Ecology of Protozoa: The Biology of Free-living Phagotrophic Protists 3030599787, 9783030599782

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Genoveva F. Esteban Tom M. Fenchel

Ecology of Protozoa The Biology of Free-living Phagotrophic Protists Second Edition

Ecology of Protozoa

Genoveva F. Esteban • Tom M. Fenchel

Ecology of Protozoa The Biology of Free-living Phagotrophic Protists Second Edition

Genoveva F. Esteban Talbot Campus Bournemouth University Poole, UK

Tom M. Fenchel Marine Biological Laboratory University of Copenhagen Copenhagen, Denmark

ISBN 978-3-030-59978-2 ISBN 978-3-030-59979-9 https://doi.org/10.1007/978-3-030-59979-9

(eBook)

# Springer Nature Switzerland AG 1987, 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Photo: Hilda Canter-Lund, with permission from the Freshwater Biological Association (UK) This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

The scope of this book remains that of the first edition: to make protozoologists as well as ecologists aware of the role protozoa play in nature. We emphasise functional properties of different protozoan phenotypes and experimental work, as well as properties of the habitats in which the different protozoan species live. During the last few decades, genomic approaches have enjoyed increasing popularity in the context of microbial ecology. There is no question that the development of molecular biology has had a tremendous impact on biological sciences—among many other aspects and in our context, a fundamentally new understanding of the phylogeny of microbial organisms and also in unravelling complexes of sibling species. But it is not—as some seem to believe—a substitute for organismal biology: the study of phenotypic traits and the environment in which the different organisms live, which is our purpose with the book: microbial ecology is more than just SSU ribosomal DNA sequencing. In this book, we have also cited many “older” works, that is, from the “pre DNA-sequencing era” which still contain many important discoveries and observations. Poole, UK Copenhagen, Denmark

Genoveva F. Esteban Tom M. Fenchel

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Acknowledgements

Special thanks to Dr. John R. Dolan, Station Zoologique, Villefranche-sur-Mer, France, who insisted that a second edition of the book should be written, and even contacted the publisher to that end. We are grateful to Carlos F. Finlay for producing some of the artwork in this edition of the book. We express our gratitude to the following colleagues for allowing us to use their micrographs and illustrations: Simon Abitabile, O. Roger Anderson, Wolfgang Bettighofer, Hilda Canter-Lund, Ken J. Clarke, Steffen Clauss, John R. Dolan, Bland J. Finlay, Hunter N. Hines, Håkan Kvarnström, Barry Leadbeater, David J. Patterson, Michael Plewka, Benedikt Pleyer and James Weiss.

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Contents

1

What Is a Protozoon? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Historical Views on the Nature of Protozoa . . . . . . . . . . . . . . . . 1.2 The Origin and Diversification of Protists . . . . . . . . . . . . . . . . . 1.3 The Species Concept in Protozoa . . . . . . . . . . . . . . . . . . . . . . . 1.4 The Structural Complexity of Protozoa . . . . . . . . . . . . . . . . . . . 1.5 Unicellularity, Death, and Sex . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 The Size Range of Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 3 5 7 9 13

2

Motility: Life in Syrup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 21

3

Orientation in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Mechanisms for Orientation in the Environment . . . . . . . . . . . 3.2 Geotaxis and Responses to Light . . . . . . . . . . . . . . . . . . . . . . 3.3 Jumping Ciliates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Dispersal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

23 23 26 28 29 30

4

Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Feeding in Real Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Raptorial Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Diffusion Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

33 33 41 44 50 51

5

Bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Balanced Growth: Efficiency of Conversion and the Relative Importance of Power Generation for Different Functions . . . . . 5.2 The Rate of Living . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Facultative Anaerobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Obligate Anaerobic Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Prokaryotic Symbionts of Anaerobic Ciliates . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

55

. . . . . .

55 57 58 59 60 61

ix

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Contents

6

Polymorphic Life Histories and Sex . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Adaptations to a Feast and Famine Existence . . . . . . . . . . . . . . 6.2 Sex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 63 67 69

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The Niches of Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Steady-state Phagotrophic Food Chains . . . . . . . . . . . . . . . . . 7.2 Patchiness and Successional Patterns . . . . . . . . . . . . . . . . . . . 7.3 Niche Differentiation and Coexistence . . . . . . . . . . . . . . . . . . 7.4 Biogeography of Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

71 71 74 77 81 84

8

Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 The Definition of Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Associations with Photosynthetic Organisms . . . . . . . . . . . . . . 8.3 Non-photosynthetic Symbionts . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 87 . 87 . 88 . 96 . 101

9

Marine Habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Marine Pelagic Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 The Sea Floor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 The Interstitial Biota of Sandy Sediments . . . . . . . . . . . . . . . . 9.4 Silt and Clay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Deep Sea Sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Algal Mats, Tidal Pools, and Sulphureta . . . . . . . . . . . . . . . . . 9.7 Detrital Matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

