the class Cubozoa [25]. Characteristic features of cubozoans are the presence of four sensory structures called rhopalia, specifically, prevalent one on each side of the bell and each bearing up to six eyes, attached by means of a pedalium and a nerve ring from all corners of the bell, which in turn possesses tentacles [25]. Cubozoans are categorized into two orders, such as Carybdeida and Chirodropida, depending primarily on their overall morphological features [25]. The characteristic attribute of Carybdeids is the presence of a single tentacle for each pedalium [25]. Moreover, the presence of nematocysts both on their tentacles and on their bell is another noteworthy diagnostic feature [25], in comparison to the presence of multiple tentacles for each pedalium. Some species, however, can possess up to 15 tentacles for each pedalium. Each tentacle can extend almost up to 3 m [26], with nematocysts seen generally present only on their tentacles, comprising the basic morphological attributes of order Chirodropida [25]. Cubozoans display a polymorphic life form, distinctive for many pelagic forms of Cnidarians [25]. Exceptions, however, are also prevalent, particularly in the development of medusa from polyp forms [25]. Rhizostoma jellyfishes usually possess monodisk or one ephyra polypoid strobila, whereas nearly all of the semaeostomes discharge polydisk or multiple ephyrae from each scyphistoma by means of transverse fission process during strobilation [25, 27]. The scyphistoma is observed to regenerate in the strobilation period last phase. Cubozoan polyps, yet, are not seen to strobilate but they reveal transformation altogether into a single medusa form [25].

2.2.4 Class Scyphozoa Phylogenetically primarily scyphozoans are divided into two main groups [28], namely, Coronatae and Discomedusae, based on recent phylogenies studied based on 18S and 28S rDNA [29], as well as transcriptomic studies [30]. Coronate polyps (stephanoscyphistoma) including sedentary forms colonize in hard substrates, underwater, inhabiting deep sea to sublittoral zones, marine caves and thrive in firm, supportive chitinous tubes [31]. Coronate medusae, on the other hand, possess deep grooves located along the bell margin, with separate thick ridges, termed “pedalia” on the bell [28, 32, 33]. It is observed that a single tentacle may or may

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not be present on each pedalium [34]. Other distinguishing features of coronate medusae that separate them from other medusae are the presence of nonpigmented oocytes [11, 33], presence of simple mouth held up by a stalk called manubrium, and in several species encircled by tentacles facing forward [28]. Discomedusae are seen to bear polyps where a chitinous tube is either lacking or have a partial chitinous cover on the aboral stalk, where the polyp is unable to retract [28]. “Podocysts” are cystic dormant stages of some polyps [33, 35]. Discomedusoid medusae are characterized by the presence of intricate oral arms, a gastric apparatus with canals [33], bells where grooves and pedalia are lacking, and in species with the presence of marginal tentacles. The tentacles are observed to droop gently beyond the organism [28]. The life cycle of scyphozoans is divided into three developmental stages [28]. Scyphomedusae give rise to reproductive units of either eggs or sperm, seldom both are produced [36], the fusion and development of which results in the formation of ciliated larval form, called as “planulae” [28]. Generally in most species in this class, a planula larva is observed to settle on benthic layer, which gradually develops into the sessile form, termed as a scyphistoma or scyphopolyp, or polyp [28]. Polyps, similar in appearance to minute sea anemones, are found to be quite generous in number, and of substantial ecological importance [28, 37]. The reason behind this is the fact that, for majority of the scyphozoans, the juvenile medusa is produced by the polyp stage, known as ephyra, by means of a unique, transformation procedure termed “strobilation” [28]. While polyp forms may seem inconspicuous, the scyphomedusae group has attained substantial attention in research, of late, due to their impact on human beings and ecosystems [28, 38–43].

2.2.5 Class Staurozoa Staurozoa, commonly termed as stalked jellyfishes, comprise a class of benthic cnidarians, represented by near about 50 species [11, 44–47, 49]. The most striking feature of class Staurozoa of phylum Cnidaria [29, 33, 48] is its peculiar life cycle, represented by creeping larvae developing into juvenile stauropolyps, which subsequently, metamorphoses into non-free-swimming form, adult stauromedusae form, while remaining adhered to the substrate with the help of a peduncle [48, 50–52]. Generally, the apical region of the metamorphosed stauromedusa termed as “calyx” bears morphological features resembling those of adult forms of scyphomedusae and cubomedusae, including the presence of structural features including circular coronal muscle, rhopalioids or rhopalia, gastric filaments, and gonads [29, 48, 53]. The basal portion termed as “peduncle,” however, retains polypoid features like gastric septa connected with four inter-radial longitudinal type of muscles [53, 54]. Hence, comprehending the body organization of a stauromedusa is rather complicated, owing to its dual nature as compared to other medusozoans [29, 55].

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Staurozoan taxonomy is chiefly based on characters concerned with the internal anatomy [55], as stauromedusae have comparatively few macromorphological features worthwhile to demarcate species [56].

2.3 Origin of phylum Cnidaria Since phylum Cnidaria is usually considered as a sister group to Bilateria, a comprehensive analysis of all characteristics of cnidarians holds paramount significance in investigating the evolution of metazoans [57]. In accordance with the molecular clock analysis, cnidarians, alongside sponges, bilaterians, and ctenophores originated in the Cryogenian Period, estimated around 720–635 million years ago, which also revealed to be a period with dynamic environmental changes, including glaciations impacting biological evolutions, continental breakdown of Rodina, and tectonic and magmatic activities responsible for rifting due to volcanoes [57–60]. On the basis of paleontological and molecular investigations, the divergence of anthozoans and medusozoans is considered to have occurred about 531.5–641.8 Ma [60] with the medusa form presumably independently acquired from polyps in various groups of medusozoans [29, 33, 53, 57].

2.4 Early diversification Current molecular clock analyses reveal phylum Cnidaria origin to the Cryogenian Period with the division between the two subphyla Anthozoaria and Medusozoa similarly taking place during this period of time [61]. Nevertheless, the primitive cnidarian macrofossils, all medusozoans, prevailed in the fossil records dating back to the late Ediacaran Period [61]. Sparsely skeletonized Corumbella werneri (C. werneri), at present reported from Brazil, Paraguay, as well as Nevada (USA), of late Ediacaran strata, has been linked with coronate and conulariid scyphozoans. However, it also reveals overall common morphological affinities with Carinachites spinatus, a probable conulariid form reported from China dating back to the Cambrian Stage 1 period and presumably comparable with Sinotubulites sp. and Wutubus annularis (W. annularis) from China, dating back to the late Ediacaran Dengying Formation period [61]. The most profound confirmation of similarity with coronate scyphozoans is displayed by Paraconularia sp. from Corumbella – bearing shale period dating back to latest Ediacaran Tamengo Formation of central Brazil [61]. Moreover, Paraconularia sp. retrieved from fossil records in rocks denotes conulariids belonging to cnidarians that traversed the Proterozoic–Phanerozoic times in the geological timescale [61]. Haootia quadriformis, prevalent in the late Ediacaran lower Fermeuse and Trepassey formations reported from southeastern Newfoundland, Canada, depicts

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fascinating overall morphological resemblances to extant staurozoans and can presumably render the earliest record of metazoan architecture [61].

2.5 Zoogeographical distribution and ecological significance 2.5.1 Class Anthozoa Class Anthozoa primarily comprises faunal groups such as corals bearing calcareous exoskeletons, sea anemones, and sea pens possessing endoskeletons [57]. Sea anemones may be quite simple with respect to their body plan, nevertheless, are of profound worth from their ecological diversity [62]. Not only are sea anemones widely distributed inhabiting varied habitat types such as shallow water mud flats, rocky shores, coral reefs, deep sea, man-made structures, and others, but they are capable of establishing noteworthy ecological associations with various organisms, like photosynthetic microbes, invertebrates, and fishes [62–66]. Symbiotic relationship between hermit crabs and sea anemones belonging to the genus Calliactis is well known [64]. Usually, the prevalence of sea anemones is comparatively less in the tropical belt, whereas in the globally temperate regions, they are capable of composing large clusters which acquire the terrain and constitute the local groups [62]. Now, while considering their ecological worth, sea anemones, particularly in areas where they are found profusely, can play a vital part in control of population size by means of the larval predation from other organisms including mollusks and fishes [62, 67, 68]. Sea anemones’ interrelation with photosynthetic microorganisms (termed as zooxanthellae) provides them another source of nutrition, being benefitted by the product of photosynthesis, in such a relationship [69], which had imparted knowledge about some notable characteristics of the diversity of the group [62]. Photosynthetic dinoflagellates mainly belonging to Symbiodinium genus constitute common symbiont forms with other anthozoans, and usually, each of the group includes genetically diverse subgroups which display an exclusive ecobiogeographical and host-specific pattern of interaction [70, 62]. 2.5.1.1 Subclass Hexacorallia Stony corals, belonging to subclass Hexacorallia, constitute the well-known reefbuilding scleractinian corals, in which they predominantly exhibit polyp form, while remaining adhered to a hard substrate and encircled by a secreted skeleton composed of calcium carbonate [71, 72]. The calcium here is derived from the seawater where it inhabits and is used for composing its structural framework, defense, and growth [72]. An intricate web of intercommunication prevails between the microbial holobiont [72]. A holobiont concept of host–microbe interaction in ecosystems has revealed the rapid adaptation of corals to specific pathogens. A

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hologenome comprises two components including genome of host and microbiome. Genomic alterations can either lead to variations leading to selection for or against the organism and can influence evolution [72, 73]. Symbiotic associations generally prevail among the coral as hosts, and their symbionts including algae and microbes [72, 74]. Corals possess a diverse assemblage of microorganisms comprising archaea, bacteria, and fungi, and specific microbial processes can have an impact on the coral hosts physiology and ecosystem of coral reef. Microorganisms associated with corals have a crucial role to play in coral health and coral reef resilience, in addition to nutrient cycling [72, 75, 76]. Coral-associated microbes have been reported to play a role in biogeochemical cycling, primarily in nitrogen (N), sulfur (S), and phosphorus (P) cycle that fosters coral health and maintains the resilience of ecosystem of coral reef [72]. Perhaps the best known instances of symbiotic relationships in the marine environment have been put forward among Cnidarians, namely, sea anemones, jellyfish, hard and soft corals and hydrocorals, and algae under phylum Dinoflagellata, belonging to Symbiodinium genus, usually known as zooxanthellae or commonly termed as single-celled dinoflagellates [77–79]. Such dinoflagellates generally thrive inside the gastrodermal cells of the host cnidaria, which forms the innermost tissue bordering the gastrovascular cavity, bound by complex membrane structure arranged as series of membranes bearing algal origin, together with an outermost membrane originating from the host [77, 80–82], and this entire unit is termed as symbiosome [77]. The system of cnidarians dinoflagellate association and symbiosis is prevalent across the latitudes of temperate and subtropical zones [77, 83, 84]. The photosynthetic products contributed by the symbiont dinoflagellate sustains metabolism, growth, reproduction, and survival of the coral host [85, 86] in a habitat that is comparatively wanting in terms of exogenous food reserves [77]. Moreover, these symbiont dinoflagellates foster the conservation and enable recycling of vital nutrients [88, 89], thereby assisting survival of the coral host in the waters that is poor in nutritional content. This is observed in many coral reefs, and augment rates of coral skeletogenesis [89, 90], thus permitting the total persistence of the architecture of coral reef amid the challenges of erosion from biological and mechanical sources. As a response to these various beneficial services, nutrients present in the waste products of the coral is accessible to the dinoflagellates, in addition to achieving a secured position within the water column for retrieving light, and extended defense from grazers [77]. 2.5.1.2 Subclass Octocorallia Soft corals embrace quite a varied number of species, belonging to subclass Octocorallia or Alcyonaria including gorgonians exhibiting sessile colonial existence inhabiting oceans, and sea pens grouped under order Pennatulacea, living in colonial

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marine habitats [15]. The demarcating feature of soft corals is that they do not develop a rigid skeleton of calcium carbonate and are not reef-forming corals, even though they may be seen to occur in a reef ecosystem [15]. Well-diversified distribution patterns are exhibited by octocorals, inhabiting seas and oceans globally, their distribution extending from shallow waters and reaching up to depth of 6,400 m, occupying both reef environments and soft bottoms, although a few cosmopolitan ones, usually composed of pennatulaceans, are also present [15, 91]. Even though octocorals are prevalent in all oceans, yet, they are quite varied in terms of their distribution locally, as they are affected by different factors, like strong ocean currents, organic matter suspended in water, and the distance from the coast [92]. In terms of distribution of scleractinian corals, the Indo-Pacific dominates, as this region is known for the greatest diversity as well as endemism of octocorals [15, 93]. Octocoral species, like pennatulaceans, characterized by their wide depth ranges, tend to inhabit more areas, quite similar in case of various abyssal species, cosmopolitan in distribution, whereas species more geographically constrained are the ones colonizing continental margins [91]. A common notion while considering the distribution and diversity of shallow water octocoral species is that, as shallow water zooxanthellate octocorals rely on light, their distribution pattern is more limited as compared to the azooxanthellate ones [15]. Certain factors such as significant fluctuations in temperature and salinity and exposure to river runoffs, often faced by octocoral species thriving in shallow water and coastal habitats, can lead to speciation and a greater extent of endemism, as a result of formation of geographic barriers [15]. Speciation is persistent in deep sea conditions as in the habitats of shallow water, indicated by their high diversity observed in the habitats of deep sea environment, as more than 75% of octocoral species are reported in waters at a depth of more than 50 m [93, 94]. Nevertheless, the endemism prevalent in shallow waters is readily noted [95, 96]. Characteristic features of octocorals inhabiting deepwater distinguish them from those inhabiting shallow water environments, involving high longevities, slower growth rates, low water temperature tolerance, and insubstantial accessibility to food [15]. Cold water corals are chiefly prevalent in environmental conditions, where temperature extends from 4 to 12 °C. Yet, some species that can withstand even more cold temperatures are also reported [15, 97–99].

2.5.2 Class Hydrozoa Hydrozoans are considered as crucial planktonic forms prevalent in the medusa stage and benthic forms observed in the polyp stage predators devouring crustaceans, fish larvae, as well as other benthic and planktonic organisms [100], from ecological point of view [18]. Hydrozoans have diverse feeding habits. Some species

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can thrive on phytoplankton, bacteria, and protozoans, while others live on dissolved organic matter [101]. Some hydrozoan species are reported to harbor symbiotic intracellular algae [102]. Under appropriate environmental conditions, such strong seasonal conditions, hydrozoans [103], can become profuse in numbers, resulting in seasonal blooms in planktonic communities [18, 104–106]. Together with seasonal conditions, regular vertical shifts between epipelagic and mesopelagic waters are often carried out by many organisms [107]. Apart from this, hydromedusae is considered as yardsticks of upwelling systems [108–110] and known to play pivotal roles as biological indicators of environmental conditions, being ubiquitous constituents of the benthic fauna along rocky shore [111].

2.5.3 Class Cubozoa Although cubozoans are jellyfishes predominantly inhabiting the tropical belt, a few species have been recorded outside the tropical belt. A few taxa have been reported at higher latitudes, ranging from 42°N and 42°S [112]. In terms of diversity of cubozoans, the biodiversity-rich zones of the Coral Sea and Indo-West Pacific with predominance of 10 and 12 accepted species reported, respectively [112]. Various species have been reported from such regions of Caribbean Sea, Gulf of Mexico, Philippine Sea, and east and west limits of the Atlantic Ocean [112]. The distribution of cubozoans is not only confined to continental coastlines, as they are also documented from island waters, some set apart by nearly about 4,000 km stretch of oceanic waters, encompassing Hawaii, Society Islands, Samoa and New Zealand in the Pacific Ocean, in addition to Bermuda and Saint Helena in the Atlantic Ocean [112]. Taking into consideration their ecology, box jellyfishes are keen predators, with an array of both invertebrates and vertebrates as prey [112]. Considering the evolutionary perspective, cubozoans have developed species and age-specific toxins and injection processes for targeting particular prey types [113, 114]. In fact, toxins from some cubozoan species including Chironex fleckeri and Chironex yamaguchii are reported to be so powerful that they can be life-threatening to human beings [112]. Prey capture of the entangling predators, the cubomedusae, is executed by injection of venom by nematocysts on their tentacles [113, 115]. Majority of the species display feeding or foraging or gathering food behavior which is a characteristic feature of order cubomedusae of box jellyfishes [112]. Cubozoans chiefly prey on planktonic crustaceans [112]. Often having an influence on the abundance of planktons, jellyfishes are considered as chief organisms responsible for direct predation on fish larvae [116, 117]. Furthermore, reduction in the abundance of small plankton can subsequently have an impact on the survivability of other plankters [118].

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2.5.4 Class Scyphozoa Among cnidarians, the scyphomedusae are notable for their profound impacts on ecology, economics, and human health [28]. From the ecological perspective, scyphomedusae play pivotal roles both as predators and as prey, in many marine ecosystems [119]. Natural predators of the scyphomedusae include the leatherback turtle Dermochelys coriacea [120] and the sunfish Mola mola (M. mola) [121], in addition to the economically significant fish species, for instance, the Mediterranean Boops boops [122]. Another point worth mentioning is that scyphomedusae have significant consequences on human health and industry, especially while forming mass aggregations or blooms [28]. Moreover, scyphomedusae are noteworthy cnidarians, as potent substitutes for fish, as highest pelagic predators in ecosystems that are overfished [40]. Their stings can be painful and can obstruct power plant intakes [38, 41].

2.5.5 Class Staurozoa Considering the diversity aspects of staurozoans, it is observed that their diversity patterns deviate from the usual patterns of diversity, wherein species richness is higher near the Equator and diminishes toward the poles. Staurozoan diversity is reported to be greater in the temperate belts [49, 123, 124], although the current comprehension of global marine diversity is still inadequate [125]. Nevertheless, deviations from this usual pattern exists, as seen in sea anemones [124] and benthic marine algae [126], which display higher species richness at midlatitudes [49]. Distribution of benthic marine algae is especially fascinating, since algae are presumed to be the favorable substrate of stauromedusae [49]. Biodiversity of the algal genera displayed is higher in the temperate belts, diminishing in richness toward the tropics and polar region [126], alike to that observed in staurozoans [49]. The present-day distribution of stalked jellyfishes could be interpreted on account of the biotic interrelationships between algae and stauromedusae, outweighing the physical variables and emerging in an obvious close relationship, and the life cycle of some staurozoans [127] is presumably influenced by the life cycle of the algae [49].

2.6 Regenerative capacity of cnidarians One of the remarkable attributes of cnidarians lies in their potentiality to regenerate [4, 128]. For instance, when cleaved into two halves, a Hydra polyp will regenerate the missing oral and aboral framework in a matter of 2–4 days’ timespan [4, 129].

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Interestingly, Hydra bears the potential to regenerate each cut portion into a complete polyp, after being split into ~20 fragments [130]. Furthermore, strikingly, Hydra can be segregated into a cell suspension, which, when recombined, is potent of regenerating polyps de novo, and is in accordance with a reaction–diffusion-based mechanism of pattern formation [4, 131–133]. Regeneration in Cnidaria occurs by means of morphallaxis, in which the existing tissue repatterns itself in the absence of cell proliferation [4, 128]. Comparably, the process of regeneration in Hydra is of morphallactic type, involving a procedure devoid of growth and proliferation [4, 134]. Presumably due to the plasticity of the tissue with high morphogenetic potential, neither local dedifferentiation formation nor blastema formation forms the necessities in the process of regeneration in cnidarians [128]. Interestingly, at the cellular level, the epithelial stem cells seem to find importance in enabling morphogenesis at the injury site and able to differentiate [128]. In cnidarians, the head organizer or apical signaling center holds considerable evidence to be the prime force leading to the process of regeneration [128].

2.7 Cnidarian immunity: an overview Progress in research domains like cell biology and genomics has strengthened the concept on cnidarian immunity by facilitating investigators in unraveling the underlying mechanisms and differentiating them with those studied in other phyla [135–137]. Innate immunity comprises the major driving force of cnidarian defense strategies, as being invertebrates, and the potentiality in generating resistance to a specific pathogen is lacking in them [137]. Yet, of late, interestingly, this contradiction among innate and adaptive immunities is rather questionable, owing to the fact that existence of immune memory has been discovered in cnidarians [137, 138]. So far, the memory response in the immune system of Cnidaria is based on two lines of evidences [137]. Primarily, experimental approaches based on transplantation of tissues, studies of member organism of the same species or conspecific organisms, and rejection among the heterospecific organism or organism of a different species have led to the presumption of existence of immunological memory [139]. The other fact empowering the concept of prevalence of immunological memory in cnidarians is the discovery of different kinds of receptors that are capable of differentiating between molecular patterns of various pathogens [138]. Thus, no doubt, this crucial attribute of recognition of variations among various types of pathogen and binding with the receptor empowers the concept of memory response to that pathogen [137]. Cnidarian immunity can be categorized into four basic mechanisms (as depicted in Fig. 2.2), which forms the chief components of the immune system, including

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immune recognition, followed by signaling reaction mediated by intracellular signaling cascades, effector responses, and repair of damaged tissue mechanisms. Cnidarian Immune system Pathogen/ PAMP Immune Recognition

PRRs

TLRs

Lectins

NLRs Intracellular signalling cascade

TLR Pathways

Associated proteins

Complement Pathway

Opsonization

Effector responses

AMP

Bactericidal response

ROS

Phagocytosis

Proteases PO/PPO

Melanin ROS Cellular responses

Encapsulation

Fig. 2.2: Schematic diagram displaying the basics of cnidarian immunity.