107 107 115 117 121 123 123 126 128

10

Protozoan Communities in Freshwater Habitats . . . . . . . . . . . . . . 10.1 Benthic Protozoan Communities . . . . . . . . . . . . . . . . . . . . . . 10.2 Planktonic Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Running Waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Organic Enrichment and Polluted Waters . . . . . . . . . . . . . . . . Appendix: Protozoan Names in Fig. 10.7 . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

133 135 140 148 150 152 153

11

Protozoan Communities: Terrestrial Habitats . . . . . . . . . . . . . . . . . 157 11.1 The Role of Protozoa in Soil Ecosystems . . . . . . . . . . . . . . . . . 167 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

12

Symbiotic Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

13

Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

1

What Is a Protozoon?

1.1

Historical Views on the Nature of Protozoa

The question as stated by the chapter heading is not trivial. Many standard textbooks define protozoa as “unicellular animals”, but this is not satisfactory. The idea that protozoa are unicellular in the sense that they correspond to a single cell of a multicellular organism was first conceived about 200 years after their discovery by Leeuwenhoek in 1674. The term “Protozoa” was coined by Goldfuss in 1817 to mean “original animals” and he included the coelenterates. The title of Ehrenberg’s memoir: “Die Infusionsthierchen als volkommene Organismen” (“The Infusoria as Complete Organisms”) from 1838, which includes small multicellular organisms such as rotifers, alluded to the idea that protozoa were quite comparable to real animals with, e.g. the feeding vacuole being a stomach and so on (Esteban and Finlay 2002). D’Orbigny, who coined the name Foraminifera in 1826, considered them to be a kind of cephalopod because of the resemblance between the test of (some) foraminifera and the shell of the mollusc Nautilus. It was only in the middle of the nineteenth century, after the cell was recognized as a building unit of animals and plants, that the idea of protozoa as single cells comparable to cells of multicellular organisms became generally accepted. This idea then led to the theory that multicellular organisms originated as protozoan cell colonies. In a certain sense, we have in this book adopted Ehrenberg’s concept as “complete organisms”. Protozoa interact with their environment and with other organisms just like multicellular organisms and the individual, whether uni- or multicellular, is the basic unit of natural selection. So, in this sense protozoa are “complete organisms”. Traditionally, life was considered to belong either to the plant or to the animal kingdom—although the botanists have more recently yielded the fungi and the bacteria to mycologists and microbiologists, respectively. With respect to eukaryote microorganisms, some groups consist exclusively of obligate phototrophs while others are exclusively phagotrophs or parasites. But several groups include species that are either phototrophs or phagotrophs and in some cases are mixotrophs. # Springer Nature Switzerland AG 2020 G. F. Esteban, T. M. Fenchel, Ecology of Protozoa, https://doi.org/10.1007/978-3-030-59979-9_1

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1 What Is a Protozoon?

Mixotrophs are species that can perform both photosynthesis and also feed on particulate food; this is the case, for example, among some dinoflagellates. As a result, botanists and zoologists both claimed ownership for these groups in part resulting in two sets of nomenclature which was not useful. In this context, the term “protists”, originally coined by Haeckel (1866), was revived to designate unicellular eukaryotes whether green or not (Corliss 1984) and sometimes the term is used to designate all eukaryotic organisms that are not metazoans, land plants, or fungi. The concept of Protozoa has no particular phylogenetic significance since they include a number of independent eukaryotic lineages (Simpson and Eglit 2016; Adl et al. 2019). We are here primarily concerned with ecology so we can define protozoa in a functional sense as phagotrophic protists and we will consider only free-living forms.

1.2

The Origin and Diversification of Protists

The most fundamental distinction which can be made in the living world is that of between prokaryotes and eukaryotes, and the protists unambiguously belong to the latter group. They possess a nuclear envelope, eukaryotic ribosomal RNA, and endoplasmic membranes. They also have characteristic eukaryotic organelles (mitochondria, chloroplasts, and flagella/cilia), histones associated with chromosomal DNA, a cytoskeleton, and the ability to perform phagocytosis although some of the above-mentioned features may be absent in some forms. Also, eukaryotic cells are in almost all cases larger than prokaryotic cells. In spite of the apparent discontinuity between prokaryotes and eukaryotes, it is evident that the latter are in some way descended from the former. The eukaryote cells have a much more complex structure than the prokaryotes and the geological record shows that the prokaryotes came into existence about 2 billion years before the origin of the eukaryotes. It was early suggested that some characteristic eukaryote organelles, namely chloroplasts and mitochondria, derived from endosymbiotic bacteria. These ideas were taken seriously by Margulis (1981) who accumulated evidence to the effect that eukaryote chloroplasts derived from endosymbiotic cyanobacteria and mitochondria from endosymbiotic aerobic bacteria and so made eukaryote cells capable of photosynthesis and of aerobic metabolism. These ideas are now firmly established (see Archibald 2014). The organelles in question have their own genome and prokaryote-like ribosomes that show the descent from cyanobacteria and α-Proteobacteria, respectively. Mitochondria serve a number of functions besides of oxidative phosphorylation and occur in all eukaryote cells—in a modified form even in obligate anaerobes (see Chaps. 5 and 8). Today, molecular evidence shows beyond doubt that these organelles originated as prokaryote endosymbionts, and it is also supported by cases where other endosymbionts are evolving into organelles (see Chap. 8). Molecular evidence indicates that the origin of eukaryotes was in the form of an archaebacterium (Archaea) which contained aerobic proteobacteria as endosymbionts. The problem is that the host cell must have had the ability to