2.7.1 Recognition of pathogen by immune system The arousal of innate immunity in vertebrates as well as invertebrates relies on recognition of pathogen by different members of proteins, collectively termed as pattern recognition receptors (PRRs). The immune system in cnidarians has been studied to include primarily three major families of PRRs, including lectins which are protein molecules that bind to carbohydrates, Toll-like receptors (TLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs) [135, 137, 140–142].

2.7.2 Toll-like receptors (TLRs) TLRs are glycoprotein in nature and composed of characteristic pathogen-associated molecular patterns (PAMPs) as the ligand, and they play a major role in activation of innate immune responses in diverse number of species [137, 143, 144]. Ample amount of data from genomic studies on cnidarians have revealed the existence of a broad

2.7 Cnidarian immunity: an overview

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range of TLR-type receptors [137], such as those reported from Nematostella vectensis (N. vectensis) and gorgonian soft coral Gorgonia ventalina (G. ventalina) expressing TLR-2, -4, and -6 [145]. Numerous TLR-type proteins were first studied in organisms under genus Acropora in scleractinian corals [137]. TLRs have been reported from members of Hydractinia [137]. Regarding the functional perspective of TLRs, assumptions are made, based on the similarities of sequences that they chiefly serve in pathogen recognition and immune system activation [137].

2.7.3 Lectins These are proteins that bind to carbohydrates. They act as important PRRs. Lectins have been reported of their diversity and being specific to lineages and play a major role in recognizing pathogens and activating the innate immune responses have been described in cnidarians [137, 146–149]. Lectins may provide the initial innate arm of immunity against pathogens in many cnidarians by localized expression of certain genes [146, 149]. Lectins on binding to potential pathogens lead to activation of complement system, a process of opsonization [150]. Primarily, four types of lectins have been reported from organisms under phylum Cnidaria including C-type lectins (CTLs), millectin, rhamnospondin, tachylectin-2 and are responsible for the activation of immune responses [137].

2.7.4 Nucleotide oligomerization domain (NOD)-like receptor (NLRs) Though NLR-like genes are reported from various cnidarian genomes, interestingly, it is also found to be highly variable across various lineages [151, 152]. Acropora digitifera (A. digitifera) genome contains around 500 NLR loci including NODs [137]. Moreover, NLRs reveal a greater variety of protein motifs in comparison to those reported from vertebrate genomes [152]. NLR diversity is lacking in Nematostella and Hydra, specifically as compared to scleractinian corals [137]. Assumptions are that the necessity in maintaining a diversified microbial population seems to be responsible for the diversification of NLRs [152].

2.7.5 Intracellular signaling cascade After the binding of receptor, the activation of immune responses and triggering of signal reactions occur [137]. These signaling pathways involving molecules that function as a cascade with interrelated signaling events assist in signal propagation [137].

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In addition to TLRs, complement cascades, mitogen-activated protein kinases (MAPK), and prophenoloxidase (PPO) cascade comprise the chief components of the intracellular signaling cascade [137].

2.7.6 TLR pathways and TIR domains Various constituents of the Toll pathway have been reported in cnidarians [137], among which four Toll/interleukin-1 receptor (TIR) domain comprising proteins [153], including IL-1 R-like, Myd88, TLR-like, and TIR, have been reported. IL-1Rlike and myeloid differentiation primary response 88 (Myd88) proteins have been identified from three anemone species and from Caribbean scleractinian and IndoPacific corals [137]. Myd88 proteins have been identified from Hydra magnipapillata (H. magnipapillata), the freshwater Cnidarian belonging to class Hydrozoa, and their involvement in bacterial recognition has been described [154]. Transcription factor nuclear factor kappa light chain enhancer of activated B cells (NF-κB) has been reported to play a role in Toll pathways in cnidarians [137]. Activation of NF-κB modifies the target gene expression, taking into consideration innate immune factors including antimicrobial peptides (AMPs) and cytokines [155]. The upregulation of NF-κB in exposure to flagellin, as reported in Hydra, displays the function of NF-κB as a crucial immunity transcription factor [156].

2.7.7 GTPase immune-related protein mitogen-activated protein kinases (MAPK)/ extracellular signal-regulated kinase (ERK) pathway As a constituent of TLR pathways, the multileveled kinase signaling pathway comprising MAPK/ERK (extracellular signal-regulated kinases) has been reported in cnidarians [135, 137]. Till now, MAPK signaling involvement and functional perspectives have been studied to play a role in wound healing [137, 157]. GTPases comprise hydrolase enzymes, which play a role in signaling and gene expression [137]. Of late, various gene families of GIMAPs including a smaller family of GTPases have been elucidated in the transcriptome of the coral Acropora millepora (A. millepora) belonging to scleractinian corals under class Anthozoa, which undergoes upregulation when treated with viral and bacterial PAMPs [158].

2.7.8 Prophenoloxidase (PPO) signaling pathways As a crucial unit of invertebrate immunity, melanin synthesis is majorly involved in the process of pathogen encapsulation and healing of wound and tissue repair

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[137]. This cascade, being launched by different PRRs, involving lectins and TLRs, thereafter triggers various proteolytic reactions ultimately resulting in the synthesis of melanin [137]. PPO enzymes, after activation, are cleaved into phenoloxidases (PO) by trypsin-like serine proteases [137]. Biochemical and/or histological analyses conducted on many cnidarians such as hydrozoans, anthozoans like sea anemones and gorgonians, as well as alcyonacean and scleractinian corals have revealed PPO/PO activity and melanin deposits [159–162].

2.7.9 Complement pathway The lectin pathway, extensively identified among cnidarians [137, 163], plays a major role in immune defenses. Various mediators of the lectin complement pathway have been recognized in cnidarians, including complement component C3, mannan-binding lectin-associated serine protease-1 , and membrane-attack complex/perforin (MACPF) or pore-forming proteins [137]. The transcriptome studies of N. vectensis and two scleractinian corals, A. millepora and Orbicella faveolata (O. faveolata), have revealed mannose-binding proteins [137, 164, 165]. Homologous genes for the C3 complex have been reported in the transcriptomes of various Cnidarians [137], such as the anemones N. vectensis [166] and Aptasia pallida (A. pallida) [167], as well as the octocorals G. ventalina and Swiftia exserta [168, 169] and also the scleractinian corals, namely, A. millepora [135] and O. faveolata [170]. Toxic nematocyst venoms (TX-60A), components of the MACPF protein family, reported from the anemones Actineria villosa and Phyllodiscus semoni, lead to pore formation in target cell membranes and take part in feeding and antipredator defense mechanisms [171, 172] but are also potent enough in destroying probable pathogens [137].

2.7.10 Effector responses Quite some heterogeneity has been reported concerning effector responses in cnidarians [137] as stated below. 2.7.10.1 Surface mucus layer Surface mucus layer (SML), primarily a dynamic complex involving polysaccharide, protein and lipid embracing the animal’s external surface is considered as the primary line of defense against invading pathogens in members of Class Anthozoa and Hydrozoa [reviewed by 173, 174].

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Interestingly, the potentiality of Cnidarians in controlling the expression and synthesis of SML and the infecting bacteria may serve as a vital unit of immunity [138]. Antibacterial compounds generated by the host, and its concerned microbial association, are released in the mucus and have key roles in regulating the mucusassociated microbial community [73, 76 [174–178],]. 2.7.10.2 Antimicrobial activity AMPs play a major role in vertebrate and invertebrate innate immune system. Diverse forms of AMPs have been identified in phylum Cnidaria [137, 179]. Damicornin was the AMP reported for the first time from a scleractinian coral [180], structurally comparable to the jellyfish AMP aurelin which displays antibacterial potential against both Gram-positive and Gram-negative bacteria [181]. With respect to hydrozoans, chiefly three families of AMPs have been reported in Hydra by various genome-based studies and biochemical-based methods [182–184].

2.7.11 Cellular responses in cnidarians Preserving an intact epithelium layer is crucial in providing defense from pathogens and other stressors [137]. In cnidarians, cellular responses primarily comprise the phagocytic cells, which have the ability to engulf and lyse pathogens, external agents or foreign cells, by antimicrobial mediators or reactive oxygen species (ROS) generated by phagocytes. They can also encapsulate foreign cells and remove them from the body [159].

2.7.12 Mobile cytotoxic cells: ROS and reactive nitrogen species (RNS) Compared to the members of class Hydrozoa, members of class Anthozoa possesses circulating amebocytes that have the ability to reach injury sites and attack and phagocytose foreign cells, thereby triggering exposure to free radicals and proteolytic enzymes [137, 173]. Moreover, amebocytes have been reported to have key roles in generating cellular immune response to disease in gorgonians [137], as seen in the sea fan G. ventalina where an inflammatory response is distinguished by upregulation in granular acidophilic amebocytes [137]. A key feature of mobile cytotoxic cells, characterized by amebocytes or phagocytic cells, is the discharge of ROS and RNS (reactive nitrogen species), enabling killing and removal of pathogen [137]. Hydrogen peroxide has been identified as key players of ROS, leading to oxidative burst, in gorgonian corals [185]. With respect to RNS, nitric oxide synthase is rather predominant in cnidarians, such as the sea anemone A. pallida, A. millepora, and the soft coral Lobophytum pauciflorum [137, 186].

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31

2.7.13 Encapsulation Quite an interesting cellular response mechanism has been reported in scleractinians and gorgonians, characterized by encapsulation of microbes by expanding deposition of organic material and skeleton and biochemically proteinaceous in nature, including collagen or gorgonin, in order to construct a nodule embracing the foreign organism [173, 185]. Melanin is also reported in the encapsulation mechanism, chiefly studied in G. ventalina, where it occurs as a band along the axial skeleton in adjoining regions of fungal hyphae, and helps not only in strengthening the skeleton but also in encapsulating the fungal hyphae, ensuring that they are incapable of penetrating the surrounding tissue [173, 187, 188].

2.8 Repair mechanisms In cnidarians, rather intricate and dynamic repair mechanisms have been reported [189], involving various cellular components and events [190], among which the best categorized are antioxidants, apoptosis, and wound healing [137].

2.9 Antioxidants Oxidative stress (OS) is a consequence of disparity between ROS production and antioxidant defenses [191–194], and in order to restrain ROS levels and prevent OS, diverse enzymatic and nonenzymatic antioxidants are utilized by the cell [191, 195–197]. Antioxidant genes, including catalase genes, and superoxide dismutase (SOD) including CuZn-SOD and Mn-SOD were reported from the transcriptomes of the corals A. digitifera [142], Acropora palmate [198], and O. faveolata [199]. Antioxidant genes have also been identified from G. ventalina in response to infection by unicellular parasite Aplanochytrium [137, 169]. In Hydra vulgaris (H. vulgaris), genes for catalase have been reported [200].

2.10 Apoptosis Multiple genes concerned with the process of apoptosis, involving both pro- and anti-apoptotic regulators were distinctively affected in white band disease-infected Acropora cervicornis [137]. While caspase 3 (CASP-3) and tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) genes were upregulated, caspase 8 (CASP-8) was downregulated in corals infected with white band disease [201].

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2.11 Wound healing Most cnidarians, being sessile in nature, face continuous exposure to damage by both biotic and abiotic stress factors; hence, wound healing is utmost crucial to them [190]. The process of healing of wound involves various phases, including inflammation, plug formation, tissue proliferation, and maturation [202–206]. In-depth mechanisms of the wound healing process in cnidarians are now being deciphered with the help of genomic analyses [190, 207]. In N. vectensis, the cells surrounding a site of injury are observed to congregate, which subsequently provokes the gene expression related to wound healing and tissue regeneration [137]. The MAPK/ERK and Wnt signaling pathways are reported to be associated with this mechanism [207]. Wnt3 signaling is presumably the chief tissue regeneration mechanism in H. magnipapillata [2, 208]. Amebocytes have crucial roles in the wound healing process by clustering together near the site of injury and commencing the generation of connective mesogleal fibers for sealing the wound [160, 209]. Such mechanisms were analyzed in depth by higher resolution studies in Porites cylindrical and A. millepora [137].

2.12 Overlap between immunity and symbiosis: anthozoan and symbiotic dinoflagellates Many Cnidarians, generally anthozoans including sea anemones and corals, are seen to establish mutualistic associations with dinoflagellate symbionts which contributes the nutritional requirements for the host in return for defense [210]. Owing to the fact that the symbiont is intracellular, host immunity is a part of both the establishment and the breakdown of the symbiosis [211]. The term “holobiont” is assigned to denote the entire set of organisms, which includes the cnidarian host, algal symbionts, and microbial associates [73]. Despite the fact that the host is presumed to mainly contribute to cnidarian immune responses, there are escalating evidences suggesting the crucial role played by symbiont algal and microbial [137]. As a matter of fact, the very well-being of the cnidarian holobiont depends on the balance involved in symbioses, and upsetting such an organization can result in coral death [137]. The term “bleaching” refers to the disorganization of the symbiotic relationship between the cnidarian host and the dinoflagellate symbiont [137]. Bleaching usually takes place when the holobiont is in stressful conditions, owing to high temperature, excess ultraviolet radiation, or pathogens [210]. Moreover, changes in the communities of microbes of corals due to abiotic stress factors like dissolved organic carbon, temperature, pH, and nutrients can result in proliferation of disease causing pathogens [137]. Interestingly, several of the cnidarian host responses to the process of bleaching and altering communities of microbes bear the potential to provoke immune responses [137].

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2.13 Nanoparticles and Cnidaria 2.13.1 Cnidarians in nanowaste decontamination In spite of the numerous promising socioeconomic impacts of nanomaterials globally, still, discharge of such materials in the natural environment raises crucial issues as these are potentially destructive to both human health and the ecosystem [212–214]. In this context, a novel approach had been undertaken by Patwa et al. [212] in developing a notion for a probable role of glycoprotein and/or glycans in the mucus in entrapping minute particles, namely nanoparticles (NPs), and the decontamination of aquatic suspensions, especially those contaminated with nanowaste [212]. Glycoproteins, important part of mucins, play a key role in entrapping NPs by interactions of electric charges [212]. It is a well-known fact that mucus, a colloidal material which develops from the cells in the epidermis and gastrodermis, is produced in copious amounts in jellyfishes [212, 215]. Nonetheless, meager knowledge is available regarding its biochemical nature [212]. Investigations on the biochemical composition of mucus of Aurelia aurita (A. aurita) showed the presence of carbohydrates, lipids, and proteins which is quite comparable to the mucus composition from other members of phylum Cnidaria [216]. Regarding the biochemical composition of mucus discharged by Pelagia noctiluca (P. noctiluca), investigations conducted by Patwa et al. [212] revealed an expression of mucin-type O-linked glycans, high-mannose-type N-linked glycans, and high levels of pentose-containing oligomers. A noteworthy approach taken by Patwa et al. [212] in investigating the jellyfish for its recycling potential or cultured specifically for industrial purpose to decontaminate wastewaters revealed promising outcomes. The study was conducted on moon jellyfish A. aurita, the mauve stinger P. noctiluca and Mnemiopsis leidyi. Patwa et al. [212] successfully executed the removal of NPs from aqueous colloidal suspensions containing nanowastes by utilizing the mucus secreted by jellyfish, at room temperature. The probable implications of the study lie in removing NPs from aqueous samples and create new perspectives on waste management.

2.13.2 Nanoparticle-mediated environmental toxicity and Cnidarians Toxicity assessment is crucial in investigating the potential risk of newly developed NP pollutants in the environments [217]. Such a study recently conducted by Yamindago et al. [217] elucidated zinc oxide NP-mediated toxic effects affecting morphology and regeneration in H. magnipapillata [218], leading to the formation of extraordinary number of bifurcated tipped tentacles, clubbed tentacle, formation of slender body, and retracting tentacles and body column [217]. Silicon is present in ample amounts in the Earth’s crust and biota [219]. Amorphous silica of micrometer size has applications

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in oral and cosmetics, food, dermatological formulations, and medicines; thus SiO2 NPs reveal relatively low toxicity and biocompatibility [219] but a dose-related toxicity was reported by SiO2 NPs [220]. When administered on cnidarian H. vulgaris, they will affect their morphology and behavior [219], resulting in gastric region paralysis, disorganization and depletion of tentacle specialized cells, increased apoptotic and collapsed cells, reduced epithelial cell proliferation rate, increased stress response, cuticle renovation, and affected gene expression [219]. Exposure of copper oxide nanorod to H. magnipapillata revealed toxic effects at cellular, organismal, and molecular levels; morphological disabilities in feeding; affected population growth; regenerative capacities; OS; genotoxicity; increased apoptotic cell death; and affected cell cycle progression [221].

2.13.3 Nanoparticles in augmenting our understanding of the anatomy and physiology of cnidarians Freshwater cnidarian H. vulgaris has found importance as a potent model organism for determination and study of environmental toxicity [222–224]. Living polyps on exposure to quantum rods (QRs) caused neuronal activation [223, 224]. Utilizing H. vulgaris revealed toxic effects when exposed to amino-PEG-coated CdSe/CdS core/shell QRs and affected annexin XII, family of proteins [225–227]. Transport of NPs through coral tissues [228] has been less studied. Several types and different sized NPs, including dextrans, latex beads, gold NPs, were studied in the coral Stylophora pistillata (S. pistillata) [229], where NP movement could be tracked from the surrounding seawater to the different tissue layers of coelenterons, passage through paracellular pathway followed by entry into cell cytoplasm, by means of macropinocytosis being a major endocytic pathway in sea anemone Anemonia viridis and octocorallian Corallium rubrum. Mucus acted as a mesh prior to particle entry into oral ectoderm [228].

2.13.4 Anti-inflammatory potential and applications Till date, a broad spectrum of natural compounds with potential antiviral, antimicrobial, antimalarial, antitumor, as well as bearing antioxidant, and anti-inflammatory properties had been dispensed from marine organisms [230, 231]. Amidst them, soft corals, inhabitants of coral reefs, in fact, are regarded as biochemical storerooms of crucial secondary metabolites, such as sesquiterpenes [232], diterpenes [233], and sterols [234], being equipped with antiviral, antifouling, antitumor, and anti-inflammatory potentials [235, 236].

References

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Even though, in the last few decades, the green synthesis of NPs have attained quite some attention, still, till date, no such studies have been carried out to assess the anti-inflammatory activity of soft corals [237].

2.14 Discussion Cnidarians comprising mostly marine forms of life reveal primitive body form, but also an innate system that protects the body against pathogens and environmental stress factors. NPs are a major environmental contaminant. Released in the marine environment, they contribute to the toxic environment in the ocean and affect the life, physiology, and morphology of the Cnidarians. Thus, the understanding of the impact of NP contamination on Cnidarian life finds importance as it finds its application as toxicity markers of a contaminated marine environment. Research in this domain of biology is very intriguing and extremely important in conservation of the marine fauna. NPs designed from agents and extracts from Cnidarians with anti-inflammatory potential also have found importance and reveal biomedical applications. Research in this branch of biology is focused on deriving better and effective natural compounds with better and effective biomedical and therapeutic applications.

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[131] Gierer A, Berking S, Bode H, David CN, Flick K, Hansmann G, Schaller H, Trenkner E. Regeneration of hydra from reaggregated cells. Nat New Biol. 1972;239:98–101. [132] Technau U, Cramer von Laue C, Rentzsch F, Luft S, Hobmayer B, Bode HR, Holstein TW. Parameters of self-organization in Hydra aggregates. Proc Natl Acad Sci USA. 2000;97: 12127–31. [133] Technau U, Holstein TW. Cell sorting during the regeneration of Hydra from reaggregated cells. Dev Biol. 1992;151:117–27. [134] Cummings SG, Bode HR. Head regeneration and polarity reversal in Hydra attenuata can occur in the absence of DNA synthesis. Rouxs Arch Dev Biol. 1984;194:79–86. [135] Miller DJ, Hemmrich G, Ball EE, Hayward DC, Khalturin K, Funayama N, Agata K, Bosch TC. The innate immune repertoire in cnidaria – Ancestral complexity and stochastic gene loss. Genome Biol. 2007;8(4):R59. [136] Miller DJ, Ball EE, Forêt S, Satoh N. Coral genomics and transcriptomics – Ushering in a new era in coral biology. J Exp Mar Biol Ecol. 2011;408(1–2):114–19. [137] Mydlarz LD, Fuess L, Mann W, Pinzón JH, Gochfeld DJ. Cnidarian immunity: From genomes to phenomes. In: Goffredo S., Dubinsky Z. (eds) The Cnidaria, Past, Present and Future. Springer International Publishing, Switzerland, 2016, 441–66. [138] Netea MG, Quintin J, van der Meer JW. Trained immunity: A memory for innate host defense. Cell Host Microbe. 2011;9(5):355–61. [139] Rinkevich B. Allorecognition and xenorecognition in reef corals: A decade of interactions. Hydrobiologia. 2004;530:443–50. [140] Schwarz RS, Hodes-Villamar L, Fitzpatrick KA, Fain MG, Hughes AL, Cadavid LF. A gene family of putative immune recognition molecules in the hydroid. Hydractinia Immunogenet. 2007;59 (3):233–46. [141] Schwarz RS, Bosch TC, Cadavid LF. Evolution of polydomlike molecules: Identification and characterization of cnidarian polydom (Cnpolydom) in the basal metazoan. Hydractinia Dev Comp Immunol. 2008;32(10):1192–210. [142] Shinzato C, Shoguchi E, Kawashima T, Hamada M, Hisata K, Tanaka M, Fujie M, Fujiwara M, Koyanagi R, Ikuta T, Fujiyama A, Miller DJ, Satoh N. Using the Acropora digitifera genome to understand coral responses to environmental change. Nature. 2011;476(7360):320–U382. [143] Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406(6797):782–87. [144] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol. 2004;4(7):499–511. [145] Burge CA, Mark Eakin C, Friedman CS, Froelich B, Hershberger PK, Hofmann EE, Petes LE, Prager KC, Weil E, Willis BL, Ford SE, Harvell CD. Climate change influences on marine infectious diseases: Implications for management and society. Annu Rev Mar Sci. 2014;6: 249–77. [146] Schwarz RS, Hodes-Villamar L, Fitzpatrick KA, Fain MG, Hughes AL, Cadavid LF. A gene family of putative immune recognition molecules in the hydroid. Hydractinia Immunogenet. 2007;59 (3):233–46. [147] Wood-Charlson EM, Weis VM. The diversity of C-type lectins in the genome of a basal metazoan. Nematostella Vectensis Dev Comp Immunol. 2009;33(8):881–89. [148] Hayes ML, Eytan RI, Hellberg ME. High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2). BMC Evol Biol. 2010;10:150. [149] Kvennefors EC, Leggat W, Kerr CC, Ainsworth TD, Hoegh-Guldberg O, Barnes AC. Analysis of evolutionarily conserved innate immune components in coral links immunity and symbiosis. Dev Comp Immunol. 2010;34(11):1219–29. [150] Dunn SR. Immunorecognition and immunoreceptors in the Cnidaria. Invertebr Surviv J. 2009;6(1):7–14.

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3 Caenorhabditis elegans and nanoparticles Shyamasree Ghosh Abstract: Caenorhabditis elegans belongs to phylum Nematoda and is the first multicellular organism known for whole genome sequencing. It is an excellent invertebrate model of study of human diseases, genes, and mechanisms involved in disease pathology and research on therapeutic targets and drug screening studies, including neurodegenerative diseases and diabetes. C. elegans immune system is robust with antiviral and antibacterial immune systems and associated pathways. We discuss in this chapter in brief the C. elegans immune system and the impact of nanoparticles on the C. elegans physiology and immune system. Keywords: C. elegans, bivalve, nanoparticles, immune system Abbreviations C. elegans R. elegans RNAi AD Aβ PD CNS ALS ds RIG-I E. faecalis P. aeruginosa S. typhimurium TLR nsy-1 sek-1 pmk-1 PCD ROS ER UPR ER GPCR AgNPs GO QD

Caenorhabditis elegans Rhabditides elegans RNA interference Alzheimer’s disease beta-Amyloid Parkinson’s disease Central nervous system Amyotrophic lateral sclerosis Double stranded Retinoic acid-inducible gene I Enterococcus faecalis Pseudomonas aeruginosa Salmonella typhimurium Toll-like receptor Neuronal symmetry family member 1 SAPK/ERK kinase 1 p38 MAPK family member 1 Programmed cell death Reactive oxygen species Endoplasmic reticulum Endoplasmic reticulum unfolded protein response g-protein coupled receptor Silver nanoparticles Graphene oxide Quantum dots

Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India https://doi.org/10.1515/9783110655872-003

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3.1 Introduction Caenorhabditis elegans (C. elegans) (Table 3.1, Fig. 3.1) belongs to phylum Nematoda and includes free-living transparent forms of size around 1 mm. They are mostly found in temperate soil environments. The history of its nomenclature dates back to early records of around 1900. Initially named as Rhabditides elegans (R. elegans), it was renamed as Caenorhabditis elegans (C. elegans) as early as in the 1900s by Maupas. In 1952, it was placed under subgenus Caenorhabditis by Osche, while in 1955, it was placed under genus Caenorhabditis by Dougherty. They have an unsegmented body lacking respiratory or circulatory systems, mostly comprising hermaphrodites with a few males having specialized tails for mating with spicules [1]. Since 1960s, neuronal development and, around 1974, studies related to its molecular biology and developmental biology became an important focus of research. Few years later, C. elegans began to find application in studies as model organisms. The genome sequence initially published in 1998 revealed small gaps, which was later complete by 2002. In 2019, the whole genome was sequenced, and this finds importance as it was the first multicellular organism to be sequenced. In the same year, it was also the only organism with a complete connectome also called as neuronal wiring diagram. Tab. 3.1: Taxonomic position of C. elegans. Kingdom

Animalia

Phylum Class Order Family Genus Species

Nematoda Chromadorea Rhabditida Rhabditidae Caenorhabditis C. elegans

1 mm long Head Tail

Fig. 3.1: C. elegans external.

3.2 Phylum Nematoda: a brief study

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3.2 Phylum Nematoda: a brief study Phylum Nematoda includes more than 12,000 described species, which are multicellular organisms. They include free living forms found in sea, soil, and freshwater habitats. They exist over a wide range from the polar region, desserts, high mountains, and oceanic depths. Nonparasitic, benthic forms are reported to exist in the algal mats, soil, and aquatic sediments. They are also reported from hot springs, surviving in temperatures as high as 53 °C [2]. The body is slender, elongated with gradual tapering ends in most species, and is in cylindrical form. Cuticle covers the body, pharynx, hindgut, and other openings of the body. Cuticle comprises three layers, composed of collagen, and includes a thin epicuticle, exhibiting quinine tanning, which covers the outer cuticular cortex comprising annules and sometimes comprising outer and inner parts. The outer layer is annulated or ringed and may comprise outer and inner parts. The median layer reveals uniformly granular structure of struts, skeletal rods, canals, and fibers, which varies with the species. The basal layer may contain fibers in crossed and helical arrangement, and may be striated or laminated [2]. Mouth is located in the anterior end, surrounded by lips and different types of sensilla. In some marine forms, the six liplike lobes, tree on each side, border the mouth. However, sometimes due to fusion, only three lips are observed in terrestrial and parasitic species. In the primitive forms, lips are observed to bear 18 sensilla and carry different cuticular projections. Members of the marine family Stilbonematidae have blue green alga on its body and reveal hairy appearance [2, 3]. In the free living marine forms, a caudal gland opens at the posterior end of the body which may be drawn out to a tail-like structure. Growth in nematodes involves four cuticular molts. This occurs till the worm becomes adult. During molting at the anterior end, the old cuticle separates from the underlying epidermis, sheds into fragments, and a new cuticle is secreted. In the adult stage, molting does not occur but growth of cuticle occurs. The epidermis lying beneath the cuticle is cellular and is syncytial in some species. The epidermis in its inward expansion forms ridges, four in number called as longitudinal cords located along the mid-ventral, mid-dorsal, and mid-lateral lines of the body. The epidermis secretes the cuticle, stores nutrients, and bears fibers that link the cuticle to muscles. In endoparasitic forms, it forms the surface for the host nutrient absorption. The pseudocoel of the nematodes is small in free living species and large and voluminous in large forms like parasitic Ascaris, and the cavity may extend from musculature to gut wall surrounding the reproductive organs. The fluid in the pseudocoel plays a role as a hydrostat, containing organic metabolites, like hemoglobin. It lacks cells. It may contain phagocytic cell that plays a role in immune defense.

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The nematodes move like the eel, with eel-like body undulations in dorsoventral plane, moving in forward and rearward directions. This is mediated by alternate dorsal and ventral longitudinal muscle contraction. Cuticular annulations enable flexibility, and crossed helical fibers prevent kinking and hibernation when the body flexes with a rise in hydrostatic pressure. Many free living forms are carnivorous that feed on small metazoans and other nematodes. Some species reveal phytophagy. Marine freshwater forms feed on algae, fungi, bacteria, and diatoms. Many terrestrial forms survive on plant juice. Nematodes excrete nitrogenous wastes as ammonium ions which diffuse out of the body wall. Specialized excretory structures of excretory cells, gland cells, and excretory canal system enable excretion, osmoregulation, and ionic regulation gland and canal system and share a pore in common to both. The nervous system is intraepithelial, located within epidermis, hindgut, and pharynx. The brain comprises circumpharyngeal nerve ring. Nerves originate from the brain and extend toward the anterior end and move into the cephalic sensilla and amphids. Dorsal, lateral, and ventral nerves run within the longitudinal cords. Ventral nerve is the largest nerve containing motor neurons. Muscle cells are in contact with dorsal and ventral nerves. The principal sensilla include amphids, papillae, setae, and phasmids.

3.3 C. elegans The nematode C. elegans shares the habitat in soil, fruit, compost, and snails. The worm develops from embryo to adult in 3 days at 20 °C and can survive for 2–3 weeks. Within 3 days, an adult hermaphrodite worm can produce around 300 genetically identical individuals. Its genome has been sequenced. Mutants and knockdown forms can be established easily and therefore they serve as excellent animal models of study in developmental biology, apoptosis, RNA interference (RNAi), regulation of life span, and study of innate immune responses [1–4].

3.4 C. elegans as animal model C. elegans finds importance as a model organism for neurodegenerative diseases like Alzheimer’s disease (AD) with pathological manifestations of beta-amyloid (Aβ) peptide and tau protein depositions due to protein misfoldings. Since Hsp70 and Hsp90 are known to regulate protein folding, studies on its role in Aβ formation and associated toxic effects have been studied in knockdown Hsp90 in C. elegans model [5]. In other neurodegenerative diseases, including AD, Parkinson’s disease (PD), and amyotrophic lateral sclerosis, with pathophysiology of proteinaceous deposits

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in their central nervous system (CNS) with the involvement of prions lead to loss of neuron and are almost incurable, and development and progression of the disease are poorly understood. C. elegans has been used as a model system to study the transmission of prions [6] in neurodegenerative disorders (NDs) [7, 8], role of metals in NDs [9], associated pathophysiology in neuroinflammation [10], and screening of drugs and agents in targeting NDs [11, 12]. Use of transgenic C. elegans is also used in AD research [13]. It also finds its research importance in diabetes mellitus [14]. In AD research, it finds important applications as modes for understanding the role played by genes in AD [15, 16]. It is used as a model system to study mechanisms in PD and to identify therapeutic targets [17]. Model system of C. elegans has been used to study the effect of alcoholism [18]. C. elegans has found a major role as a model system in drug discovery [19].

3.5 Immune system in C. elegans Several human microbial pathogens infect the nematode intestine. The immune system of C. elegans is primitive having ancient signaling networks. Mechanisms involved in stress responses and regulation of life have been studied in C. elegans. Recently, studies are being conducted on the role of nervous system in regulation of innate immune responses.

3.5.1 Antiviral immune defense Intracellular RNAi plays a role in the antiviral immune responses (Fig. 3.2). Viral replication produces double-stranded (ds) RNA intermediates that are sequestered by the RDE-1/4 dsRNA binding complex. RNA molecules are unwind by the DExD box RNA helicase. DRH-1 unwinds and facilitates their acquisition by the Dicer complex and small viral RNAs generated by DCR-1 get amplified by RNA-directed RNA polymerases. This produces antisense viral siRNAs in large numbers. Degradation of the target mRNA is mediated by RNase MUT-7 and Argonaut proteins. RNA + Orsay virus of family Nodaviridae is a natural pathogen for C. elegans that alters the morphology of the worm intestine. Several viruses can evade the immune surveillance by blocking MHC class I antigen processing and presentation, and eventual recognition and killing by cytotoxic T cells. Viral proteins like Flock house virus protein B2 in C. elegans escape the RNAi surveillance by inhibiting the RNAi machinery. C. elegans DRH-1 reveals homology with human RIG-I (retinoic acid-inducible gene I) RNA helicase that can sense mammalian dsRNAs. The DExD/H-box helicase domain that binds RNA bears the maximum homology.

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Viral ss RNA

Transposons

replication

transcription

Viral ds RNA

ds RNA MUT-14

RDE-1, RDE-4 DRH-1 DCR-1

Viral small RNA RNA-directed RNA polymerases (RdRP) Antisense viral siRNA Argonautes/RISC MUT-7 Target RNA degradation

Fig. 3.2: Antiviral response in C. elegans.

3.5.2 Antimicrobial immune responses C. elegans are exposed to microbes, from environment, like soil, water, habitat, and food sources. Some are pathogenic to C. elegans including Gram-positive organisms like Enterococcus faecalis (E. faecalis) and Staphylococcus aureus, and Gram-negative organisms like Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhimurium (S. typhimurium), and Serratia marcescens, causing intestinal infection and leading to deleterious phenotypes. Microbes are pathogenic to the active virulence factors producing worms. P. aeruginosa strain PA14 can kill C. elegans by fast killing mediated by toxins like phenazines or slow-killing-mediated living microbes. Salmonella infection can cause programmed cell death (PCD) involving ced-4 and ced-3, human homologue of Apaf1 and caspase, respectively. Gram-positive Microbacterium nematophilum can colonize the rectum and anal region of C. elegans, while Yersinia pseudotuberculosis can produce biofilms rich in polysaccharides attached to the cuticle of the head inhibiting feeding and growth. The mechanism of pathogen recognition remains yet to be completely understood in C. elegans. They produce a single Toll-like receptor (TLR) homologue TOL-1 but its exact role in microbial defenses remains far from being completely understood. Molecules like NF-κB transcription factors and TLR adaptor MYD88 have not been found.

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p38 MAP kinase-related signaling pathway has been reported to play a role in antimicrobial defense. The cascade is activated on pathogen exposure, activating downstream signaling by cascade components of neuronal symmetry family member 1 (nsy-1), SAPK/ERK kinase 1 (sek-1), followed by p38 MAPK family member 1 (pmk-1) in linear phosphorylation cascade and acts orthologous to the apoptosis signal-regulating kinase 1–MAPK kinase 3/6 (MKK3/6)–p38 MAPK cascade in mammals that trigger innate immune responses. Heavy metal stress can activate MAPK kinase MEK-1 and MAP kinase phosphatase VHP-1 and also reported to regulate phosphorylation events in PMK-1. Microarray studies have revealed a role of MAP kinase in antimicrobial immune defenses (Fig. 3.3). Proteins with C-type lectin domain can bind to specific carbohydrates on the bacterial surface. Proteins with CUB (C1s/C1r complement components) domain and antimicrobial peptides play a role in immune defenses. The transcription factor ATF-7 controls the expression of innate immune genes downstream of PMK-1 and can induce PCD on S. typhimurium infection [20, 21].

Fungi, infections TIR-1

Infection

DBL-1

Lin-45

NSY-1

SMA-6 DAF4

MEK-2

SEK-1

SMA-2 SMA-3 SMA-4

MPK-1

PMK-1

C Type Lectins AMPs

ATF-7 Antimicrobial action

Fig. 3.3: C. elegans antimicrobial response.

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3.5.3 Immunity and stress pathways In C. elegans, immune activation is associated with stress. Generation of reactive oxygen species (ROS) in the intestine has been associated with resistance to E. faecalis infection [22]. Protein refolding cascades have been associated with stress and infection tolerance. The heat stress-inducible chaperone system regulated by transcription factor HSF-1 aids in toleration of variety of C. elegans pathogens [23]. During antimicrobial defense generation, generation of peptides may lead to stress in protein folding in the endoplasmic reticulum (ER). A functional ER unfolded protein response (UPR ER) acts as the key regulator to combat infection, ensuring survival, and defective or absence of UPR ER, the XBP-1 transcription factor, causes reduced life span due to infection by pathogenic P. aeruginosa [20, 23]. The xbp-1 mutant worms reveal the affected PMK-1-mediated induction of immune peptides. The abu (activated in blocked UPR) or noncanonical UPR-mediated pathway is reported to play a role in infection tolerance [20, 24]. CED-1, a phagocytic receptor’s response to infection, is known to regulate abu gene expression and plays a role in engulfing apoptotic bodies. This is revealed by ced-1 loss of function immunocompromised mutants rapidly killed by pathogenic bacteria [25, 26].

3.5.4 Immunity and neuronal control In C. elegans, 302 neurons have been identified to play a role in neuronal connectivity and find application in neuroimmune studies. npr-1, a g-protein coupled receptor (GPCR) gene, has been reported to play a major role in combating infection and enable nematode survival in response to bacterial infection. In response to PA14 infection, an npr-1 polymorphism could cause death by inhibiting oxygen-dependent pathogen avoidance [26]. OCTR-1, a GPCR, is known to act on Amphid neurons (ASH and ASI) sensory suppressing infection-driven PMK-1 activation and induction of noncanonical UPR in peripheral tissues. Mutants with octr-1 deficiency show resistance to infection with P. aeruginosa [27]. OCTR-1 is a vertebrate adrenergic catecholamine receptor with noradrenalin as the ligand that mediates responses to acute stress in mammals and causes immune suppression [28]. A nonconventional innate immune response and synthesis and release of immune peptides against intestinal infection and germline DNA damage have been reported in C. elegans. Germline DNA damage can activate MPK-1/ERK MAP kinase pathway, and innate immune response activates the ubiquitin proteasome system enabling their toleration of heat and oxidative stress while stress resistance involved the activity of PMK-1/p38 kinase pathway [20, 29].

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3.6 Nanoparticles and the immune system in C. elegans C. elegans has been used as an animal model to assess the environmental risk of NPs, affecting their growth, development, intestinal function, immune response, neuronal function, reproduction, oxidative stress, alterations of several signaling pathways, and impacts of metal oxide NPs on the ecosystem [30]. C. elegans, infected with pathogen P. aeruginosa on exposure to chemicals, revealed increased tolerance to silver nanoparticle (AgNP)-mediated toxicity, involving the PMK-1/p38 MAPK pathway in defense [31]. Graphene oxide (GO) exposure caused immunotoxicity and adverse effects on development of worms, expression of innate immune gen, p38 MAP kinase, and PMK-1 conferring protection from chronic toxicity. On the other hand, aminofunctionalized GO exposure has not been reported to cause detrimental effects or activate innate immune response revealing its biocompatibility [32]. CdTe quantum dots (QDs) are fluorescent probes and find application in biomedical research and imaging of disease. CdTe QDs were found to be more neurotoxic as compared to CdTe@ZnS QDs in C. elegans as probably protection conferred by ZnS [33]. Zinc oxide nanoparticles negatively impacted both larvae and adult forms with intestinal accumulation of C. elegans as compared to Zn and decreased survival of C. elegans exposed to P. aeruginosa PA14 infection and suppress the innate immunity of C. elegans [34]. AgNP exposure could induce DNA damage, ROS generation, and induction of p38 MAPK and PMK-1 in C. elegans [35].

3.7 Discussions C. elegans finds importance as an animal model and in research in diverse domains in biology, including developmental biology, neurotoxicity, and parasitological research. There are more questions than answers in the domain of parasitism evolution and resistance associated with host defenses. They find particular importance as animal models in aging, microbiome research model, and environmental risk assessment and detection of the impact of nanotoxicity on the environment. The adverse effects of nanoparticles on C. elegans health, development, life, and immune system have enabled our study on understanding their associated risks in application to human health and is thus an important domain of research.