1.3 The Species Concept in Protozoa

3

phagocytize another bacterium. But so far no extant prokaryote with the capability to phagocytize particular matter has been found. The fossil record of Precambrian protists is very limited and difficult to interpret. Only forms that build tests or skeletal structures will leave traces. Such remains have been found throughout the Proterozoic period, but provide very limited information with respect to the taxonomic position of these organisms (Glaessner 1984; Javaux and Lepot 2018). From the Phanerozoic period there is a considerable amount of evidence for protozoa that produced tests, cell walls, or skeletal material and these can often be assigned to extant groups of protozoa. Molecular methods have provided a real insight into the phylogenetic relationships among the eukaryotes (Burki et al. 2020; Keeling et al. 2005; Pawlowski 2014; Simpson and Eglit 2016). Briefly, the eukaryotes include five “supergroups”. The supergroup Archaeplastida (Plantae) includes the red algae, the green algae, and “land plants”. They are characterized by the fact that their chloroplasts derive directly from endosymbiotic cyanobacteria and that all extant representatives have lost the ability of phagocytosis. Photosynthetic forms also occur in other supergroups, but in these cases, the chloroplasts are secondary in the sense that they derive from endosymbiotic unicellular green or red algae. The remaining four supergroups all include what we refer to as protozoa, and what is traditionally referred to as flagellates occur in all the groups. The supergroup referred to as opisthokonts include among other groups the fungi, the naked amoebae, and the choanoflagellates that again constitute a sister group to the animals. Major groups in the traditional classification of the protists such as zoo- and phytoflagellates and sarcodines have no phylogenetic basis. But some lower-level groups like, e.g. the dinoflagellates, ciliates, foraminifera represent real monophyletic unities. The new tree of the eukaryotes shows that the deepest branching took place early in the Precambrian period.

1.3

The Species Concept in Protozoa

The ability to recognize species and the information which is built into the taxonomic system are both vital components of field and experimental ecology. Without the confident identification of organisms, the interpretation of experimental results and observations would become imprecise and possibly not reproducible. Unfortunately, the species systematics of many protozoan groups still poses problems and its degree of resolution is probably quite uneven. It is also important to emphasize that there is no theoretical-based species concept for asexual organisms and many protists are asexual and so species can only be based on phenotypic traits and the fact that organisms tend to occur in discrete groups with respect to their phenotype. In common with other eukaryotic groups of organisms, protozoan species are primarily based on morphological traits. However, the amount of detail that can be observed in protozoa varies tremendously from group to group. The fact that the two protozoan groups in which most species have been described are the foraminifera

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1 What Is a Protozoon?

and the radiolarians and this is probably not accidental. Both groups include relatively large organisms that produce skeletal material or tests that can be easily preserved, and also reveal much detail in the light microscope. Conversely, small amoebae show comparably little detail and the species systematics is accordingly cruder. In the case of many small heterotrophic flagellates, a satisfactory species taxonomy based on morphological traits often requires electron microscopy. Molecular methods and in particular the sequence of the gene for the small subunit of 18S ribosomal RNA have played an increasing role in supporting species identifications, and particularly in the detection of protists that are challenging to identify by light microscopy (Hines et al. 2018; Stern et al. 2018). However, this gene may also show polymorphy within nominal species (e.g. Finlay et al. 2006). In outbreeding forms it is in principle a question of establishing species according to the biological species concept, that is, a population of organisms that share a common gene pool—a definition that, of course, applies to sexual outbreeding forms. Such an analysis was first attempted by Sonneborn on the ciliate Paramecium aurelia (Sonneborn 1957, 1975). Mating experiments showed that this “species” is really a complex of fourteen species that are genetically isolated in terms of the biological species concept. Closer studies have revealed that these sibling species (or syngenes as Sonneborn called them) do in fact show slight morphologic differences and may to some extent be identified microscopically. A similar system has been demonstrated by Nanney and co-workers (Nanney 1982; Nanney et al. 1980) for the popular ciliate Tetrahymena pyriformis. This complex has been shown to consist of seventeen isolated groups. Among these, however, there are four nonsexual, amicronucleate strains, characterized only by isozyme patterns while the others are outbreeders and thus true species according to the biological species concept. Based on rRNA genes and the “molecular clock” it could be shown that the age of the Tetrahymena pyriformis complex is 30–40 million years (Nanney 1985). This suggests that protozoan phenotypes have been conserved over long geological periods. Such SSU rRNA genotypic variation may correlate with particular adaptations. The ciliate Cyclidium glaucoma is known to be very euryhaline and can be found in all salinities from freshwater to hyperhaline conditions. Finlay et al. (2006) found among 31 isolates collected in different continents correlation between the SSU rRNA and their tolerance range to different salinities, independently of the geographical location. It is not known if these are syngenes (in the sense of Sonneborn) and if/or how many are sexual. It has been suggested that the members of complexes of genetically isolated forms should be considered as “real species” and the members should be named as has already been the case of the members of the Paramecium aurelia complex (Corliss and Daggett 1983; Catania et al. 2009). The phenomenon is interesting and maybe widespread among outbreeding protists, but providing these sibling species with a name may from a pragmatic point of view perhaps be a questionable practice.

1.4 The Structural Complexity of Protozoa

1.4

5

The Structural Complexity of Protozoa

The concept of protozoa as single-celled organisms gives many people the impression of primitive, archaic organisms, the paradigm of which is the seemingly structure-less slime that makes up an amoeba. It is true that the constraints on size at the protozoan level of organization set certain limits on structural complexity. Yet protozoa have evolved over a time span which exceeds that of metazoan evolution by hundreds of millions of years. During this time span, different kinds of protozoa have adapted to all types of environments and to different prey, predators, and symbionts. As a result of this, protozoa show specializations, complex morphological adaptations, and life cycles that have no counterpart in the individual cells constituting multicellular organisms. Figure 1.1 of the external morphology of the hypotrich ciliate Euplotes exemplifies this complexity. It shows the cirri (bundles of individual cilia) with which the organism can walk on surfaces and the row of ciliary membranelles with which the cell filters suspended food particles. Also seen are the dorsal “bristles”: short, immobile cilia which have been speculated to serve as mechanoreceptors. Figure 1.2a shows the “Müller vesicle” of the ciliate Loxodes. This organelle serves as a gravity sensor, informing the geotactic ciliate what is up and what is down (Fenchel and Finlay 1984, 1986; Finlay and Fenchel 1986; see also Chaps. 3 and 10). The dinoflagellate Erythropsidinium has a structure called the ocellus (Fig. 1.2b)

Fig. 1.1 The complex surface structure of the hypotrich ciliate Euplotes moebiusi seen with the scanning electron microscope. The ventral side (left) shows bundles of cilia (cirrie) with which the cell walks on solid surfaces, and the peristome with the ciliary membranelles. The dorsal side (right) shows the short “sensory” cilia. Scale bar: 10 μm

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1 What Is a Protozoon?

Fig. 1.2 Complex structures in protozoa. (a) The Müller vesicle of the ciliate Loxodes striatus, an organelle that functions as a gravity sensor. The statolith (a membrane-covered conglomerate of barite granules—inset photograph) is held by a rigid stalk connected to a pair of basal granules which are invaginated from the cell surface. The figure shows the two possible orientations of the Müller body for the four ways in which Loxodes orientates in the environment (Illustration courtesy of Bland J. Finlay). (b) The planktonic dinoflagellate Erythropsidinium (¼Erythropsis) pavillardi with an “eye” (arrowhead) consisting of a lens and a pigmented cup. Scale bar: 50 μm. Redrawn from Greuet (1968)

which is a light-sensitive receptor that, in all respects, resembles an eye, including a pigment capsule and a lens, and it is usually assumed that it allows phototactic behaviour in this oceanic plankton organism (Greuet 1968; Hayakawa et al. 2015). A final example of structural complexity in protozoa is illustrated by the lorica of acanthoceid choanoflagellates (Fig. 9.3e, here represented by Diaphanoeca grandis). These organisms which abound in marine plankton build complex siliceous loricas that vary considerably in size and structure among different species. The function of the lorica has so far been a matter of speculation.

1.5 Unicellularity, Death, and Sex

1.5

7

Unicellularity, Death, and Sex

The definition of protozoa as unicellular organisms is not as simple as it might sound. With respect to some definitions of protists, these include multicellular organisms such as large algae, but even among the protozoa, we find many examples in terms of cell colonies that have arisen independently. Many large protozoa (e.g. large ciliates, amoebae, and foraminifera) are multinucleate. Functionally, no doubt, this reflects the need for a large nuclear surface through which enough RNA can be transported to supply a large cell. This situation could have been brought about by endomitosis or, in some cases, by fusion of single cells into a syncytium as in some acellular myxamoebae (the so-called slime moulds). Other species may form cell colonies which obviously originated from incomplete cell divisions, e.g. the dinoflagellate Polykrikos, and colonial peritrich ciliates (Fig. 1.3). There would seem to be another fundamental difference between true multicellularity and protozoa that form cell colonies. In the latter case, all cells may potentially form new colonies and so remain potentially immortal (but see below). In multicellular organisms, some cells (somatic cells) form tissue with some special function, but they are mortal. Other cells (the germ line) are cells that produce gametes, eggs, seeds, or spores and they are potentially immortal. The resulting phenotype is a cell colony which is the result of mitotic cell divisions of the fertilized egg that determines the Darwinian fitness of the individual, but it is mortal. Only the gonadal cells are immortal while the somatic part of the phenotype is doomed.