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[18] [19] [20]

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[21] Pees B, Yang W, Kloock A, Petersen C, Peters L, Fan L. et al., Effector and regulator: Diverse functions of C. elegans C-type lectin-like domain proteins. PLoS Pathog. 2021 17(4): e1009454. [22] McCallum KC, Garsin DA. The role of reactive oxygen species in modulating the Caenorhabditis elegans immune response. PLoS Pathog. 2016 12(11):e1005923. https://doi. org/10.1371/journal.ppat.1005923. [23] Kumsta C, Chang JT, Schmalz J, Hansen M. Hormetic heat stress and HSF-1 induce autophagy to improve survival and proteostasis in C. elegans. Nat Commun, 2017 8, 14337. [24] Richardson CE, Kooistra T, Kim DH. An essential role for XBP-1 in host protection against immune activation in C. elegans. Nature., 2010 Feb 25 463;7284:1092–95. [25] Haskins KA, Russell JF, Gaddis N, Dressman HK, Aballay A. Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity. Dev Cell, 2008 15(1):87–97. [26] Liu Y, Sun J. G protein-coupled receptors mediate neural regulation of innate immune responses in Caenorhabditis elegans. Receptors Clin Investig, 2017 4(1):e1543. [27] Cao X, Kajino-Sakamoto R, Doss A, Aballay A. Distinct roles of sensory neurons in mediating pathogen avoidance and neuropeptide-dependent immune regulation. Cell Rep, 2017 Nov 7 21;6:1442–51. [28] Aballay A. Role of the nervous system in the control of proteostasis during innate immune activation: Insights from C. elegans. PLoS Pathog, 2013 9(8):e1003433. [29] Kim DH, Ewbank JJ. Signaling in the innate immune response. In: WormBook: The Online Review of C. elegans Biology [Internet]. WormBook, Pasadena (CA), 2005–18. [30] Wu T, Xu H, Liang X, Tang M. Caenorhabditis elegans as a complete model organism for biosafety assessments of nanoparticles. Chemosphere, 2019 Apr;221:708–26. [31] Kim Y, Choudhry QN, Chatterjee N, Choi J. Immune and xenobiotic response crosstalk to chemical exposure by PA01 infection in the nematode Caenorhabditis elegans. Chemosphere, 2018 Nov;210:1082–90. [32] Rive C, Reina G, Wagle P, Treossi E, Palermo V, Bianco A, Delogu LG, Rieckher M, Schumacher B. Improved biocompatibility of amino-functionalized graphene oxide in Caenorhabditis elegans. Small, 2019 Nov 15;45:e1902699. [33] Wu T, Liang X, He K, Liu X, Li Y, Wang Y, Kong L, Tang M. The NLRP3-mediated neuroinflammatory responses to CdTe quantum dots and the protection of ZnS shell. Int J Nanomedicine, 2020 May 6;15:3217–33. [34] Li SW, Huang CW, Liao VH. Early-life long-term exposure to ZnO nanoparticles suppresses innate immunity regulated by SKN-1/Nrf and the p38 MAPK signaling pathway in Caenorhabditis elegans. Environ Pollut, 2020 Jan;256:113382. [35] Chatterjee N, Eom HJ, Choi J. Effects of silver nanoparticles on oxidative DNA damage-repair as a function of p38 MAPK status: A comparative approach using human Jurkat T cells and the nematode Caenorhabditis elegans. Environ Mol Mutagen, 2014 Mar 55;2:122–33.

4 Insects and nanoparticles Rashmi Bhattacherje, Shyamasree Ghosh, Dhriti Banerjee Abstract: The insects have been studied for having a robust innate immune system and recently reported for properties of specificity and memory-like response. Different pathways that contribute to the insect immunity include TOLL, immune deficiency, mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 kinase pathways. Other pathways including autophagy and RNA interference pathways have been implicated in antiviral immune responses. Furthermore, different types of antimicrobial peptides also reported in insects that function in diverse ways to combat pathogenic infections. Beneficial insects are directly or indirectly involved in providing various ecosystem services, whereas harmful groups of insects are responsible for crop and stored product damage as well as transmit several types of pathogenic organisms. The induction of cellular and humoral immune responses in insects against diverse array of microbes has been studied with deeper insights with the assistance of various types of nanoparticles. Nanoparticles for insect control and against insect vectors have shown promising results and can be bioengineered in different ways for getting beneficial outputs; thus, they are worth exploring. Keywords: Insects, Arthropoda, nanoparticles, apoptosis Abbreviations ZnO NP CPMV VLNPs eCPMV B mori RSV Sf9

Zinc oxide Nanoparticles Cowpea mosaic virus Virus-like nanoparticles Empty CPMV Bombyx mori Respiratory syncytial virus Spodoptera frugiperda

CuO

Copper oxide

Dhriti Banerjee, Diptera Section, Zoological Survey of India, Ministry of Environment, Forest and Climate Change, M-Block, New Alipore, Kolkata 700053, India Rashmi Bhattacherje, Diptera Section, Zoological Survey of India, Ministry of Environment, Forest and Climate Change, M-Block, New Alipore, Kolkata 700053, India; Departmentof Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal,India Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India https://doi.org/10.1515/9783110655872-004

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SOD CAT GSH-PX SiNPs Drosophila melanogaster

Superoxide dismutase Catalase Glutathione peroxidase Silicon nanoparticles D. melanogaster

4.1 Introduction Arthropods represent up to 80% of animal phylum [1] and make up for the largest proportion of species richness at any spatial scale [2]. Insects are the most diverse groups of animals on the Earth with more than 1,000,000 species described and approximately 5,000,000 extant species [3, 4]. They perform major roles as herbivores, pollinators, seed dispersers, predators, detritivores, and vectors, thereby bestowing to the fundamental biological basis of terrestrial ecosystems [5, 6]. The insects being the amazingly diverse and ancient group of arthropods have grappled most of the challenging environmental conditions ranging from the Arctic tundra to Alpine mountain peaks in addition to warmer tropical rainforest and coastal mangrove as well as extreme environmental temperatures [7]. The success of insect evolution lays in their ability of using the wings to fly and invade various types of habitats. The study of insects is known as entomology. Insects provide a diverse array of ecosystem services and are essential for several types of ecosystem functions such as nutrient recycling and soil turnover, via leaf litter and degradation of wood, dispersal of fungi and plant propagation by pollination and seed dispersal, disposal and decomposition of carrion and droppings. They also aid in the maintenance of the composition and structure of plant community, via phytophagy and animal community structure by transmitting diseases, parasitism, and predation; insects are food for a wide range of insectivorous animals, such as birds, mammals, fish, and reptiles [8]. The appearance of first insect probably occurred before the Devonian period (400–360 million years ago), and by the Carboniferous period (360–285 million years ago) they have adapted flight and by the Permian period (285–245 million years ago) insects attained their utmost diversity to shape earlier ecosystems on the Earth. To date, the most primitively discovered fossil evidence for insects is 400 million years old, though recent research approaches using molecular genetic techniques to substantiate reviews that they evolved much earlier (https://www.nhm.ac.uk/dis cover/news/2014/november/intricacies-insect-evolution-revealed.html). The major characteristic feature of insects includes a hard, jointed exoskeleton. The exoskeleton is lined by the cuticle which is continuous throughout the outer length of the body and entails of a series of hard plates known as sclerites. These sclerites are further hinged together by cuticular flexible membranes and at times are articulated together for the execution of specific movement of one on the following. The dorsal side of each segment of the body is lined by a dorsal sclerite called

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tergum, joined to a ventral sclerite, the sternum, by lateral membranous areas, the pleura. From the sternopleural region, a jointed appendage arises on each side [9]. The class Insecta is primarily classified into five subclasses: Apterygota, Palaeoptera, Polyneoptera, Paraneoptera, and Endopterygota (Fig. 4.1). These subclasses are further divided into about 30 orders.

4.1.1 Apterygotan assemblage The subclass Apterygota includes two orders of wingless insects: Archaeognatha and Zygentoma. Both of the orders share remarkable morphological similarities but the presence of dicondylous mandibles sharply differentiates Zygentoma from Archaeognatha (https://www.royensoc.co.uk/entomology/orders/silverfish-andfirebrats).

4.1.2 Palaeopteran assemblage Order Ephemeroptera includes most primitive winged insects, which are generally recognized by their triangular fore wings held vertically above the body at rest (https://www.royensoc.co.uk/entomology/orders/mayflies-or-upwing-flies). Mayflies are unique in having a subimago stage in their metamorphic cycle which is an active and mobile stage occurring between the ultimate larval instar and the mature adult stage (imago), when present in the life cycle [10]. Both the subimagoes and imagoes are extremely short-lived; thus, they are capable of providing only few ecosystem services [11]. The imagos, in certain cases, subimagos are specialized for dispersal and reproduction with their nonfunctional mouthparts and digestive systems. There are two pairs of wings, with the hind wings being smaller than the forewings in altate individuals [11]. Odonata include dragonflies and damselflies with a tractable record of about 6,000 described species [12]. The wings of dragonflies are larger and their suspended wings held out to the sides of the body, whereas damselflies are slender bodied, and the wings are held over the abdomen during resting [13]. Their ancient phylogenetic rank, widespread phenotypic and ecological diversity, and effectiveness as bioindicators for freshwater ecosystems are remarkable on a global scenario [12].

4.1.3 Paraneopteran assemblage Hemiptera is the most diversified group of non-endopterygote insects [14] belonging to a monophyletic group [15, 16] that includes approximately 82,000 described species [17]. In hemiptera, the mandibles and maxillary laciniae are modified into

Family : Pyrgomorphidae Order : Orthoptera Genus : Aularches Sc. name : Aularches miliaris Common name : Ghost grasshopper/ Northern Soptted grasshopper POLYNEOPTERAN ASSEMBLAGE

APTERYGOTAN ASSEMBLAGE

Family : Entomobryidae Order : Entomobryomorpha Genus : Lepidocyrtus (Cinctocyrtus) Sc. name : Lepidocyrtus (Cinctocyrtus) satkosiaensis

Family : Nymphalidae Order : Lepidoptera Genus : Acraea Sc. name : Acraea terpsicore Common name : Tawny coster ENDOPTERYGOTAN ASSEMBLAGE

Family : Libellulidae Order : Odonata Genus : Orthetrum Sc. name : Orthetrum sabina Common name : Green March Hawk PALAEOPTERAN ASSEMBLAGE

Family : Calliphoridae Order : Diptera Genus : Chrysomya Sc. name : Chrysomya megacephala Common name : Blow fly ENDOPTERYGOTAN ASSEMBLAGE

Family : Scutelleridae Order : Hemiptera Genus : Chrysocoris Sc. Name : Chrysocoris stollii Common name : Green Jewel Bug PARANEOPTERAN ASSEMBLAGE

64 4 Insects and nanoparticles

Fig. 4.1: Insects of subclasses – Apterygota, Palaeoptera, Polyneoptera, Paraneoptera, and Endopterygota. Image reproduced with permission from Director, Zoological Survey of India, Kolkata 700053, India.

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concentric stylets, the mandibular stylets enfolding the maxillary ones in such a way that both are engaged in the formation of the food and salivary channels; the multisegmented labium covering the mandibular and maxillary stylets is sheet like in appearance; and there is no maxillary and labial palpi [14, 18–22]. Lice (Insecta: Phthiraptera) are obligate, permanent ectoparasites of birds and mammals, entirely spend all the stages of their life depending on their vertebrate host [23] and feed upon the skin (feathers, fur, and dander) and often the blood of their hosts [24]. Four suborders of Phthiraptera include the chewing louse suborders Amblycera, Ischnocera, and Rhynchophthirina, and the sucking louse suborder Anoplura [25]. Morphologically, they are wingless, dorsoventrally (DV) flattened, and their adaptive tibiotarsal claws aid them to cling to host hair, whereas feeding is assisted by modified piercing mouthparts [25]. Being highly evolved hematophagous group, these ectoparasitic insects possess highly derived mouthparts to feed directly from host blood vessels [26]. Psocoptera represents paraphylectic assemblages of (psocids, booklice, or barklice) nonparasitic members of the order Psocodea [25, 27–29], comprising roughly 5,500 described species [30]. Morphologically, they are about 1–10 mm in length and possess well-developed postclypeus, long antennae, pick-like lacinia, reduced prothorax, well-developed pterothorax, and so on [31]. Phylogenetically speaking, Psocoptera constitute a monophyletic group member of the order Psocodea with parasitic lice Phtiraptera (biting lice and sucking lice) [25, 27–29] and is also related to Thysanoptera (thrips) and Hemiptera (bugs, cicadas, etc.) [32, 33]. The characteristic apomorphic feature of the thrips (Thysanoptera) includes the asymmetric structure of their piercing–sucking mouthparts which have no right mandible [34]. Both the adult and larval morph of the suborders Terebrantia and Tubulifera include one unique structural feature. Among the two mandibles only the left one is completely developed, whereas the right mandible is resorbed during the embryonic stage. The sharp jaws of the left mandible are used for penetrating tissues [34]. The salivary fluids run down through the feeding tunnel formed by the maxillary stylets and are used for sucking food [34].

4.1.4 Polyneopteran assemblage The Polyneoptera includes orders Plecoptera, Mantodea, Blattodea, Isoptera, Grylloblattodea, Mantophasmatodea, Orthoptera, Phasmatodea, Embiidina, Dermaptera, and Zoraptera, which might form a monophyletic group based on the unique shared attribute of tarsal plantulae (except Zoraptera) and molecular taxonomic analysis of specific nucleotide sequences [35].

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4.1.5 Endopterygotan assemblage Endopterygota represents higher orders of insects with holometabolous development in which immature (larval) morphs are conspicuously different from that of their respective adults, and the adult wings and genitalia are suppressed in their pre-adult appearance, evolving into evaginated imaginal disks at the penultimate molt [35]. There are three possible ancestral relationships between different orders of endopterygota which are as follows [35]. Sister group association is called Amphiesmenoptera between Trichoptera (caddisflies) and Lepidoptera (butterflies and moths). Sister group connection is called Neuropterida between Neuroptera, Megaloptera, and Raphidioptera. Another strong sister group relationship termed Antliophora connects Diptera (true flies), Siphonaptera (fleas), and Mecoptera (scorpionflies and hanging flies).

4.2 Contribution of insects to ecosystem functioning Nonetheless, despite insects are involved in shaping most of the terrestrial ecosystems on the Earth, their significance in nutrient cycling has been neglected since long time which might be for the reason that the total biomass of insects (the standing crop) seems to be trivialin comparison to plant/animal biomass [36]. There are few evidences of insects having a large role on ecosystem functioning including the outbreaks of specific species such as the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae) or Epirrata autumnata (Bkh.) (Lep., Geometridae). In such instances, a huge proportion of leaf area is removed by the exceptionally high inhabitants outbreaking herbivore species densities which can reach up to 100% and show rapid and immense effects on nutrient fluxes [37–40]. For this kind of outbreak situations, herbivorous insects are sometimes called as cybernetic regulators for ecosystem [41, 42], which in turn maintains ecosystem functioning on spatiotemporal scale within sustainable ranges [42]. Revolutionary mesocosm studies including mycorrhizal fungi [43], freshwater insects [44, 45], terrestrial insects [46, 47], and soil fauna [48] have opened a door to connect to biodiversity–ecosystem functioning research [36]. Furthermore, insect interacts both directly and indirectly with the primary producers in various ways, thus affecting nutrient recycling, and herbivory and mutualism are the major types of interactions that regulate ecosystem functioning, specifically pollination, seed dispersal, and plant protection [36].

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4.3 Insect immunity The immune response in insects includes innate or adaptive immune responses (AIRs). The innate immune response (IIR) is conserved in organisms including insects revealing broadness, nonspecificity, and robustness. Studies on immune priming in insects involving insects exposed to dead or a sublethal dose of microbes lead to immune response, and subsequent infections in primed insects revealed that the innate immune system has properties of AIR of memory-like feature and capable of generation of a stronger response to the pathogen. The pattern recognition receptors (PRRs) of the IIR are encoded in the germline and they play a role in distinguishing different microbial pathogens broadly and trigger responses to remove the infection [49], while those of the AIR are not encoded in the germline. Somatic recombination in B and T cells, initiated by recombination-activating genes, enables the formation of adaptive immune system [37]. Tobacco hornworm Manduca sexta (M. sexta) caterpillar infected by Escherichia coli (E. coli) nonpathogenic strain revealed upregulation of microbial PRR and antimicrobial peptides (AMPs) [50], and similar responses are observed when challenged with Photorhabdus luminescens (P. luminescens) an insect pathogen, wherein the immune primed insect has better chances of survival, indicative of memory-like response (MLR) in insects. Both specificity and MLR have been shown in insects. A study in Drosophila melanogaster (D. melanogaster) primed with Streptococcus pneumoniae (S. pneumoniae) at sublethal dose and heat-killed bacteria has been reported to show specificity and long-term immune response [51].

4.3.1 Pathogen recognition Immune system of insects reveals activated pathogen recognition mechanism when exposed to pathogens. The carbohydrate binding moieties of insect hemolymph proteins are called hemagglutinins or lectins as they agglutinate mammalian erythrocytes [52]. The surfaces of most of the bacteria and fungi contain carbohydrate moieties including β-1,3-glucans in the walls of many fungi, and lipopolysaccharides (LPS) or peptidoglycans in the bacterial cell walls, which in turn induces phagocytosis or triggering of the prophenoloxidase (proPO) cascade to execute opsonization [52]. In Bombyx sp., the interaction of a specific protein that resides in the granules of granulocytes and in the hemolymph with β-1,3-glucanplasmatocytes is attracted to the site of pathogen invasion [52, 53]. In the larvae of the greater wax moth, Galleria sp., the occurrence of granulocytes significantly boosted the phagocytic efficacy of plasmatocytes [54], which in turn produces a factor that rouses phagocytic bustle in other plasmatocytes [52, 55].

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4.3.1.1 TOLL and IMD signaling in Drosophila Drosophila has evolved complex TOLL pathways including Toll receptor and Toll signaling pathway and evolutionarily conserved signaling immune deficiency (IMD) pathways (Fig. 4.2) activating NF-κB (nuclear factor kappa light chain enhancer of activated B cells) that play a role to combat microbial infection. The TOLL pathway detects microbes including fungi, Gram-positive bacteria, ad virulence factors and can cause degradation of maternal effect gene coding for cytoplasmic protein Cactus and member of rel transcription factor family or Dorsal and cause localization of Dif in the nucleus. Cactus and Dorsal play a role in maintaining the DV polarity in fly. On binding of cleaved Spätzle to Toll receptor, the Toll signaling is activated, leading to dimerization of intracytoplasmic TIR domains, which in turn induces binding of the adaptor protein MyD88 leading to recruitment of the Pelle, a protein kinase which triggers events of autophosphorylation, and degradation of IκB inhibitor, cactus, and translocation of the NF-κB, Dif, and dorsal. Receptors including peptidoglycan recognition protein (PGRP)-LC and PGRP-LE on binding to cell wall meso-diaminopimelic acid-type peptidoglycan of Gram-negative bacteria trigger the Imd pathway. It controls the expression of the Drosophila AMPs. It can identify Gram-negative bacteria, confer immunity, and cause activation of Relish. After binding to peptidoglycan, receptors undergo dimerization, followed by recruitment of adaptor protein Imd, which in turn recruits dFADD (Drosophila FADD) and the DREDD caspase and then cleaves Imd, which undergoes activation by ubiquitination of K63 and inhibitor of apoptosis 2 (IAP2), thereby recruiting and activating TAK1 by TAK1-associated binding protein 2 (TAB2). TAK1 then activates IKK complex releasing Relish. Dorsal, Dif, and Relish play a role to upregulate AMPs in nucleus ([56], https://www.genome.jp/dbget-bin/www_bget?pathway+dme04624). 4.3.1.2 Other pathways Other pathways that play a role in apoptosis, mitosis, metabolism and motility in Drosophila include mitogen-activated protein kinase (Fig. 4.3) signaling pathway, cJun N-terminal kinase (JNK), extracellular signal-regulated kinase pathway, and p38 kinase pathway. Environmental stress factors can activate JNK and p38 pathways ([56], https://www.genome.jp/dbget-bin/www_bget?pathway+dme04013). The autophagy pathway has been reported to be activated in response to infection by virus. RNA interference (RNAi) pathway has also been implicated in viral infections. RNAi pathway On viral infection, the RNAi pathway gets activated. Viral double-stranded (ds) RNA genome is detected by R2D2 and Dicer-2, RNase III endo ribonuclease which cleaves the dsRNA into small fragments of 21 nucleotides followed by duplex unwinding. The guide strand is then attached to the RNA-induced silencing complex. This includes the RNase Argonaute that degrades the target viral RNA paired with the guide strand.

Fig. 4.2: Toll and Imd signaling pathway – D. melanogaster (fruit fly) dme04624. Image produced with permission from KEGG pathway database.

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Fig. 4.3: Mitogen-activated protein kinase signaling pathway – fly – D. melanogaster. https:// www.genome.jp/dbget-bin/www_bget?pathway+dme04013 Image produced with permission from KEGG pathway database.

JAK‐STAT pathway JAK‐STAT pathway plays a major role in mediating antiviral responses, thereby preventing infection functioning like the interferon pathway in mammals. They are also activated in response to bacterial infection triggering synthesis of AMP downstream effector molecules. In fruit flies, infections by Drosophila C virus and Sindbis virus lead to upregulated synthesis of Vago, with a single von Willebrand factor type C motif. In Culex quinquefasciatus , infections by West Nile virus has been reported to upregulate synthesis of Vago which in turn is dependent on Dicer‐2 leading to the activation of JAK‐STAT pathway. However, this pathway is not completely understood.

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The Autophagy pathway Autophagy pathway induced in response to stress plays a role in eliciting antiviral mechanism in insects independent of Imd, JAK‐STAT, and Toll pathways. Autophagosomes formed inside cells fuse with lysosomes degrading the content, which aids in nutrient recycling and regulates homeostasis. It involves the activation of the phosphoinositide 3‐kinase (PI3K)‐Akt pathway, which upregulates TOR, a negative regulator of autophagy.

4.3.2 Immune responses Normally in the larvae and adults of hemimetabolous insects, wound causes a drastic degeneration of hemocyte count, which shifts to its normal level within a very short period of time but the entry of a pathogen during this crucial time may longer the span of normal recovery of the hemocyte count [52]. In the larva of Galleria, Bacillus cereus infection causes heavy destruction of plasmatocytes from circulation, whereas the count of other types of hemocytes remains unaltered [52, 57, 58]. Furthermore, the reduction in the number of plasmatocytes may lure more pathogenic organisms including bacterial cells to bind hemocytes associated with nodulation or capsule formation [52, 59]. For example, in grasshoppers, infection by the fungus, Beauveria, causes increased probability of hemocytes to bind pathogen’s lectin, and mortality rate of hemocytes increases by toxins secreted by the pathogenic invaders [52].

4.3.3 Humoral response In insects, humoral response is evoked by a damage to epidermis, even without the involvement of pathogenic infection which in turn upregulates the amounts of hemolymph proteins, but the immune response is greatly boosted by the peptidoglycans of the bacterial cell wall, no matter whether the bacteria is alive or dead [52]. It is a member of the immunoglobulin family of proteins that are important in the immune systems of vertebrates. Bacterial infection causes a sharp rise in immunogenic peptidoglycans which in turn leads to an elevation of a small protein in the hemolymph, called hemolin of molecular weight 48 kDa, and recruits about 15 different groups of proteins in Hyalophora and 25 in Manduca larvae including 2 major families of proteins, the cecropins and the attacins. These include two major families of bactericidal proteins known as cecropins (35–37aa moieties, weighing about 4 kDa) and attacins (188aa moieties, weighing about 20 kDa [52]). A suite of equivalent proteins belonging to different protein families are often found in other group of insects [60].

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Furthermore, in the house cricket, Gryllus, an increased level of lysozymes was observed in response to pathogenic invasion, which actually digests the bacterial cell walls to dispose them from the body [52]. Among Diptera, humoral encapsulation occurs without the interference of hemocytes in Chiromonidae and some other groups of lower Diptera in which hemocyte counts are low and have shown efficacy to digest the huge amount of bacteria and fungi and nematode pathogens [49, 61, 62].

4.3.4 Antimicrobial peptides (AMP) Insects exert antimicrobial effect by induction, synthesis of AMPs by hematocytes, and fat body in response to microbial infection and thereafter its release in the hemolymph. A lysozyme from Galleria mellonella (G. mellonella), the honeycomb moth or greater wax moth, was the first AMP reported in insects and is predominantly active against Gram-positive bacteria, and also Gram-negative bacteria and fungi. Insect defensins are predominant against Gram-positive bacteria. Defensins from Lepidoptera reveal antifungal and antibacterial activities. They comprise two groups, one including peptides with both α-helix/β-sheet structures and the other group comprising peptides with triple-stranded antiparallel β-sheet. Cecropins, peptides of 31–37 amino acid length with an amphipathic α-helix structure, can cause damage to the pathogen membranes and were reported from silkworm hemolymph Hyalophora cecropia (H. cecropia), which is the largest moth of North America. Moricins, peptides with amphipathic α-helix structure, were reported first from silkworm B. mori. Moricin reported from G. mellonella has been reported to be active against yeast, fungi, and bacteria. H. cecropia AMPs include attacin, bactericidin, lepidoptericin, and sarcotoxin. Drosocin reported from Drosophila includes a cationic AMP of 19 peptide length with an O-glycosylated threonine with antimicrobial activity. Attacins include an AMP of 20 kDa with acidic and basic forms, and on binding to LPS increases permeability and causes lysis of E. coli. Gloverins and lebocins have been reported from Lepidoptera that play a role in inhibiting bacterial growth, and antibacterial and antifungal activities [63]. PSK (Fig. 4.4) is an AMP with bactericidal activity that has been isolated and reported from Chrysomya megacephala larvae [64]. Another class of AMP called Diptericin is a basic heat‐stable peptide with high glycine content and synthesized by insects immediately after Gram-negative bacterial infection or wound. Diptericin damages the cytoplasmic membrane of invading bacteria, therefore, eliminating it from the body [68]. Drosomycin (Fig. 4.4) is an antifungal peptide consisting of an α‐helix, and a three‐stranded β‐pleated sheet stabilized by four disulfide bridges [69] is synthesized by fat body cells and released into the hemolymph but is inactive against bacterial particles. However, recent research reports suggest that recombinant

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Fig. 4.4: (A) 7DTL crystal structure of PSK, an antimicrobial peptide from C. megacephala [65]. (B) 1MYN solution NMR structure of drosomycin, inducible antifungal peptide [66]. (C) 1ZRV solution structure of spinigerin in H20/TFE 50% [67]. (D) 1ZRX solution structure of stomoxyn in H20/TFE 50% [67].

drosomycin expressed in E. coli revealed both antiparasitic and anti-yeast actions [70]. Metchnikowin is an antibacterial and antifungal proline-rich peptide expressed in an elevated titer in the fat body of Drosophila after infectious invasion induced by the Toll or the Imd pathways [71, 72].

4.3.5 Receptors PRRs are chiefly responsible for innate immune functions involving PGRP [73], β‐1,3‐glucan recognition protein (βGRP), hemolin, and C‐type lectins. 4.3.5.1 Peptidoglycan recognition proteins (PGRPs) The receptiveness and responses of the innate immune system are conquered by a group of proteins known as PGRPs, which are conserved from lower to higher group of metazoans. There are almost 19 PGRPs found in insects, which are subdivided into short (S) and long (L) forms. Short forms are particularly found in hemolymph, cuticle, and fat body cells, while hemocytes chiefly express the long forms [74, 75].

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4.3.5.2 β‐1,3‐Glucan recognition proteins (βGRPs) Another important PRRs in insects include plasma proteins and β‐1,3‐glucan recognition proteins which bind to β‐1,3‐glucans on bacterial surface and eventually induce proPO cascade. βGRP1 is found to be constitutively expressed in the fat body cells of Manduca sexta, whereas βGRP2 proteins elevate in titer before/after pupal development or during an immunogenic attack [76]. 4.3.5.3 Hemolins Hemolins belong to a plasma protein family with four immunoglobulin domains capable of binding to bacterial LPS and d lipoteichoic acid [77]. Hemolins are generally found in order Lepidoptera, including Bombyx mori [79] and Antheraea mylitta [78]. 4.3.5.4 C‐type lectins (CTLs) This group of PRRs contains carbohydrate-binding moieties that adhere to microbial surfaces and are common among lepidopteran insect. There are several forms of lectins found in Lepidoptera which binds to either bacterial LPS or lipoteichoic acid inducing agglutination [80, 81].

4.3.6 Cellular immune response 4.3.6.1 Hemocytes Hemocytes play a role in conferring cellular immunity by which immediate immunogenic responses arise after pathogenic attack known as hemocytes. There are quite a lot of variations of hemocytes found in millions of insect groups which are yet to be understood [82, 83]. Morphologically, the insect hemocytes include granular cells, crystal cells, oenocytoids, and plasmatocyte, which are usually capable of adhesion and execution of phagocytosis [83, 84]. However, crystal cells, plasmatocytes, and lamellocytes were described with much deeper insights in D. melanogaster than the other group of insects [85]. Furthermore, crystal cells include bulky crystal inclusion and synthesize a zymogen proPO, which in turn gets activated in the course of melanization, whereas plasmatocytes comprising 95% of the hemocyte pools are small long-lived cells but spread out large lamellipodial projections and develop dynamic filopodia [85, 86]. The lamellocytes can be distinguished from other hemocytes of D. melanogaster by only being traceable during the larval stages when parasites invade them and perform encapsulation of the parasitoid wasp eggs [87]. In the sequence of immunogenic events, it is well established that binding to hemocytes leads to phagocytosis of the foreign particles that invade the insect body.

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4.3.6.2 Nodule formation Formation of nodule is a prime indicator of entry of pathogenic organisms in many insects, and within a very short time period, huge number of bacteria/fungal spores get attacked and trapped in the coagulum produced by a population of defensive granulocytes. On the arrival of plasmatocytes, it forms a layer externally covering the periphery of the clump of cells and induces phagocytosis on a small scale [52]. The immunogenic reactions and its associated responsiveness depend on the pathogenicity of the invading organisms which may be correlated with the weak or strong rapid response depending on whether the invader is pathogenic or not [58]. Eicosanoids have been reported to modulate nodule formation [59]. 4.3.6.3 Encapsulation According to Chapman [52], parasitoid larvae or nematodes are larger invaders that induce a third type of response during which they are encapsulated by huge numbers of hemocytes. As a response of this type of pathogenic attack, granulocytes release their cellular contents at the invader’s surface within 5 min of the pathogenic entry and around 30 min later, plasmatocytes get attracted and accumulated forming layers of cells around the outside of the capsule so that the pathogen gets entrapped within it and the cells contiguous to the object turn out to be necrotic [52]. 4.3.6.4 Phagocytosis It is the process by which large foreign pathogenic particles are recognized, adhered, engulfed, and ingested by a subset of hemocyte population [83, 88]. In most of the insect species, including Diptera and Lepidoptera, plasmatocytes and granulocytes are regarded as the major hemocytes responsible for phagocytosis [83, 88–91]. There are two modes of induction of phagocytosis: involvement of specific cell-surface receptors that recognize large immunogenic foreign particles, and opsonization during which opsonins bind and cover the immunogenic particle making it recognizable for phagocytic receptors. Additionally, reliable research reports suggest that scavenger receptors Croquemort [92] and Draper [93] have been reported to be involved in recognizing many dying cells that are eventually killed by phagocytic hemocytes during development of insects. In the larva and adult insects, recognition of pathogen is mediated by the Nimrod family receptors Eater [94] and NimC1 [95], are solely responsible for recognizing invading microorganisms in the larva and adult forms of the insects cytokines and chemokines are also involved in inducing hemocytes. A chemotactic peptide member of lepidopteran ENF peptides from Pseudaletia separata has been reported to induce f migration and aggregation of hemocytes [96].

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4.3.6.5 Melanization The process of melanin production and deposition during the healing of wound and nodule and capsule formation against large pathogens or parasites is known as melanization, and this has been reported in quite a few groups of insects [97, 98]. Phenoloxidase (PO) being the key player of this process gets activated by a cascade of enzymatic reactions during the first phase, of which proPO is converted to PO [99] by a serine proteinase cascade [100]. The latter requires pattern‐recognition proteins such as PGRP or βGR to execute the process of PO activation which in turn binds to foreign surfaces including hemocyte membranes [101], resulting in melanin formation.

4.3.7 Proteasome Proteasome (Fig. 4.5) comprising a core called as 20S particle and other factors plays a role in regulation of cell cycle, cell differentiation, signal transduction pathways, antigen processing, signaling in stress, inflammatory responses, and apoptosis. It degrades different cellular proteins by ubiquitination. The 26S proteasome (Fig. 4.5) includes one 20S core particle together with two 19S regulatory particles that degrade ubiquitinated proteins in an ATP-dependent manner. During immune responses, immunoproteasome comprising two 11S regulatory particles, PA28 αand PA28 β, are induced by interferon gamma. PA28 γ and PA200 act as regulatory particles ([56], https://www.genome.jp/dbget-bin/www_bget?pathway+dme03050).

4.3.8 Apoptosis Apoptosis enables maintenance of homeostasis in multicellular organisms, thereby removing old unwanted damaged cells. In Drosophila, caspases play a central role in apoptosis. Dark activates the initiator caspase Dronc. But both Dronc and effector caspases Drice and Dcp-1 can be inhibited by Drosophila inhibitor of apoptosis protein 1. In Drosophila, members of the RHG protein family including Grim, Hid, Jafrac-2, Rpr, and Sickle lead to apoptosis. Cytotoxic stress factors and DNA damage can activate P53, which lead to RHG gene expression. Eiger by JNK signaling can initiate cell death by an IAP-sensitive cell death pathway ([56], (Fig. 4.6).

4.4 Nanoparticles and insect immune system A research report showed that in Tenebrio molitor nanodiamonds can drift through the insect cuticle in such a way that it did not hinder cellular and humoral immune responses in larvae, pupae, and adults of the respective species, and the applied

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Fig. 4.5: Dme03050 proteasome pathway of D. melanogaster (https://www.genome.jp/dbget-bin/ www_bget?pathway+dme03050). Image reproduced with permission from KEGG pathway database.

doses were not hemocytotoxic [102]. Furthermore, the distribution of nanodiamond aggregates was collected in hemocytes to a larger extent as a result of phagocytosis, whereas less amount detected in fat body cells and in malpighian tubule cells nanodiamonds were untraceable [102]. In addition for the attainment of new pest control approaches, the conjugate of nanodiamonds with Neb-colloostatin which is an insect hemocytotoxic and gonad inhibitory peptide can be used as it passes through the insect cuticle into the hemolymph, where the conjugate induces apoptosis of hemocytes, which in turn affect the insect’s cellular and humoral immune responses in all developmental stages of life cycle [102].

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Fig. 4.6: dme04214 pathway apoptosis of D. melanogaster (https://www.genome.jp/dbget-bin/ www_bget?pathway+dme04214). Image produced with permission from KEGG pathway database.

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Research on zinc oxide (ZnO) nanoparticles (NPs) has gained much attention in recent years. A recent research data showed that ZnO NPs can transmigrate the gut barrier in the lepidopteran insect, Bombyx mori (B. mori), and by interacting with the immune-competent cells in hemolymph it can activate innumerable toxic responses including a reduction in hemocyte viability, reactive oxygen species (ROS) production, morphological changes, and apoptotic cell death [103]. In response to this, there is an alteration in hemocyte dynamics including an increase in the production of granulocytes and plasmatocytes, which in turn could reverse the toxic effects so that the insects could return to normal physiological states after the clearance of these NPs from their body [103]. Cowpea mosaic virus (CPMV) with its multiple biomedical and nanotechnological as well as immunotherapeutic applications is used in two key platforms: virus NPs constructed on the complete CMPV virion, including the genomic RNA, and virus-like NPs (VLPs) involving empty CPMV (eCPMV) virion [104]. The antiviral effector responses as well as the chemophysical and immunostimulatory properties of wild-type CPMV (RNA containing) and eCPMV (RNA-free VLPs) generated from baculovirus-insect cell expression were studied and find the importance of each CPMV and eCPMV as novel adjuvants to overcome immunosuppression and tumor regression in ovarian cancer and other types of cancerous proliferation [104]. Recently, using a novel approach for designing respiratory syncytial virus (RSV) vaccine, modified RSV fusion (F) surface glycoprotein was cloned into a baculovirus vector for the attainment of effective expression in Spodoptera frugiperda (Sf9) insect cells [106]. The glycosylated RSV F cleaved into covalently linked F2 and F1 polypeptides generating homotrimers, and the extracted purified form of RSV F from insect cell membranes assembled into 40 nm protein NPs containing multiple RSV F oligomers that seem to be rosette like appearance [105]. It was also reported that the protection to lower and upper respiratory tracts against both RSV strain A and strain B infection given by recombinant RSV F enhanced polyclonal palivizumab competing antibodies, thus providing stronger protective platform [106]. In another study, the effects of dietary CuO NPs on metabolism and immune system of the greater wax moth G. mellonella (family Pyralidae) were reported, where toxic effects of CuO NPs on metabolic enzyme activity, biochemical parameters, and total counts and differential counts were estimated in G. mellonella larvae [107]. The negative impact of CuO NPs in varying concentration on metabolic enzyme activity and biochemical parameters in larval hemolymph was measured, and the result finds significance to relate the accumulated effects on insect hemocytes with mammalian blood cells which in turn could affect the mammalian immune system [107]. Recent research strategies in malarial research involve NPs that not only target the asexual stages of Plasmodium sp. in the peripheral circulation but also the itinerant stages of the protozoan parasite between humans and mosquitoes [108]. There is a report of curing of mice infected with Plasmodium yoelii (P. yoelii) after using orally administered polyamidoamine NPs combined with chloroquine, which effectively

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disposes the parasite from blood circulation by enhancing the activity of the free drug and inducing the immunity of the animal against malarial infection [108]. A study on Alexa Fluor 647-h-BN-OH-n finds that it can pass through the insect cuticle barrier and was killed by the increasing titer of hemocytes in the circulation [109]. Short-term responses induce low cytotoxic activity in insect hemocytes, while long-term immunoassays revealed that h-BN-OH-nanoflakes interfere with proper nodulation resulting in reduced hemocyte capacity to recognize bacteria, which ultimately causes dysfunction of the immune system during pathogen attack and inhibits the most important cellular immune defensive response in insects [109]. Speaking about the heavy usage of nanoparticles in industry involves applications of zinc oxide NPs (ZnO NPs) which is associated with the increasing risk of ecotoxicity as it releases into the environment. In a recent research report, the silkworm was used as an insect model to evaluate the potential risk of using ZnO NPs, as it was capable to show effective immune response along with pharmacokinetics related to mammals [110]. The accumulation, biodistribution, and toxicity of Zn in silkworms scrutinized after a subcutaneous injection and at different time intervals showed highest ZnO NP aggregate in the midgut as well as a set of antioxidant enzymes including superoxide dismutase, catalase, plasma glutathione peroxidase were found to be expressed in the midgut cells in response to ZnO NPs. Additionally, to omit the discrepancies between the in vitro and in vivo use of NPs, in vivo polystyrene microsized particle phagocytosis assay was performed in Drosophila melanogaster (D. melanogaster) by a reputed research group to compare the effect with an in vitro assay involving the same immune cells in culture and exposure to the same NPs [111]. In the in vitro assay, the phagocytized beads per cell were much decreased in comparison to the in vivo assay, which probably indicates the involvement of reduced amount of membrane in culture [111]. Silicon NPs (SiNPs) also have impending applications in biomedical industries. As an invertebrate model, the silkworm was used to evaluate the effects of SiNPs on hemocytes and hematopoiesis by a research group [112]. Varied effects of SiNPs were perceived, including a rapid uptake in the immune cells including granulocytes, oenocytoids, and spherulocytes in the circulating hemolymph, while very lower amount was observed in prohemocytes [112]. The SiNP-induced autophagy and apoptosis were observed in hemocytes where their entry was nonprohibited but can also be postulated that a high-dose of SiNPs in adequately roused lysosomal activity in hematopoietic organs was incapable of inducing the production of ROS to execute autophagy or apoptosis in the tissues [112]. Thus, from the above study it was clear that the SiNP-induced damage to hematopoietic tissues in the organ was found to be restricted. During the past few years with the growth of NP-based industries, gallium phosphide nanowires were also tested for their toxicity evaluation in selected invertebrate model organism. The application of gallium phosphide nanowires on D. melanogaster was executed by exposure to gallium phosphide nanowires during feeding [113]. The

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evaluation of toxicity of gallium phosphide nanowires was not found to be significant in relation to evoking concrete immune response or alterations in genome-wide gene expression and also not as worthy to affect the life span or somatic mutation rate of the selected insect model organism [113].

4.5 Discussion This chapter basically sheds light on the importance of NPs and their assessment in insects. Insects with gargantuan global diversity are adapted to various environmental conditions and include different assemblages. Different species of insects having different ecological niches play key roles in human welfare and must be studied in-depth. In response to pathogenic invaders, the components of insect’s cellular and humoral immune system gets activated and leads to the elimination of the invading pathogens either by phagocytosis or opsonization. We all are aware of the fact that the possible roles of agricultural insecticides for the control of insect pests have become backdated with lesser amount of fruitful results. The development of nanotechnology has spread their wings out with beneficial results in diverse fields of science and technology. Thus, the use of NPs in controlling insect pests has superseded the use of conventional agricultural pesticides that have harmful effects on soil. The nanotechnological applications also minimize the potential risks associated with decreased soil fertility and productivity [114]. Furthermore, several research data reveal the use of nanomaterials against potential insect vectors like Au NPs that have shown potential toxicity against insect vectors [115]. Additionally, many viruses with their potential nanotechnological applications tested for their immunomodulatory and immunostimulatory activities are synthesized in insect cell expression platform.