Fig. 1.3 Colonial protozoa. (a) Polykrikos schwartzi, a phagotrophic dinoflagellate that may have evolved through incomplete cell divisions in an ancestor that looked like a typical dinoflagellate. It has four nuclei but eight pairs of flagella; typical dinoflagellates have only one pair of flagella and one nucleus (redrawn from Grassé 1952). (b) Zoothamnium hiketes, a colonial peritrich found on the body surface of the amphipod Gammarus. Each individual colony develops from a single cell through repeated cell divisions. Scale bars for A and B: 50 μm

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1 What Is a Protozoon?

However, the tendency to evolve multicellular traits and cell specialization and “built-in” death can also be found among protists. The best-known example is Volvox, a photosynthetic colonial flagellate that is not really a protozoon in the present sense. It forms spherical colonies and new colonies develop inside the mother colony. The young colonies can escape and start their independent life only after the mother colony breaks open and succumbs. And hence death was introduced in the protistan world. This is beautifully illustrated in a cartoon in the book by Hegner (1938) which shows young Volvox colonies mourning at the wake of their burst mother. Many peritrich ciliates form cell colonies. According to Wesenberg-Lund (1925), the colonies of Zoothamnium geniculatum, with colonies that may grow to several millimetres in height, have a restricted life length, but only one kind of cells in the colonies produces free-swimming cells that can establish new colonies. This was later confirmed for the congener Z. alternans (Fauré-Fremiet, 1930), and more recently for the cosmopolitan Z. niveum (Bright et al. 2014) where three cell types are described in detail; macrozooids (which become the swarmers that disperse and give rise to new colonies), microzooids (in charge of feeding the macrozooids), and the terminal zooids (one at the tip of the colony and of each branch, and in charge of mitosis to produce the microzooids)—thus, in this sense, true multicellularity does occur among ciliates. The tendency to develop multicellularity has occurred independently among other protistan lineages. Molecular and structural evidence makes it highly probable that vascular plants evolved via colonial green algae. There is also strong evidence that metazoans evolved from choanoflagellate-related organisms. Many extant choanoflagellates form cell colonies and they are very similar to a cell type (the choanocytes) in sponges. This was suggested more than a century ago and is supported by similar structural details at the cellular level as well as molecular data (e.g. Leadbeater, 2015). But there is no evidence of a distinction between a germ line and somatic cells in extant colonial choanoflagellates. Members of many groups of protozoa apparently do not have sexual processes. This applies to amoebae, to several flagellate groups, and to several minor taxa within most major groups of protozoa, while other groups include members that have sex as an obligatory part of their life cycle or under certain environmental conditions; ciliates, for example, may enter into conjugation (where two cells attach to each other and exchange haploid micronuclei, Fig. 6.3) in response to a change in the environment. The origin of sex is probably primarily to adopt diploid cell nuclei because this reduces the effect of recessive deleterious mutations, and also the origin of recombination that implies DNA repair. The distribution and variation of sexual processes among different major taxa would suggest that sexual processes arose independently in different groups. The adaptive nature of sex is discussed further in Chap. 6.