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5 Nanoparticles and the immune system in Mollusca Shyamasree Ghosh Abstract: Mollusca are an invertebrate phylum leading to an aquatic life either in freshwater or in marine environments and are in continuous exposure to pathogens. They have evolved robust innate immune system comprising cellular and humoral components that confer them immunity against pathogens and toxic agents and enable their survival. Due to anthropogenic exposures, the marine environment is exposed to nanoparticles which may affect the health of the molluskan members. Many of the members of phylum Mollusca finds importance as human feed and thus their health is of utmost importance. In this chapter, we discuss the phylum Mollusca immunity in brief, and then the impact of nanoparticle exposure on health and immunity of members of phylum Mollusca. Keywords: Mollusca, bivalve, nanoparticles, immune system

Abbreviations A. granosa A. fulica A. niger AFL AgNO3 AgNPs AMP B. alexandrina c(ALL) C. gigas C. hongkongensis C. hortensis C. virginica C. virginica CdS/Cd-Te CINP CTL CuO CuSO4

Anadara granosa Achatina fulica Aspergillus niger Aspergillus fumigatus (A. fumigatus) lectin Silver nitrate Silver nanoparticles Antimicrobial peptide Biomphalaria alexandrina Childhood acute lymphoblastic leukemia Crassostrea gigas Crassostrea hongkongensis Cepaea hortensis Crassostrea virginica Crassostrea virginica Cadmium sulfate/cadmium telluride Cuttlefish ink Cytotoxic T lymphocytes Copper oxide Copper sulfate

Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India https://doi.org/10.1515/9783110655872-005

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DMSO E. complanata E. leei EDS EPp Fc HS L. flavus LMS LPO M. arenaria M. edulis M. galloprovincialis M. modiolus MAPK MDR NaTeO3 NCB nCuO Neu5,9Ac2 Neu5Ac Neu5GC NPs NR n-SiO2 n-TiO2 O. mykiss P. globosa P. variotii PCA PCR PRR PS-NH2 PTT QDs R. philippinarum RER ROS S. agalactiae: S. grandis SABL SOD TEM TLR V. anguillarum V. philippinarum V. tapetis ZnO

Dimethyl sulfoxide Elliptio complanata Ensis leei Energy-dispersive X-ray spectroscopy Extrapallial protein precursor Fragment of crystallization Hemolymph serum Limax flavus Lysosomal membrane stability Lipid peroxidation Mya arenaria Mytilus edulis Mytilus galloprovincialis Modiolus modiolus Mitogen-activated protein kinase Multidrug resistance Sodium telluride Nanosized carbon black Nano-CuO N-Acetyl-9-O-acetylneuraminic acid N-Acetylneuraminic acid N-Glycolylneuraminic acid Nanoparticles Near-infrared Nanosilica Nano-titanium oxide Oncorhynchus mykiss Pila globosa Paecilomyces variotii Passive cutaneous anaphylaxis Polymerase chain reaction Pattern recognition receptors Amino-modified nanopolystyrene Photothermal therapy Quantum dots Ruditapes philippinarum Rough endoplasmic reticulum Reactive oxygen species Streptococcus agalactiae Solen grandis Sialic acid-binding lectin Superoxide dismutase Transmission electron microscope Toll-like receptor Vibrio anguillarum Venerupis philippinarum Vibrio tapetis Zinc oxide

5.2 Phylum Mollusca: an overview

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5.1 Introduction Nearly 70% of the Earth’s surface is occupied by oceans wherein greater than 90% of habitats of life forms exist. The earliest life on the Earth records back to greater than 3,500 million years ago, which originated in the ocean. Evolutionary processes have enabled the survival of marine organisms in a hostile environment of temperature extremes, fluctuating salinity, pressure, and overcoming the mutation effects. They have also evolved combating infection by bacteria and virus. The diversity of species in undisturbed natural marine habitats is found to be very rich [1, 2]. Marine organisms in the course of evolution reveal extremely complex biological mechanisms revealing cross-phylum activity with terrestrial biota. The sea is richer than the terrestrial ecosystem in terms of evolution and biodiversity, and marine species contribute to half of the biodiversity, thus being a potential source for naturally occurring therapeutics. Phylum Mollusca including clams, oysters, octopods, snails, and squids forms the largest insect phyla, second to the arthropods with more than 50,000 living species and 35,000 fossil forms. It is intriguing to understand and to know how the organisms have survived and evolved. Although we, the humans, have an immune system that is highly complex in its function with many components and signaling pathways, the phylum Mollusca, even in its primitive existence, reveals an immune system that offers protection of its health even when exposed to harsh conditions of the environment. We discuss in this chapter the overview of phylum Mollusca, its immune system, and how exposure to nanoparticles (NPs), as environmental contaminants, affects their physiology and overall health [1, 2].

5.2 Phylum Mollusca: an overview Phylum Mollusca (Fig. 5.1, Fig. 5.2) comprises class Monoplacophora with single symmetrical cap-like shell in Laevipilina antarctica, Aplacophora lacking shell-like Spathoderma californicum, Polyplacophora with shield-like shell with overlapping plates, like Cryptochiton stelleri, Scaphopoda with tusk shell with hollow tube-like structure like Paradentalium disparile, Gastropoda with single coiled shell with diversity in patterns and colors like in African apple snail, Achitina fulica (A. fulica), Bivulvia with shells comprising two separate symmetrical valves that are joined by ligament, and Cephalopoda lack any shell or internal shell with an exception of Nautilus sp. Despite large diversity within the group, a few commonly observed characteristic remains are that they are aquatic animals that graze on hard substratum, and their body being bilaterally symmetrical, ovoid in shape, ranging several centimeters in length, with flat ventral surface and muscular feet. For the shelled mollusks, the dorsal surface is covered by a shell, oval and convex, protecting internal organs or visceral mass, with an underlying epidermis or mantle. Pedal retractor

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Radix sp

Unio

Mussel

Snail

Snail Indoplanorbis sp

Snail

Fig. 5.1: Few molluscs. Picture with permission from Director, Zoological Survey of India, Kolkata, 700053, India.

muscles occur in pair that helps to pull down the shell against substratum. Presence of mantle cavity underlying the shell and in between the mantle bearing a pair of gills on opposite sides of the mantle cavity that is held in position by dorsal and ventral membranes and presence of a pair of nephridia. Each gill or ctenidium could be bipectinate which has long flattened axis, containing blood vessels and nerves. Triangular filaments are attached to each side of the broad surface of the axis placed in alternate positions with filaments on the opposite side of the axis. Monopectinate gills reveal one-sided filaments. The lower posterior part of the mantle cavity is the route of entry of water that moves upward between gill filaments, moving back outside the cavity. The beating of the lateral cilia located on the gills enables propulsion of water through the mantle cavity. Sediments and large particles that enter into the body are trapped by mucous on the gills, carried upward toward the axis by frontal cilia and then by abfrontal cilia, which is removed by the exhalant current. On the roof mantle, two patches of hypobranchial glands trap sediments in the existing current water. The afferent blood vessel carries blood to the gills, and the efferent blood vessel drains the gills and blood flows from afferent to the efferent blood vessels.

5.3 Phylum Mollusca: a brief study

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The mouth opens into the buccal cavity, thickened by elongated, muscular, cartilaginous mass called odontophore. Radula with rows of teeth extends over odontophore and arises from the radula sac. Digestive system comprises one pair of salivary glands, esophagus, stomach, digestive glands, and intestine. The posterior end of the stomach or style sac is ciliated. Other than cephalopods, they house an open type of circulatory system. They develop from a trocophore larva [1, 2].

Monopl acophora

Aplaco phora

Polypla cophora CLASS Scapho poda

Mollusca

Gastrop oda

Bivulvia

Cephal opoda

Fig. 5.2: Classification of phylum Mollusca, as in [1].

5.3 Phylum Mollusca: a brief study Many marine organisms reveal the morphology of being sessile and soft bodied. And the most interesting observation in this domain lies in the robustness of the physiology and immune system that confers them protection against predators, pathogens, and environmental agents. Secondary metabolites produced by these marine organisms are endowed with the property of chemical defense that contributes to their protection. Organisms under phylum Mollusca reveal extreme richness in bioactive secondary metabolites ranging in active compounds as amino acids, nucleosides, macrolides, porphyrins, terpenoids, aliphatic cyclic peroxides, and sterols that enable to deter predators, keep competitors away, or paralyze their prey.

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Mollusks encompass the largest phyla of marine invertebrate comprising gastropods, bivalves, scaphopods, cephalopods, aplacophorans, monoplacophorans, and polyplacophorans, many of which are edible and act as human food. Shells of gastropods and bivalves have economical and aesthetic value and used in designing tools and ornaments. Gastropods are also known to synthesize defensive like polygodial synthesized from mevalonates by nudibranchs under genus Dendrodoris and a rare class of marine natural products called as polypropionates. Tyrian purple or royal purple derived from gastropods, superfamily Muricacea, is used in the eastern Mediterranean and in China and is the earliest marine biotechnological application of mollusks. Gastropods and bivalves are known to sequester harmful chemicals from food or environment; and bivalves can accumulate toxins from phytoplanktons or harmful algae (HABs) and cause food poisoning in humans and marine mammals devouring them. Opisthobranchs are shell-less gastropods that use chemicals for defense. Peptides isolated from herbivorous opisthobranchs have applications as anticancer agents and are under human clinical trials. Cone snails reveal small peptides or conotoxins with different pharmacological activities showing therapeutic potential. Omega-conotoxin MVIIA is a 25-residue, disulfide-bridged polypeptide isolated from sea snail Conus magus venom, which can bind to neuronal N-type calcium channels and can act as a calcium channel blocker with potential pharmaceutical application. Bivalve and mussel produced biopolymers like glue proteins that have biotechnological applications. Cephalopods are mollusks that can swim fast and use ink for defense. Several antimicrobial peptides (AMPs) and proteins play a role in antimicrobial defenses and are reported from bivalves and gastropods. We discuss the immune molecules in phylum Mollusca in detail in the next paragraphs [1, 2].

5.4 Immune molecules in Mollusca A study of molluskan immunity as marine invertebrates and understanding their responses to pathogens finds importance. The innate immunity in mollusks is conferred by the protective anatomical and chemical barriers like shell and mucus covering soft parts of the body, preventing loss of body fluid, and inhibiting infections by pathogens and parasites. Blood clots and wound healing prevent havoc caused by injury. Host defenses to incoming pathogens include atrophy, encapsulation, nodule and pearl formation, phagocytosis, necrosis, and tissue liquefaction. Granular hemocytes including cellular components are functionally professional phagocytes and humoral components including nitric oxide (NO), lectins, lysozymes and products of prophenyloxidase (PpO) system, mercenenes, antimitotic in nature, antivirals like paolins, acute-phase reactants, and antiprotease activity of alpha 2macroglobulins that can entrap different proteinases [3] play an active role in cellular defenses. Encapsulation and nodule formation enable the removal of intruders

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by cellular or humoral immune responses [3–5]. Bivalve mollusks including clams, mussels, oysters, and scallops have been studied for immunity, and further studies on hematopoiesis, hemocyte subpopulation characterization, genetic and molecular mechanisms controlling immune reactions, and hematopoiesis need to be done [3–5]. An interplay of endocrine system, nervous system, and immune systems has been reported in Octopus vulgaris [6] when exposed to environmental and internal stress factors.

Internal stress factors

Sensory Systems

Environmental stress factors

Nervous System Neurohormones Endocrines

Hormones

Immune System Target Tissues

Altered behaviour Fig. 5.3: Interplay of nervous, endocrine, and immune systems due to environmental and internal stress factors in Octopus vulgaris. Image adapted with modification from [6], under CCBY license.

As early as in 1905, Metchnikoff identified phagocytes with a role in starfish immunity. The mollusks, including bivalves, cephalopods, and snails, represent a highly diverse phylum and the lophotrochozoan protostomes. Immune biology of mollusks finds importance in not only knowing the importance as combating infections caused by pathogens but also understanding the evolution of immune function in metazoan phylogeny. Lectins are proteins capable of recognition and binding to carbohydrates acting as pattern recognition receptors (PRRs) and play a role in innate immune responses against pathogen. Together with lectins, activation of PpO is required for mediate pathogen removal [7]. C-type lectins in mollusks have been reported to comprise a common bi-looped structure with two conserved disulfide linkages with a Ca2+-

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5 Nanoparticles and the immune system in Mollusca

A

B

Fig. 4: (A) Prediction of structure of S. grandis SABL. (B) Prediction of structure of SABL from L. flavans. Images reproduced with permission from [9].

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dependent carbohydrate binding ability revealing specificity toward mannose and galactose with roles in pathogen recognition [7, 8]. Sialic acid-binding lectins (SABLs) have been reported from phylum Mollusca revealing affinity toward Nacetyl- D -galactosamine, N-acetyl- D -glucosamine, N-acetyl- D -mannosamine, Nacetylneuraminic acid, and glycan residues and have been reported from classes Bivalvia and Gastropoda [8, 9]. Structural studies have revealed a conserved C1q complement domain with a probable role in recognition of bacteria. Solen grandis (S. grandis) SABL structure reveals an additional domain bearing structural similarity to prefoldin with a molecular chaperon-like activity, and Limax flavus (L. flavus) reveals domains with homology to fibrinogen-related domains superfamily with a probable role in blood clotting. Hemocytes in mollusks may be of a single or different types, synthesized in gastropods from connective tissue or amebocyte producing organ or in cephalopods from the white body organ. Lectins act as PRRs that identify and bind to pathogenassociated molecular patterns like lipopolysaccharide, bacterial peptidoglycan and play a role in nonself-recognition and immune activation. Agglutinins or cytophilic receptors act as humoral factors or are expressed on hemocyte cell surface. Although records of some immunological memory have been reported from gastropods but unlike vertebrate immune system, lymphocytes like T and B cells were not yet known. Hemocytes on exposure to pathogens and environmental toxic substances can release reactive oxygen species (ROS). Hemocytes of Mytilus edulis (M. edulis) commonly called as blue mussels on exposure to pathogens and environmental agents can release cytotoxic factors that play a role in lysis of target cells. Biomphalaria alexandrina (B. alexandrina) expresses glycan-binding lectins. Activity of lectins differed in Pacific oysters, Crassostrea gigas (C. gigas), that can render them susceptible or resistant to infection by parasites of Perkinsus marinus. Biomphalaria glabrata, a gastropod on exposure to flatworms Echinostoma paraensei, leads to overexpression of hemolymph lectins of different mol weight including G1M, G2M, and 65 kDa lectins. Molluskan immune system has been studied more since the start of immunogenomics dating back to the early 1990s and has enabled cataloguing of immune system in bivalves and gastropods [10, 11]. Oysters and clams find value as food that is prone to be affected by anthropogenic changes and opportunistic pathogens. Extracellular vesicle from Mollusca species, including M. edulis, Mya arenaria (M. arenaria), Crassostrea virginica (C. virginica), and Ensis leei (E. leei), reveals a role in cellular communication and in immunity and host–pathogen interactions [12]. Argopecten purpuratus of economic importance revealed hemocytes that play a key role in its immunity. Challenged by β-glucan zymosan and bacteria Vibrio splendidus, the molluskan immune system revealed an activity with a pathophysiology of infiltrating hemocytes, generation of ROS, expression of PRRs including ApCLec and ApTLR and AMPs including ferritin and big defensin [13].

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Tab. 5.1: SABL in Mollusca: source, specificity, and function. Species

Lectin

Specificity

Function

S. grandis

SgSABL- and SgSABL-

Sialic acid, NeuAc

PRR, pathogen recognition

C. hongkongensis

Ch-salectin

Bactericidal effects

V. philippinarum

VpSABL

PRR, Gram-negative bacteria, Vibrio anguillarum recognition

R. philippinarum

MCsialec

Antibacterial in role, overexpressed during infection by V. tapetis

M. modiolus

Modiolin H and modiolin E

Antibacterial in role

C. gigas

Hemagglutinin gigalin H (human) and gigalin E (equine)

Agglutination of equine and human RBC

C. virginica

Hemagglutinin

Antibacterial in role; agglutination

M. edulis

Occurs in hemolymph

Antibacterial in role

A. granosa

AFL

NeuGc

C. hortensis

Agglutinin

Neu,Ac

A. fulica

Achatinin H

P. globosa

PAL

NeuGc

L. flavus

LFA

More specificity toward NeuAc than NeuGc

Ca+-dependent agglutination of erythrocytes

Reproduced with permission from [9] with modification.

5.5 Nanoparticles and the immune system in Mollusca Bivalves predominant in coastal environments are exposed to random environmental fluctuations and altered anthropogenic involvement affecting their immunity. Release of atmospheric anthropogenic carbon dioxide (CO2) can cause ocean acidification which can cause NP bioaccumulation in marine organisms. Titanium dioxide (TiO2), NP exposed edible bivalves, revealed their accumulation, organ edema, and altered hematologic indices and blood chemistry [14], thus posing a threat to the health of consumers.

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The robust immune system of Mytilus spp. also commonly called as mussel can combat potential pathogens and environmental stress factors. Mytilus on exposure to nano-carbon black (NCB), C60 fullerene (C60), nano-titanium dioxide (n-TiO2), and nanosilica (n-SiO2) revealed affected the gills and digestive system, causing ROS generation, release of lysosomal enzyme, lysosomal membrane destabilization by most of NCB followed by C60, followed by n-TiO2, followed by n-SiO2, induced accumulation of lysosomal lipofuscin, increase in catalase, mostly by n-SiO2 followed by NCB and n-TiO 2 followed by C60, followed by NCB. n-TiO2 stimulated production of glutathione transferase (GST). Gills revealed altered catalase and GST activities revealed affected immune parameters in hemocytes, concentration-dependent lysozyme release, NO and ROS generation, and inflammatory effects mediated by stress-activated p38 MAPK [15]. ZnO-NPs in the coastal areas are reported to be toxic to marine lifeforms. A study on M. edulis revealed that both salinity and ZnO-NPs affected the innate immunity, observed in Baltic Sea brackish area which was concentration dependent. ZnO-NPs could increase hemocyte death, reduce their adhesion, and increase in phagocytosis and lysosomal volume. Together with exposure to salinity 15, expression of innate immune molecules including Toll-like receptors (TLR) b and c, C-lectin, and C3q complement component were altered indicating decreased pathogen recognition property but defensin–AMP expression increased. Exposure of different levels of salinity together with ZnO-NPs revealed upregulation of C1, C3q complement components and TLR-a, b, and c, while low salinity conditions revealed the physiology of immunosuppression [16]. Immunotoxicity of cadmium sulfate/cadmium telluride (CdS/CdTe) mixture quantum dots (QDs) dissolved cadmium chloride (CdCl2)/sodium telluride (NaTeO3) salts, and a CdCl2/NaTeO3 mixture studied on M. edulis revealed QDs more toxic than dissolved metals [17]. Mytilus galloprovincialis (M. galloprovincialis) treated with amino-modified nanopolystyrene (PS-NH2) or nanoplastics led to, affected parameters of hemocyte mitochondria, lysosome, proliferation/apoptosis marker expression, upregulation of extrapallial protein precursor (EPp), lysozyme, and mytilin B downregulation, indicative of pathophysiology of stress conditions, increased hemolymph bactericidal activity, most of the immune-related genes [18]. The host microbiome contributes to the health of all organisms and reveals association with the host innate immune system maintaining the homeostasis. Titanium dioxide NPs or TiO2-NPs are known to affect the gut microbiome of marine bivalve M. galloprovincialis. In bivalves, exposure to TiO2-NPs revealed immunomodulatory effects, pathophysiology of stress, increased bacteriocidal activity in hemolymph, decreased bacterial population of Shewanella, Kistimonas, and Vibrio and increased population of Stenotrophomonas [19]. Exposure to silver NPs (AgNPs) on M. galloprovincialis revealed cytotoxicity of hemocytes by lysosomal membrane stability (LMS) assay, affected clathrin- and caveolae-mediated endocytosis, and the immunotoxic effects were largely based on size and duration of exposure [20]. M. galloprovincialis exposure to NPs could lead to their uptake and cause toxicity. PS-NH2 exposed Mytilus hemocytes revealed cellular damage, lysosomal membrane

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instability, ROS generation, phagocytosis, morphological changes, dysregulation of p38 MAPK and protein kinase C, formation of protein corona, and involvement of putative C1q domain containing protein (MgC1q6) [21]. Release of engineered NPs into the aquatic environment affects the immunity and toxicity of aquatic biota and those that feed on suspensions like marine invertebrates and bivalves as they can be uptaken. Uptake of NPs including TiO2, SiO2, ZnO, and CeO2 have been reported to induce toxicity in bivalve M. galloprovincialis affecting the hemocytes. Studies on uptake of NPs by bivalves from artificial seawater revealed affected hemocyte lysosomal, mitochondrial parameters, pathophysiology of stress, induction of ROS and NO production, and phagocytic activity [22]. Bioaccumulation of TiO2-NPs in M. galloprovincialis in the gills and digestive glands altered tissue organization, hemocyte infiltration, activated immune system, increased lipid peroxidation (LPO), DNA damage, overexpression of superoxide dismutase (SOD1), increased toxicity, ROS production, multilamellar bodies, fragmentation of RER, vacuoles in cytoplasm with increased dense granules, residual bodies, lipid inclusions, cellular inflammatory response, and apoptosis [23]. Melanin is the dark pigment in many organisms. Melanin NPs derived from squid ink when targeted on cell line RBL-2H3 revealed partial attachment to cell surface and partial intake by cells from TEM studies. In their solubilized form, they revealed antiallergic effects, inhibiting antigen-IgE-FcεRI-mediated IgE-sensitized mast cell degranulation, events of phosphorylation, calcium influx, reduced FcεRIbound IgE molecules in number, reduced plasma membrane and cytoplasmic fluidity, and increased viscosities. Passive systemic anaphylaxis and passive cutaneous anaphylaxis (PCA) assays in mouse models revealed to increase the reduced body temperature post antigen infection and suppressed antigen-induced extravasation in PCA [24]. NPs from cuttlefish ink (CINPs) revealed antitumor efficacy and find their application in tumor immunotherapy and PTT [25]. Naturally occurring melanin NPs find their application in treating primary and abscopal breast cancers [26]. Nanoparticles of copper and copper oxide (CuO) in the marine environment can impact the innate immune system, overall health of the mussels, increased susceptibility to bacterial infection, increasing generation of ROS, cellular toxicity, increased lysosome, decreased activity of multidrug resistance (MDR) transporter with soluble copper accumulated in gills and hemolymph, and CuO-NPs causing gill damage and accumulation in hemocytes, decreasing activity, decreased phagocytosis, and increased bacterial proliferation on exposure to bacteria Vibrio tubiashii post copper exposure [27]. Lives in coastal environments suffer from challenges of pollutants like HAP, heavy metals, pesticides, PCB, and pharmaceuticals threatening marine life like bivalves, affecting their innate immune system and causing immunotoxicity [28]. Freshwater mussel, Elliptio complanata (E. complanata), on exposure to AgNPs caused immunotoxicity, affected hemocyte viability, phagocytosis, cellular toxicity, affected metallothioneins levels, LPO in gills, and aggregation and accumulation of

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AgNPs [29]. Capped CdS/CdTe QDs lead to aggregation in M. edulis and E. complanata, which led to size and concentration-dependent toxicity [30]. Mollusks of bivalve genus when exposed to carbon, copper, chromium, and cobalt NPs reveal toxicity studied by neutral red retention time assay revealing decreased LMS of hemocytes as compared to gold and TiO2-NPs [31]. AgNPs from fungal isolates including Paecilomyces variotii (P. variotii) and Aspergillus niger (A. niger) exposed to B. alexandrina snails revealed toxic effects, affecting albumin, lipids, decreased expression of steroid hormone progesterone, increase in testosterone and estradiol, upregulation of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase liver enzymes, total protein, glucose, increased total count of hemocytes and altered differential count with reduced number of granulocytes and increased number of hyalinocytes and their affected morphology, increased number of oocytes and sperms, increased apoptotic hemolymph cells with pathophysiology of cellular fragmentation and cytoplasmic degeneration, formation of large fat vacuoles, shrunked nucleus, increased phagocytosis, abnormal division of nucleus, and atretic oocytes, dead and necrotic sperm head, isolated sperm tail, abnormal morphology of sperms, dead sperms, and hyperplasia [32]. Marine life and environment can be contaminated due to exposure to metal NPs. Exposure of CeO2 NPs did not affect the lysosome membrane stability, phagocytosis, or ROS production [33]. Gold NPs from the environment on exposure to early larval stage of the Japanese oyster C. gigas led to NP cellular uptake and permeation by alimentary pinocytic or phagocytic routes, its subcellular distribution, bioaccumulation, mechanisms, and storage in residual bodies [34].