1.6 The Size Range of Protozoa

1.6

9

The Size Range of Protozoa

The following chapters concentrate on the functional biology of protozoa, that is, the study of physiological and structural properties from the viewpoint and structural properties of their adaptive significance. With respect to protozoology, the subject is intimately related to cell biology in general, as regards structure, function, and biochemistry. We have tried to limit the subject to its ecological and adaptive aspects: motility, for example, will be treated with emphasis on hydrodynamics and adaptive significance, with only passing reference to the molecular basis. One theme that recurs in several of the following chapters is the problem of size and scaling. Many properties are anti-intuitive simply because these organisms are small. Problems of scaling, especially the fact that basic functional properties do not simply scale as a function of body length, have long been recognized, but in spite of this, false conclusions, based on erroneous assumptions about functional aspects of microorganisms. Here we consider the constraints of the size range of protozoa. Protozoa are considered microorganisms and it is generally understood that there are constraints with respect to the possible size range of unicellular organisms. Eukaryote cells must include organelles such as the nucleus and mitochondria and this sets a lower size limit and the tiniest protozoa measure about 3–4 μm in diameter. On the other hand, there is an upper size limit and this is usually explained in terms of the supply of oxygen that depends on diffusion from the surroundings. Metazoa that exceed millimetre sizes possess a vascular system and respiratory pigments that can provide a larger body with oxygen, and for larger organisms ventilated gills or lungs with a large surface area are important. Still, the size range in terms of length of protozoa (Fig. 1.4) covers almost four orders of magnitude in size—a range that exceeds that of all vertebrates from centimetre-long small fish to the blue whale. The classical approach to this problem was to consider the protozoan cells as spheres and then estimate the diffusive flux of dissolved oxygen from the cell surface and inside the cell, a homogenously distributed consumption rate of oxygen throughout the cell, realistic respiration rates, and a diffusion coefficient for oxygen within the cell. The maximum size limit was considered to be when anoxia prevails within the centre of the cell (e.g. Rubinow 1975). The conclusion was then that a spherical protozoon cannot exceed the size of about 1 mm. The problem with this model is the assumption that oxygen uptake takes place homogeneously throughout the cells. In very small protozoa mitochondria may seem to be randomly distributed within the cell. However, in medium- and larger-sized protozoa the mitochondria are mainly concentrated immediately beneath the cell surface and so a protozoon that respires oxygen should rather be modelled as a body with an absorbing surface (Fenchel 2014). For a sphere that absorbs a solute from the surroundings it applies that R ¼ 4πr0 DC(1), where R is the rate of uptake (respiration), D is the diffusion coefficient for the solute in water, r0 is the radius of the sphere, and C(1) is the bulk concentration at a distance from the absorbing sphere (Berg 1983). Except for the smallest species, protozoa are rarely spherical. Berg (op. cit.) provides two other examples: a disc and a cylinder; in the former, uptake is

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1 What Is a Protozoon?

Fig. 1.4 The size range (length): the range covering all organisms (left) and that of different groups of protozoa

proportional to the diameter of the disc, and for a cylinder, it is proportional to its length and also find that in general the uptake is roughly proportional to the greatest length of the absorbing object of all shapes. And so we could expect that while tiny protozoa may have a spherical shape, larger species should be very oblong or flattened, which also tends to be true (Fig. 1.5). To complete the model of protozoan respiration we must note that the model only applies to very low O2 concentrations since the capacity for cells to reduce oxygen in practice has an upper limit. So, we assume a maximum rate of oxygen uptake Rm, and so the equation above becomes R ¼ 4πr0DC(1)[1  R/Rm]. Solving for R we have: R¼

R m C ð 1Þ , ½K m þ C ð1Þ

ð1Þ

with Km ¼ Rm/(4πr0 D). An example is shown in Fig. 1.6. See also Fig. 1.7. Km ¼ has the dimension of concentration and is the bulk concentration of dissolved O2 that supports a respiration rate that is half the value of Rm. Provided that Rm as being proportional to (cell volume)3/4 which is approximately true (Fenchel and Finlay 1984) then Km should scale as L5/4. As seen from Fig. 1.5, the

1.6 The Size Range of Protozoa

11

Fig. 1.5 The ratio between length and cell surface for a variety of protozoa (filled circles: ciliates and open circles: amoebae and flagellates). If these organisms were isometric the expected slope of the regression line should be 0.67, but it is 0.77 showing that the organisms become increasingly longer and/or flatter with increasing cell volume (after Fenchel 2014)

slope of the regression line is closer to unity; this is due to the fact that larger protozoans have an oblong or flattened shape. At any rate, the graph shows two important things. In the present context extrapolating regression line that cells larger than a few millimetres would have very limited respiration rates even if at oxygen atmospheric of 100% saturations. The graph also shows metazoa exceeding the mm range are capable of attaining larger sizes caused by an internal distribution system for dissolved O2 and the efficiency of ventilated gills which can only work at a larger size scale. Figures 1.5 and 1.6 show another important fact: aerobic microorganisms can thrive at very low O2 tensions and so occupy low oxygen habitats where no macroorganisms can live; in fact, many protozoa show behavioural responses showing preferences for oxygen tension within the range of 1–10% atm. sat. (Chap. 5). There are actually some unicellular organisms that attain much larger sizes. One possibility is to live in a permanently cold environment. Considering the expression for Km (1), the maximum respiration rate, Rm is highly dependent on temperature; within the physiological range, it increases by a factor of 2–3 for an increase in temperature of 10  C. In contrast to this, temperature has a much lower effect on the

12

1 What Is a Protozoon?

Fig. 1.6 The oxygen uptake of the ciliate Euplotes balteatus as function of ambient O2-tension with date data fitted to Eq. (1). (After Fenchel et al. 1989)

Fig. 1.7 The half-saturation constant (Km) for different unicellular organisms as function of size. (Redrawn from Fenchel 2014)

References

13

diffusion coefficient D which is proportional to degrees of Kelvin. Consequently, some foraminifera that live in cold seas or at greater depths can reach substantial sizes, and the Xenophyophores that occur in the deep sea at constant temperatures of about 4  C can attain sizes in the centimetre range (Fig. 9.11). Another solution for developing larger cells is to have an internal oxygen supply in the forms of endosymbiotic organisms. An example is the foraminiferan Sorites that can reach sizes of almost a centimetre (Figs. 8.3b, c and 9.1b). It contains a photosynthetic endosymbiont (the dinoflagellate Symbiodinium), and certain extinct foraminifera reached sizes approaching several centimetres (Lee et al. 1985). These arguments, although not very precise, do show that the size range of protozoa is limited by their basic organization in conjunction with fundamental physical constraints. It is also probable that these limits have been reached by real protozoa.

References Adl SA, Bass D, Lane CE, Luke J et al (2019) Revisions to the classification, nomenclature, and diversity of eukaryotes. J Euk Microbiol 66:4–119 Archibald J (2014) One plus one equals one: symbiosis and the evolution of complex life. Oxford University Press. 205pp Berg HC (1983) Random walks in biology. Princeton University Press, Princeton Bright M, Espada-Hinojosa S, Lagkouvardos I, Volland J-M (2014) The giant ciliate Zoothamnium niveum and its thiotrophic epibiont Candidatus Thiobios zoothamnicoli: a model system to study interspecies cooperation. Front Microbiol 145:1–13 Burki F et al (2020) The new tree of eukaryotes. Trends Ecol Evol 35:43–55 Catania F, Wurmser F, Potekhin AA, Przyboś E, Lynch M (2009) Genetic Diversity in the Paramecium aurelia Species Complex. Mol Biol Evol 26:421–431 Corliss JO (1984) The kingdom Protista and its 45 phyla. Biosystems 17:87–126 Corliss JO, Daggett P-M (1983) “Paramecium aurelia” and “Tetrahymena pyriformis”: current status of the taxonomy and nomenclature of these popularly known and widely used ciliates. Protistologica 19:307–322 Ehrenberg CG (1838) Die Infusionsthierchen als volkommende Organismen. Verlag von Leopold Voss, Leipzig Esteban GF, Finlay BJ (2002) Historical encounters with a little-known ciliate (Gerda glans Claparède and Lachmann, 1858) from the ‘Jungfernheide’. Protist 153:79–86 Fauré-Fremiet E (1930) Growth and differentiation of the colonies of Zoothamnium alternans (Clap. And Lachm.). Biol Bull 58:28–51 Fenchel T (2014) Respiration in heterotrophic unicellular eukaryotic organisms. Protist 165:485–492 Fenchel T, Finlay BJ (1984) Geotaxis in the ciliated protozoon Loxodes. J Exp Bio 110:17–33 Fenchel T, Finlay BJ (1986) The structure and function of the Müller vesicles in loxodid ciliates. J Protozool 33:139–145 Fenchel T, Finlay BJ, Gianni A (1989) Microaerophily of an Euplotes sp. (Hypotricida) to oxygen tension. Arch Protistenkd 137:317–330 Finlay BJ, Fenchel T (1986) Photosensitivity in the ciliate Loxodes. pigment granules, absorption and action Spectra, blue light perception, and ecological significance. J Protozool 331:534–542 Finlay BJ, Esteban GF, Brown S, Fenchel T, Hoef-Emden K (2006) Multiple cosmopolitan ecotypes within a microbial eukaryote morphospecies. Protist 157:377–390 Glaessner MF (1984) The dawn of animal Life. Cambridge University Press, Cambridge