5.6 Discussion The health and well-being of the marine fauna remains a priority to the universe and is in direct relation to the well-being of the human life. Molluskan members find their applications as food source, having aesthetic value, and biotechnological importance. Besides, they, like every life form, have a role in maintaining ecological balance. But environmental pollutants leaching into the aquatic environment can add to pollutants and NPs in the habitat of aquatic molluskan members and thus pose a threat to their health. Although we have discussed the adverse effects of diverse types of NPs, based on nature, size, concentration, engineered on the health and immune system of the molluskan members, it is perhaps alarming now globally to minimize or control pollution in the environment so that the health and well-being of marine animals including members of phylum Mollusca can be prevented from further damages.

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Research in this domain of biology thus finds importance in imparting knowledge, highlighting the impact of NPs in damaging the health of the molluskan members and also to find and design appropriate legislation globally to minimize or prevent such leaching and dumping of NPs to water sources, eventually hampering the health of the members of the phylum Mollusca.

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6 Echinodermata and the immune system Shyamasree Ghosh Abstract: Echinoderms, belonging to the group of deuterostome invertebrates, include more than 6,000 species of marine species and bottom dwellers. Although primitive in form, they reveal immune system that confers them protection against challenges from the environment and pathogens. The exposure to nanoparticles from the environment bears a negative impact on the immune system and health of the echinoderms. In this chapter, we highlight the immune system of echinoderms and the impact of nanoparticle contamination on the health, life, and immune system of echinoderms. Keywords: Echinoderms, apoptosis, complement, C3, TLR, NOD

Abbreviations Antimicrobial peptides C-type lectin Extracellular matrix Galactose Gold nanoparticles Heat shock protein Mitogen-activated protein kinases N-Acetylgalactosamine Paracentrotus lividus Pattern recognition receptors Rreactive oxygen species Strongylocentrotus purpuratus Titanium dioxide nanoparticles Toll-like receptors

AMP CTL ECM Gal AuNPs hsp MAPK GalNAc P. lividus PRRs ROS S. purpuratus TiO2NPs TLRs

6.1 Introduction Echinoderms, members of phylum Echinodermata, belonging to the group of deuterostome invertebrates comprise more than 6,000 species, which are mainly marine species and bottom dwellers. The body reveals a pentamerous radial symmetry, divided into five parts around the central axis. Presence of internal skeleton comprised

Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India https://doi.org/10.1515/9783110655872-006

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calcareous spicules, bearing projecting spines. Body composed of water vascular system plays a role in food supply and transport and enables locomotion in some forms. Body reveals a coelom and digestive tract. Most of the members are dioecious with simple reproductive tract and external fertilization. Eggs are homolecithal, and early development reveals radial and intermediate cleavage. Blastula reveals a blastocoel, and gastrulation involves invagination by the formation of tubular archenteron. The blastopore acts as the larval anus. The gastrula develops into free-swimming larval form with planktonic life and bilateral symmetry undergoing metamorphosis to form radially symmetrical adult. Almost 40–50% of the phyla members undergo such metamorphosis while the rest have lecithotropic larva and direct development. A larva with bilateral symmetry bearing ciliated bands is a characteristic larval form of echinoderm classes. The pentamerous radial symmetrical body and endoskeleton with calcareous ossicles perhaps bear correlation with sessile life, and the water vascular system might have evolved in response to adaptation for suspension feeding. A large number of echinoderms have been known from fossil records. Echinoderms made their first appearance during early precambian times and reached abundance and most development during the Paleozoic period. Quite a few classes are extinct. Eocrinoidea include the earliest known echinoderms reported from early Cambrian to Ordovician period. Rhombifera and Diplorita classes existed from the middle Ordovician to Permian period. Class Blastoidea existed in Permian times and the Edrioasteroidea existed from Cambrian to Pennsylvanian times. The Carpoid group existed from Cambrian to Devonian times. The Holothuroids reveal few fossil records, while most records exist from echinoids. Class Asteroidea includes starfish and more than 1,500 known species; class Ophiuroidea includes basket stars or brittle stars and more than 2,000 reported species; class Echinoidea includes heart urchins, sea urchins, and sand dollars and more than 950 reported species; class Holothuroidea includes sea cucumbers and more than 900 living species; and class Crinoidea includes the sea lilies and around 80 described species, which live at 100 m depth and not very commonly seen. Order Comatulida under class Crinoidea include the feather stars, free living, and occur at greater depths from the intertidial zones with 550 reported species from the polar areas and Indo-Pacific region. Class Concentricycloidea are disk-shaped Echinoderms that inhabit at depths of thousands of meters near the coast of New Zealand [1–4].

6.2 Echinodermata: Immune system Tissue regeneration in response to wound healing and thus preventing infection has been reported from Echinodermata, and immune system–extracellular matrix (ECM) interplay has been instrumental in triggering regeneration [5].

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The innate immune system and cellular defense developed in Echinoderms belong to the deuterostome lineage. Sea urchins live long and reveal wound healing and combating major infections. Signal transductions enable combating injury and infection against pathogens [6]. Echinoderms, due to their marine life, are exposed to stress factors from the environment including predation, altered temperature and pH, hypoxia, attack by pathogens, damages due to UV radiation, and toxicity due to exposure to metals, toxicants, and nanomaterials. Paracentrotus lividus(P. lividus)finds importance as a model to study sea urchin immune cells in testing toxicity [7]. Echinoderms reveal simple complement system, lectin genes, and antimicrobial peptides (AMP) [8]. In innate immune responses, pattern recognition receptors (PRRs) like Toll-like receptors (TLRs) enable recognition of pathogen-associated molecular patterns expressed by pathogens leading to activation of signaling, and TLRs have been reported from different invertebrate phyla including Echinodermata [9]. The immune genes in purple sea urchin adult and larval form [10] reveal complexity that play a role in conferring immunity. Coelomocytes and other proteins in the coelomic fluid of echinoderms can sense environmental stress [7]. Sea urchins reveal complement proteins SpC3 and SpBf bearing homology to vertebrate complement component C3 Fig. 6.1 and factor B (Bf), respectively. Pathogen challenge in sea urchins reveals inducible SpC3 in coelomic fluid while SpBf reveals constitutive and low expression. Pathogens, particles, and foreign cells are opsonized by phagocytic coelomocytes [11]. Coelomocytes in coelomic fluid of adult sea urchins, Strongylocentrotus purpuratus (S. purpuratus), has been studied for immune reactions and gene expression [12]. Sea urchin genome plays a major role in contributing to host defense containing 222 TLR, including more than 200 NACHT domain leucine-rich repeat proteins bearing similarity to the NALP protein, vertebrate nucleotide-binding and oligomerization domain , and scavenger receptor cysteine-rich proteins [13]. Coelomocytes in sea urchins play a role in response to injuries, invasion of host, and adverse conditions, revealing chemotaxis, phagocytosis, cytotoxic metabolite generation, and comprise amebocytes that are red or colorless forms, phagocytes, and vibratile cells [15]. Coelomocytes derived from purple sea urchin, S. purpuratus, reveal Sp185/333 markers and produce more Sp185/333-positive coelomocytes in response to immune challenges [16]. In the larval form, phagocytes rapidly respond to microbes. During immune response, both up- and downregulation of genes are regulated by gene regulatory network (grns, [10]). Larval form of purple sea urchin has been used to study grns involved in developmental process and gene expression in immune cells in response to microbial challenge of the gut epithelium involving role of IL-17-mediated signaling [17]. In sea cucumbers, immune genes include AMP, clotting protein, complement C3, enzymes, humoral components, lectins, lysozyme, PRR proteins, and TLR, although

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SPU_025966-tr

SPU_006951-tr SPU_020043-tr

c3 SPU_024614-tr

SPU_017239-tr

LOC587275 SPU_000997-tr SPU_007682-tr

bf

SPU_006952-tr

c3

Complement component c3 precursor; Uncharacterized protein (639 aa)

Predicted Functional Partners: bf

Uncharacterized protein

SPU_024614-tr Uncharacterized protein loc 100892938; Uncharacterized protein; Belongs to the peptidase S1 family SPU_017239-tr Uncharacterized protein LOC587275

Uncharacterized protein

SPU_000997-tr Complement c3 isoform x2; Uncharacterized protein SPU_020043-tr Low-density lipoprotein receptor-related protein 4; Uncharacterized protein; Belongs to the peptidase S1 family SPU_006951-tr Uncharacterized protein SPU_006952-tr Insulin-like growth factor-binding protein complex acid labile subunit; Uncharacterized protein SPU_025966-tr Uncharacterized protein loc 100894023; Uncharacterized protein SPU_007682-tr Uncharacterized protein

Fig. 6.1: The complement C3 protein and its functional partners were predicted using the DB String Database for understanding protein–protein interactions (https://string-db.org/cgi/network?tas kId=b1nnqr9uCZcK&sessionId=bLtRw75z29k3 [14]).

the exact role of each component is not yet well understood [18]. Sea cucumbers reveal regeneration in response to damage in which genes and signaling pathways are activated during asexual reproduction and regeneration of anterior and posterior body parts, nervous and digestive system, revealing increased concentration of reactive oxygen species (ROS) and antioxidant enzymes, activation of cellular and humoral components, remodeling of ECM, and involvement of Wnt signaling [19]. The sea cucumber reveals a role played by microRNAs including miR-133, -137, and -2008, and their target genes have been reported to regulate innate and adaptive responses, complement system, TLR pathway, ROS generation, and induction of apoptosis [20]. Animal lectins are known to play major roles in different cellular and molecular recognition processes and play a role in immune responses and self-defense in invertebrates. Among their different families, the C-type lectins (CTLs) (Fig. 6.2) can

6.3 Echinodermata immune system and nanoparticles

A

B

C

109

D

Fig. 6.2: (A) Crystal structure of CEL-IV, 3ALS [21]. (B) 1WMY, crystal structure of C-type lectin (CTL) CEL-I from Cucumaria echinata (C. echinata) [22]. (C) 1VCL, crystal structure of hemolytic lectin CELIII [23]. (D) 3W9T pore-forming CEL-III. DOI: 10.2210/pdb3W9T/pdb, TOXIN, from C. echinata, Deposited: 2013-04-16, Released: 2014-03-19 , Depositionauthor(s): Unno, H., Goda, S., Hatakeyama, T [24]. The images are downloaded from Protein Data Bank [25].

recognize carbohydrates in the presence of Ca2+ ions, containing a C-type carbohydrate-recognition domains that can bind to carbohydrates. CEL-IV, a CTL synthesized by sea cucumber, C. echinata, bearing a Glu-Pro-Asn or EPN motif or carbohydrate-binding motif in its carbohydrate-binding site, can preferentially bind to galactose (Gal) and N-acetylgalactosamine (GalNAc) [21]. CEL-I purified from C. echinata bear higher specificity for GalNAc [22]. CEL-III, on the other hand, reveals Ca2+-dependent specificity toward galactose together with properties of hemolysis and hemagglutination [23], and CEL-III heptamer has been revealed for its pore-forming property and activity as toxin [24].

6.3 Echinodermata immune system and nanoparticles Marine life is threatened by exposure to pathogens, difference in pH, exposure to nanoparticles, and environmental pollutants. Engineered nanoparticles and their uncontrolled release into the environment and particularly marine environment bear negative impact on the health and life of echinoderms. This is revealed by nanotoxicological-based studies revealing innate immune signaling in response to stress in sea urchins [26, 27]. Release and sedimentation of zinc oxide nanoparticles, a common ingredient in sunscreens, when in contact with sea urchins (P. lividus) in marine environment impacts their health adversely causing nuclear damage of immune cells and malformed larvae [28]. Gold nanoparticles (AuNPs) of colloidal nature coated with polyvinylpyrrolidone AuNPs exposed to P. lividus leads to corona formation, metabolic effects, inflammation, and phagocytosis, TLR4-mediated activation of signaling leads to immune responses [29].

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Nanoplastics or polystyrene nanoparticles and their amino-modified form PSNH2 impact the coelomocytes, health of P. lividus,leading toformation of corona, decreased lysosomal membrane stability, nuclear membrane damage, and apoptosis [30]. Exposure of titanium dioxide nanoparticles (TiO2NPs) to P. lividus reveals immunomodulatory effects, activation of signaling, metabolism pathways, suppressed expression of genes including NF-kappa B or nuclear factor kappa light chain enhancer of activated B cells , fibroblast growth factor receptor 2, JUN, mitogen-activated protein kinase (MAPK14), FAS (Fas cell surface death receptor), vascular endothelial growth factor receptor, caspase 8 (alternatively called as FLICE or FADD-like IL-1β converting enzyme), and overexpression of antioxidant metabolic activity including cysteine-methionine, glycine-serine, and pentose phosphate pathways, acquiring immunological tolerance [31]. Reports have suggested that TiO2NPs exposure can cause corona formation with the involvement of cellular adhesion proteins including toposome, galectin-8, nectin, actin, and tubulin without any oxidative stress or activation of caspase [32]. Contrasting reports have suggested that TiO2NPs can lead to triggering of activation or inactivation of MAPKs (p38 MAPK, ERK), TLR4, heat shock protein 70 (hsp70), and interleukin-6 [33].

6.4 Discussion It is now well known that nanoparticles released into the environment are a hazard to the environment since they affect the different life forms and invertebrates including the marine forms like Echinoderms. Experimental studies are revealing how the nanoparticles affect the life and immune system of the larval and adult forms of echinoderms, leading to biocorona formation, dysregulated signaling pathways, inflammation, phagocytosis, and causing oxidative stress and activating apoptotic pathways. Research in this domain of biology is extremely intriguing and important and finds importance in understanding the harmful consequences of release and spread of nanoparticles in the environment. It also finds importance in spreading awareness among masses in prevention of rise of nanotoxicity.

References [1] [2] [3]

Ruppert EE, Fox RS, Barnes RD. Invertebrate Zoology: A Functional Evolutionary Approach. Thomson-Brooks/Cole, Belmont, CA, 2004. Barker M. Echinoderms 2000. Balkema, Lisse, 2001, 590 pp. Lacalli TC. The nature and origin of deuterostomes: Some unresolved issues. Invertebr Biol. 1997;116(4) [Wiley, American Microscopical Society]:363–70.

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Raff R, Byrne M. The active evolutionary lives of echinoderm larvae. Heredity. 2006;97:244–52. Arenas Gómez CM, Sabin KZ, Echeverri K. Wound healing across the animal kingdom: Crosstalk between the immune system and the extracellular matrix. Dev Dyn. 2020 Jul;249(7) 834–46. Smith LC, Davidson EH. The echinoderm immune system. Characters shared with vertebrate immune systems and characters arising later in deuterostome phylogeny. Ann N Y Acad Sci. 1994 Apr 15;712:213–26. Pinsino A, Matranga V. Sea urchin immune cells as sentinels of environmental stress. Dev Comp Immunol. 2015 Mar;49(1)198–205. Smith LC, Ghosh J, Buckley KM, Clow LA, Dheilly NM, Haug T, Henson JH, Li C, Lun CM, Majeske AJ, Matranga V, Nair SV, Rast JP, Raftos DA, Roth M, Sacchi S, Schrankel CS, Stensvåg K. Echinoderm immunity. Adv Exp Med Biol. 2010;708:260–301. Rauta PR, Samanta M, Dash HR, Nayak B, Das S. Toll-like receptors (TLRs) in aquatic animals: Signaling pathways, expressions and immune responses. Immunol Lett. 2014 Mar-Apr;158 (1–2)14–24. Buckley KM, Schuh NW, Heyland A, Rast JP. Analysis of immune response in the sea urchin larva. Methods Cell Biol. 2019;150:333–55. Smith LC, Clow LA, Terwilliger DP. The ancestral complement system in sea urchins. Immunol Rev. 2001 Apr;180:16–34. Smith LC, Hawley TS, Henson JH, Majeske AJ, Oren M, Rosental B. Methods for collection, handling, and analysis of sea urchin coelomocytes. Methods Cell Biol. 2019;150:357–89. Rast JP, Smith LC, Loza-Coll M, Hibino T, Litman GW. Genomic insights into the immune system of the sea urchin. Science. 2006 Nov 10;314(5801)952–56. Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, Doncheva NT, Legeay M, Fang T, Bork P, Jensen LJ, von Mering C. The STRING database in 2021: Customizable proteinprotein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res. 2021 Jan 8;49(D1)D605–D612. Buckley KM, Rast JP. Immune activity at the gut epithelium in the larval sea urchin. Cell Tissue Res. 2019 Sep;377(3)469–74. Ghosh J, Buckley KM, Nair SV, Raftos DA, Miller C, Majeske AJ, Hibino T, Rast JP, Roth M, Smith LC. Sp185/333: A novel family of genes and proteins involved in the purple sea urchin immune response. Dev Comp Immunol. 2010 Mar;34(3)235–45. Matranga V, Pinsino A, Celi M, Natoli A, Bonaventura R, Schröder HC, Müller WE. Monitoring chemical and physical stress using sea urchin immune cells. Prog Mol Subcell Biol. 2005;39: 85–110. Xue Z, Li H, Wang X, Li X, Liu Y, Sun J, Liu C. A review of the immune molecules in the sea cucumber. Fish Shellfish Immunol. 2015 May;44(1)1–11. Dolmatov IY. Molecular aspects of regeneration mechanisms in holothurians. Genes (Basel). 2021 Feb 10;12(2)250. Zhan Y, Liu L, Zhao T, Sun J, Cui D, Li Y, Chang Y. MicroRNAs involved in innate immunity regulation in the sea cucumber: A review. Fish Shellfish Immunol. 2019 Dec;95:297–304. Hatakeyama T, Kamiya T, Kusunoki M, Nakamura-Tsuruta S, Hirabayashi J, Goda S, Unno H. Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif. J Biol Chem. 2011 Mar 25;286(12):10305–15. 10.1074/jbc. M110.200576. Sugawara H, Kusunoki M, Kurisu G, Fujimoto T, Aoyagi H, Hatakeyama T. Characteristic recognition of N-acetylgalactosamine by an invertebrate C-type Lectin, CEL-I, revealed by X-ray crystallographic analysis. J Biol Chem. 2004 Oct 22;279(43)45219–25.

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[23] Uchida T, Yamasaki T, Eto S, Sugawara H, Kurisu G, Nakagawa A, Kusunoki M, Hatakeyama T. Crystal structure of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata: Implications of domain structure for its membrane pore-formation mechanism. J Biol Chem. 2004 Aug 27;279(35)37133–41. [24] https://www.rcsb.org/structure/3W9T [25] Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The protein data bank. Nucleic Acids Res. 2000;28:235–42. [26] Alijagic A, Pinsino A. Probing safety of nanoparticles by outlining sea urchin sensing and signaling cascades. Ecotoxicol Environ Saf. 2017 Oct;144:416–21. [27] Alijagic A, Pinsino A. Probing safety of nanoparticles by outlining sea urchin sensing and signaling cascades. Ecotoxicol Environ Saf. 2017 Oct;144:416–21. [28] Manzo S, Schiavo S, Oliviero M, Toscano A, Ciaravolo M, Cirino P. Immune and reproductive system impairment in adult sea urchin exposed to nanosized ZnO via food. Sci Total Environ. 2017 Dec 1;599–600:9–13. [29] Alijagic A, Barbero F, Gaglio D, Napodano E, Benada O, Kofroňová O, Puntes VF, Bastús NG, Pinsino A. Gold nanoparticles coated with polyvinylpyrrolidone and sea urchin extracellular molecules induce transient immune activation. J Hazard Mater. 2021 Jan 15;402:123793. [30] Marques-Santos LF, Grassi G, Bergami E, Faleri C, Balbi T, Salis A, Damonte G, Canesi L, Corsi I. Cationic polystyrene nanoparticle and the sea urchin immune system: Biocorona formation, cell toxicity, and multixenobiotic resistance phenotype. Nanotoxicology. 2018 Oct;12(8) 847–67. [31] Alijagic A, Gaglio D, Napodano E, Russo R, Costa C, Benada O, Kofroňová O, Pinsino A. Titanium dioxide nanoparticles temporarily influence the sea urchin immunological state suppressing inflammatory-relate gene transcription and boosting antioxidant metabolic activity. J Hazard Mater. 2020 Feb 15;384:121389. [32] Alijagic A, Benada O, Kofroňová O, Cigna D, Pinsino A. Sea urchin extracellular proteins design a complex protein corona on titanium dioxide nanoparticle surface influencing immune cell behavior. Front Immunol. 2019 Sep 20;10:2261. [33] Pinsino A, Russo R, Bonaventura R, Brunelli A, Marcomini A, Matranga V. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPKmediated signalling pathway. Sci Rep. 2015 Sep 28;5:14492.

7 Fish and nanoparticles Rashmi Bhattacherjee, Dhriti Banerjee, Shyamasree Ghosh Abstract: The use of nanoparticles to demonstrate its various uses in fish biology has become a fascinating trend in research in recent times. The fish immune system primarily involves several innate immune barriers to combat pathogenic attack. However, in conjunction with diverse components of the cell mediated and humoral immune response, a strong and substantial protection can be conquered. The presence of NK-like cells (like NK-cells of mammals) and macrophages especially augments pathogenic disposal by several forms of molecular docking with surface antigens of the intruding microbe. Apart from that, several immunological factors including cytokines, chemokines, lytic enzymes, growth inhibitors, molecules of complement pathways, agglutinins, opsonins, antibodies, and diverse types of antibacterial peptides serve as potent immunological armors of the body. The rapid intensification of the field of nanoscience has channeled a favorable way of gathering knowledge about nanomedicine research in fish accompanied by the benefits and potential hazards of nanoparticle application, which have been elaborated in this chapter. Keywords: Fish, Pisces, nanoparticles, immune system

Abbreviations ALT AST C CRP CTLs FTIR Hb HCT hpf IFN

Aminotransferase Aspartate aminotransferase Complement C-reactive protein Cytotoxic T lymphocytes Fourier-transform infrared spectroscopy Hemoglobin Hematocrit Hours post fertilization Interferon

Rashmi Bhattacherjee, Diptera Section, Zoological Survey of India, Ministry of Environment, Forest and Climate Change, M-Block, New Alipore, Kolkata 700053, India; Department of Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India Dhriti Banerjee, Diptera Section, Zoological Survey of India, Ministry of Environment, Forest and Climate Change, M-Block, New Alipore, Kolkata 700053, India Shyamasree Ghosh, School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar an OCC of Homi Bhabha National Institute, Bhubaneswar, Odisha, 752050, India https://doi.org/10.1515/9783110655872-007

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IL LPS MCH MCHC MCV MHC MMC NL NP PMN RBC RE TCR TNF

7 Fish and nanoparticles

Interleukin Lipopolysaccharide Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume Major histocompatibility complex Melanomacrophage centers Natural killer Nanoparticles Polymorphonuclear Red blood cells Reticuloepithelium T-cell receptor Tumor necrosis factor

7.1 Introduction The robust use of nanoparticles (NPs) with increasing demands in several industries has raised concerns regarding environmental hazards as well as ecological effects. However, it is quite obvious that modern technologies with their potential roles in supporting life on earth should have some shortcomings along with their beneficial effects to mankind. The increasing use of NPs in fish industries and Ichthyology research has shed light on how NPs efficiently aid in delivering and distributing potent biochemicals to different cell populations. Research reports revealed that the zebrafish (Danio rerio) has been mostly used to study NP applications and the marine fish species were poorly studied in comparison to that of the freshwater fishes [1]. The fish immune system is well armored like that of the higher vertebrates and fishes are found to be much more dependent on the innate immune system that confers nonspecific protection [2]. Additionally, innate immune cells also aids in evoking and maintaining acquired immune response and the homeostasis by performing nonspecific actions through receptor proteins that in turn recognizes pathogenic molecular moieties such as polysaccharides, lipopolysaccharide (LPS), peptidoglycan bacterial DNA, and viral RNA [3]. Not only the components of innate and adaptive immunity facilitate pathogenic disposal in these animals, but also a group of NK-like cells (like NK-cells of mammals) and macrophages works through the immunological memory response to combat pathogenic attack in fishes [4]. However, the physical barriers and molecules of innate immune system comes to interplay with the components of cellular and humoral adaptive immune system to combat pathogens and involves several immunological factors such as include lytic enzymes, growth inhibitors, the complement pathways (the classic, the alternative, and the lectin pathway), agglutinins and precipitins (opsonins and primary lectins), cytokines, chemokines, antibodies, and antibacterial peptides [3].

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Several environmental factors including fluctuations of temperature, stress controlling, and density may repress the proper functioning of the immune cells, while some groups of food additives and immune stimulants enhance the immune efficacy [3, 5].

7.2 Core immune components in fish In several teleost fishes, such as in kili (Pseudepiplatys annulatus) and rainbow trout, the ontogeny of immune system has been studied well and found that after a short period in the yolk sac, hematopoiesis transferals to the ICM has been reported [6]. Furthermore, in rainbow trout and Atlantic salmon (Salmo salar), the complete development of the anterior kidney and thymus before hatching confers immune functions at an early stage of development [7]. Although in several marine teleost species marked differences has been observed with that of the others, the development of organs of lymphomyeloid progeny includes sequences that follows kidney, spleen, and thymus [8], and the erythropoietic function of the larval spleen was found to be greater than that of the lymphopoetic function [9, 10]. Additionally, in several species, mature cells of neutrophils was observed at 72 hpf at different tissue level [11]. Though it can be proclaimed that the thymus was the first of the largest lymphoid organs to develop in freshwater teleosts, there was reports of some hematopoietic progenitors (except lymphocytes) in the anterior kidney that develop before the complete growth and differentiation of the thymus[12]. The first appearance of IgM in lymphocytes is highly variable among several fish species [13] while the first appearance of B-lymphocytes and immunoglobulins delays in marine species in comparison to the fresh water species [3] and morphologically speaking, during the first expression of IgM, larvae achieves the length of 20–30 mm [13, 14]. It was also reported that the maternal antibodies that are transferred to eggs and embryos [15] during the early stage of development gives protection to eggs against some specific groups of pathogens by hindering the vertical transmission, also maternal IgM may induce phagocytosis or the initiation of complement pathways and can even serve as a nutritional yolk protein [13]. Furthermore, in the unfertilized eggs of spotted wolfish (Anarhichas minor Olafsen), the complement component C3 was detected which confirms the process of maternal transfer in fishes [16].

7.3 Thymus These two lobed organs consisting of subcutaneously arranged thin sheet of oval lymphoid tissue, situated in the dorsal commissure of the operculum and the

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mucous tissue of the pharyngeal epithelium, lines it [17]. Principally, the thymus harbors dense population of macrophages that uphold T-cell proliferation [18]. Due to strong variability of thymic structure development in teleosts, and many other fish species, the discrimination between the cortex and medulla is found to be inconspicuous than that of the higher vertebrates [19].

7.4 Kidney In several species of teleosts, kidney serves the function of bone marrow and considered to be the principal site of hematopoiesis until maturity [6].The anterior kidney made of a complex of reticular fibers, providing strength to lymph tissue and these fibers are basically dispersedly poised among the hematopoietic cells that line the sinusoid RE [20]. The anterior kidney in fishes includes a variety of cells involved in conferring immunity such as macrophages, the aggregated forms of which are known as MMCs, and lymphoid cells, which are commonly found as Ig+ cells (B-cells) [20]. The function of lymphoid cells is principally controlled by the facets of interactions provided by reticular cells [20] and by the cells of sinusoidal endothelium [3]. The endothelial cells are specialized in performing endocytosis [21].

7.5 Spleen The spleen in most species is composed of splenic ellipsoids that are amassed together and are structured around the MMCs and lymphoid tissue [22]. These macrophages in turn are involved in phagocytosis of antigens that invades the host immune system and the antigens are held as a moiety of metabolite or antibody to build first-rate immunological memory [3]. It was revealed in a study that after 3 months post fertilization, spleen contains a much higher density of lymphoblast and the antigens are usually caught by the splenic ellipsoids.

7.6 Cells involved in immune response The lymphocytes of fishes are equivalent to T-cells, B-cells, cytotoxic cells (NK-like cells), macrophages, and PMN leukocytes of mammals [3]. The production of antibody in fishes is solely dependent on the active responses of subsets of T lymphocytes against mitogens, acute allograft reactions by B-cell, mixed leukocyte responses, and compliant communications between T-cells, B-cells, and macrophages [3]. Furthermore, in teleosts and elasmobranchs, the MHC and TCR were found most primitively among vertebrates [23].

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7.7 Nonspecific immunity The innate immune system is extremely well armored in fishes that predominantly combats pathogenic attacks as the adaptive immune system is not that well defined in comparison to other animals[3]. Additionally, their poikilothermic nature, limited stock of antibodies, as well as the proliferation, maturation, and the process of immunological memory formation were found to be very sluggish [24]. The skin, gills, and alimentary tract of fishes being constantly dipped in water contains epithelial and mucosal barrier which gives protection against potentially harmful compounds [25].

7.8 Physical barriers The first line of defense in fishes includes flakes, gills, and skin mucus [17, 26, 27] among which the mucus contains lectins, lysozymes, complement proteins, pentraxins, antibacterial peptides, and immunoglobulin M (IgM), which deters/hinders pathogenic entry [28–32]. Additionally, the epidermis combat diverse attacks including the cellular hyperplasia, aids in maintaining osmotic balance and hinders foreign particles entry [33]. Furthermore, lymphocytes, macrophages, and eosinophilic granular cells serve as the defending weapons of the immune system [17, 34, 35].

7.9 Nonspecific cellular cytotoxicity The activity of nonspecific cytotoxic cells has been observed in many fish species, including catfish, rainbow trout [34], common carp (Cyprinus carpio) [37, 38], damsel fish (Dascyllus albisella) [39], and tilapia (Oreochromis spp.) [40]. The cytotoxic responses in fishes are achieved by NK-like cells like that of the mammals and it was reported that the nonspecific cytotoxic cells of catfish are functionally similar to that of the large granular lymphocytes of mammals, whereas both are dissimilar morphologically [41]. These cells are involved in spontaneous disposal of xenogeneic targets that also includes parasites in fish [42].

7.10 Antimicrobial peptides The integument and its secretions [43] contains low-molecular-weight antimicrobial peptides that were found to play major roles in host defense for the elimination viruses and bacteria from the body [17, 44, 45] and are detected in the gill tissue, mucus, and liver of teleost fish [46]. These peptides can promote lysis of the bacterial cell walls [17].

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7.11 Phagocytosis The neutrophils and macrophages are chief cells that promote phagocytosis in fish [47] by the generation of reactive oxygen species (ROS) in the course of a respiratory burst [3]. The cytoplasmic granules of neutrophil contains myeloperoxidase activity which in conjunction with halide and hydrogen peroxide performs halogenation of the bacterial cell wall and also their lysosomes are found to be loaded with lysozymes and several types of hydrolytic enzymes [35] which facilitates phagocytosis and disposal of intruder pathogen [3]. Furthermore, as the phagocytosis is least effected by temperature fluctuation, it is one of the most important immune process found in poikilotherms [13, 48, 49].

7.12 Tumor necrosis factor Reputed research reports revealed that in several fish species including rainbow trout, turbot, sea bream (Sparus aurata), goldfish (Carassius auratus), and catfish, TNFα and -β activators lead to the activation of macrophages, leading to augmented respiratory activity, phagocytosis, and nitric oxide generation [50–52].

7.13 Interferon In vertebrates, the cytokine interferon executes its immune activities by inhibiting replication in virus-infected cells by secretion of IFNα/β [53]. The cloned INFα-1 and INFα-2 from the Atlantic salmon were characterized according to their gene structure, sequence, promoters, and stimulation of antiviral activity of interferon-stimulated genes (ISG) [54, 55] while to a greater extent the INFα-1 of Atlantic salmon was found to augment the expression of antiviral proteins MX and ISGs, which are similar to that of the mammalian INFα/β and INFγ [55]. Furthermore, it was shown in a study that rainbow trout possess three types of INFs at least [56], among which INF 3 was found to be homologous to INFα in mammals and fish, as revealed by the four conserved cysteine residues [3].

7.14 Interleukins The IL-1β, having immense role in inflammatory regulation and host defense in mammals and its homolog, was found to perform similar actions including the induction of T-cells in at least 13 species of teleost fish [25, 57]. The constitutive expression of the cloned IL-1 receptor in all body tissues of Atlantic salmon and its regulation in the anterior kidney, spleen, liver, and gills after induction with LPS

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and TNF-α, signifies the regulation of IL-1β by the IL-1 receptor during the inflammatory reaction [58, 59].

7.15 Protease inhibitors There are different types of protease inhibitors found in the body fluids of fish [60] which in turn maintains fluid homeostasis [3]. These protease inhibitors act by inhibiting the action of proteolytic enzymes secreted by invader pathogens and also participate in acute phase reactions [25]. The α-2 macroglobulin is found to be the most widely studied protease inhibitor [61].

7.16 Lysozyme This bacteriolytic enzyme was mostly studied as being distributed throughout all the body parts in most organisms and has a profound role in maintaining host defense. Several research reports have shown that in salmonids, the serum and other body fluids, secretions, mucous membranes, and tissues with high density of leucocytes, notably the kidney and intestine contain huge amounts of lysozyme in order to provide host defense response [62, 63] while monocytes/macrophages, neutrophils, and recently the eosinophilic granular cells of the intestine [34] were found to be the chief sources of lysozyme production [3]. It executes its action by hydrolyzing the peptidoglycan of bacterial cell walls with subsequent killing of the bacterial cells and are also found induce the complement system by stimulating an opsonin and phagocytes [5].

7.17 Natural antibodies Natural antibodies are one of the major components of fish immune system, which provides wide range of protection to fishes against viral, bacterial, and other pathogenic attacks. They are similar to B1 cells and perform its action without the need for induction by an antigenic stimulant [64].

7.18 Pentraxins During tissue injury, trauma, infection, as well as inflammation, the production and secretion of C-reactive protein (CRP) becomes upregulated which in turn shows immune-modulatory activity by going through the acute phase inflammatory response [65, 66]. Different groups of fish including rainbow trout [67], Atlantic salmon,

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common cod (Gadus morhua), catfish [68], dog fish (Hoplias malabaricus) [68], and halibut (Hippoglossus hippoglossus) are known to express diverse pattern of CRP during which either the amount of active CRP is decreased in serum (negative acute phase protein) [70, 71] or increased in serum (positive acute phase protein, [72]).

7.19 Transferrin Another glycoprotein called transferrin is found in serum and exudations of all groups of vertebrates and shows highly specific iron chelating efficacy. However, due to high rate of genetic polymorphism in transferrin gene, the genotypes of bacterial isolates that absorb this iron transporting protein become hugely restricted [3]. As an example, in coho salmon (Oncorhynchus kisutch), originated in Oregon, among the three genotypic determinants (AA, AC, and CC) associated with transferrin expression, only the C allele of transferrin have shown increased resistance in case of bacterial kidney disease with the [73].

7.20 Specific immune response The specific immune system in fish involves an intricate network of functionally derived cells, proteins, and signaling cascades that impart strong immune protection via several components of the cell-mediated and humoral immune responses including antigens, antibodies, and effector cells with greater affinity and responsiveness. In majority of teleost population, the major immunoglobulin is a tetramer of the IgM class that possess eight antigenic determinants [74] while the expression factors for monomeric IgM in some of the teleost are still indefinite [75]. The second effective immunoglobulin isotype in fish was found to be IgD, specifically in catfish [76]. In fish, the immunological memory response is also noteworthy and is generally caused by the increase of memory B-cell populations after first interaction with antigenic counterpart [77]. However, it was revealed that B-cells with elevated receptor affinity at suboptimal antigen levels are usually designated as memory Bcells [78]. The cellular cytotoxicity reactions augmented by leucocytes have been investigated in various fish species. However, due to the scarce knowledge about the factors that induces cytotoxicity in fish, the detailed method of activation of cytotoxicity is not well studied [78]. The adaptive immune response in mammals includes CD8+ cytotoxic T lymphocytes to combat viral pathogens using class I MHC molecules and there was several evidence indicating analogies with this mechanism of cytotoxic killing that found in fish [3].

7.21 Benefits and hazards of nanoparticles in fish

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7.21 Benefits and hazards of nanoparticles in fish Research reports revealed that the intraperitoneal injections of different doses of biocompatible zinc oxide NPs (ZnO-NPs) by means of sol–gel method Catla catla fish and cumulative effect on its hematological indicates an increase in WBCs, RBCs, hemoglobin (Hb), and hematocrit (HCT) as well as differential outcome on monocyte count while the serological analysis shown an elevated urea concentration and a reduced levels of creatinine, alanine aminotransferase, and aspartate aminotransferase [80]. Due to the vast potential in biomedical and many other industrial fields, the application of metal and metal oxide NPs is growing hugely with time. However, the potential risks to human and aquatic ecosystems associated with the usage of metal NPs, including Au, Ag, Cu, and metal oxide NPs such as TiO2, Al2O3, CuO, NiO, and ZnO. Zebrafish (Danio rerio) has not been investigated well [81]. An eminent research group has reviewed the use of Zebrafish as an animal model owing to its short life cycle and high fecundity and highlighted the acute and the chronic toxicity in zebrafish after induction with NPs and also evaluated toxicity such as immunotoxicity, developmental toxicity, neurotoxicity, reproductive toxicity, cardiovascular toxicity, and hepatotoxicity in different target organs [81]. Another review has shed light on hasty rise of the usage of metal, metal-oxide and carbon-based NPs and the potentials hazards that influences the aquatic life [81]. It was revealed that the application of nonfunctionalized engineered NPs such as titanium dioxide on immune system of fish and invertebrates have very low/negligible direct toxicity while it has hidden sublethal effects on the immune system with severe consequences, phagocytosis, and change in function of the phagocytic cells, which decrease the ability of animals to defend themselves against pathogens and infectious diseases. As the aquatic life is greatly victimized for being the major receiver of NP wastes and several other contaminants, freshwater fish Rhamdia quelen, has been used as an animal model to show the toxic effects of Titanium dioxide NPs (NPTiO2) as well as the poisonousness interrelations between NPTiO2 and lead (Pb), in regards to genetic and biochemical biomarkers [83]. The genotoxicity in blood samples and kidney was evident while no effect of NPTiO2 or in NPTiO2-Pb co-exposure on liver genotoxicity was detected [83]. However, antioxidant imbalance and increased synthesis of lipid peroxides were also observed [83]. In a research report the effect of ageing of ZnO NPs that were applied to soil and as a result of the transfer of the contaminants from soil to water toxicity hazards to aquatic organisms were evaluated by spiking the soil samples with two types of bare NPs: b1ZnO NPs (rod- and elongated-shaped) and b2ZnO NPs (nearspherical shaped) and ZnO NPs coated with (3-aminopropyl) triethoxysilane (cZnO NPs) within the soil dose range of 0–800 mg Zn kg−1 , and were left for 0, 30, 60, and 90 days to detect the effect of ageing [84]. It was investigated that b1ZnO NPs

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cause various adverse changes in fish cells including damaging the membrane function, reductase enzyme activity, and, to some extent, ROS levels whereas b2ZnO NPs and cZnO NPs influenced ROS generation on a broad scale [84]. In adult tilapia, Oreochromis mossambicus, using probit analysis, the acute toxicity of ZnO (47 nm) and TiO2 (30 nm) NPs have been analyzed and compared based on Hemato-immunological factors and also found that ZnO NPs possess higher toxicity than TiO2 NPs [85]. The effect includes reduced levels of RBC, Hb, and HCT accompanied by diminished oxygen carrying capacity of RBC and other erythrocyte indices including mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration in NPs treated groups while elevated levels of WBC, neutrophils, and monocytes and lowered lymphocyte levels were observed with increasing NP concentration [85]. In another study, the extracellular metabolites of marine bacteria (Rastrelliger kanagurta, Selachimorpha sp., and Panna microdon) were used to synthesize gold NPs and their antibacterial and antimycobacterial activities have been evaluated [86]. The outcomes of the Fourier transform-infrared spectroscopy has shown that diverse functional groups are of extracellular leads to the bio-reduction of gold ions and as an animal model exponentially growing zebrafish larvae to show potent antimicrobial antimicrobial activity [86]. Thus, it can be concluded that due to its prospective biocompatibility and a lesser amount of toxicity, gold NPs will be optimized for potential usage in pharmaceutical and biomedical companies as drug molecule [86]. The environmental consequences of small plastic particles (