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Grassé P-P (1952) Traité de Zoologie, I. Protozoaires. Masson et Cie, Paris Greuet C (1968) Organisation ultrastructurale de l’ocelle de deux peridiniens Warnowiidae, Erythropsis pavillardi Kofoid et Swezy et Warnowia pulchra Schiffer. Protistologica 4:209–230 Haeckel E (1866) Generelle Morphologie der Organismen. 2 vols. G. Reimer, Berlin Hayakawa S, Takaku Y, Hwang JS, Horiguchi T, Suga H, Gehring G, Ikeo K, Gojobori T (2015) Function and evolutionary origin of unicellular camera-type eye structure. PLoS One 10: e0118415 Hegner RW (1938) Big fleas have little fleas or who’s who among the protozoa. Williams & Wilkins, Baltimore. (Reprinted 1968 by Dover, New York) Hines HN, Onsbring H, Ettema TJG, Esteban GF (2018) Molecular investigation of the ciliate Spirostomum semivirescens, with first transcriptome and new geographical records. Protist 169:875–886 Javaux EJ, Lepot K (2018) The Paleoproterozoic fossil record: implications for the evolution of the biosphere during Earth’s middle-age. Earth-Sci Rev 176:68–86 Keeling PJ, Burger G, Durnford DG, Lang BF, Lee RW, Pearlman RE, Roger AJ, Gray MW (2005) The tree of eukaryotes. Trends Ecol Evol 20:670–676 Leadbeater BSC (2015) The Choanoflagellates. Evolution, biology and ecology. Cambridge University Press, Cambridge Lee JJ, Hutner SH, Bovee EC (eds) (1985) An illustrated guide to the protozoa. Society of protozoologists, P.O. Box 368, Lawrence, Kansas Margulis L (1981) Symbiosis in cell evolution. W.H. Freeman and Company, San Fransisco Nanney DL (1982) Genes and phenes in Tetrahymena. Bioscience 32:783–788 Nanney DL (1985) The tangled tempos underlying Tetrahymena taxonomy. Atti Soc Tosc Sci Nat Mem Ser Ser B 92:1–13 Nanney DL, Cooper LE, Simon EM, Whitt GS (1980) Isozymic characterization of three mating grooups of the Tetrahymena pyriformis complex. J Protozool 27:451–459 Pawlowski J (2014) Protist evolution and phylogeny. In: eLS, John Wiley & Sons, Ltd. https://doi. org/10.1002/9780470015902.a0001935pub2 Rubinow SI (1975) Introduction to mathematical biology. John Wiley & Sons, New York Simpson AGB, Eglit Y (2016) Protist diversification. Ency Evol Biol 3:344–360 Sonneborn TM (1957) Breeding systems, reproductive methods, and species problems in Protozoa. In: Mayr E (ed) The species problem. AAAS Publication, Washington, DC, pp 155–324 Sonneborn TM (1975) The Paramecium aurelia complex of fourteen sibling species. Trans Am Microsc Soc 94:155–178 Stern R, Kraberg A, Bresnan E, Kooistra WHCF, Lovejoy C, Montresor M, Morán XAG, Not F, Salas R, Siano R, Vaulot D, Amaral-Zettler L, Zingone A, Metfies K (2018) Molecular analyses of protists in long-term observation programmes—current status and future perspectives. J Plankton Res 40:519–536 Wesenberg-Lund C (1925) Contributions to the biology of Zoothamnium geniculatum Ayrton. D. Kgl. Danske Vidensk. Selskab. Skrifter. Naturvidenskab og Matematik. Afd. 8. række X. 1: 1–53 + plates I–XIV

2

Motility: Life in Syrup

All protozoa show some sort of motility: practically all forms move freely in the environment during at least some part of their life cycle. Even species that are normally sedentary show motility in the form of contraction or the ability to generate water currents from which food particles may be strained. All protozoa, of course, show a sort of motility during phagocytosis, during the process of “cyclosis” (the intracellular movement of food vacuoles and other organelles) and during cell division. These last-mentioned aspects of protozoan motility will receive little attention here. Instead, we will concentrate on swimming, the generation of feeding currents, creeping motility on surfaces, and, in a following chapter, motile behaviour that allows the organisms to orientate themselves with respect to environmental gradients of different sorts. Swimming in protozoa has often been illustrated by macroscopic analogies. As an example, ciliary swimming has been compared to that of a human swimmer—the effective stroke of a cilium being likened to the effective stroke of arms and legs. But this, in a sense, is a wrong analogy. The swimmer moves because when he or she forces parcels of water in one direction the preservation of momentum will cause the swimmer to move in the opposite direction. While moving the arms forward again, coasting occurs. So, in this case the swimmer moves along by inertial forces. This is not the case for a swimming ciliate: it is propelled forwards through viscous (frictional) forces acting on the cilia; inertial forces and momentum play no role. It can be calculated (see Berg 1983) that a spherical ciliate (radius: 50 μm) swimming at a velocity of 1 mm s
1 would, if the cilia suddenly stopped beating, come to a halt within 10
5 sec, allowing it to coast for about 5  10
2 μm. Thus, coasting is not a factor in protozoan swimming. Microorganisms live in a viscous world and they are always surrounded by a sticky coat of water. This is really an experience everyone knows: when washing hands in a bathroom without a towel one can try to wave the hands vigorously using inertial forces to get rid of the water. This works for most of the water, but a thin film of water persists and so one can then only rely on evaporation. The same applies to the protozoa, only what we consider a thin film of water is to the microorganism a # Springer Nature Switzerland AG 2020 G. F. Esteban, T. M. Fenchel, Ecology of Protozoa, https://doi.org/10.1007/978-3-030-59979-9_2

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thick layer. The so-called “no slip condition” means that water molecules adjacent to a surface do not move relative to a moving surface, and so above the surface, there will be a linear velocity gradient and the swimming protozoan is always surrounded by a sticky coat of the surrounding water in which water movement that is not parallel to the surface is not possible and in which turbulence cannot occur. Physicists express this by saying that these organisms live at low “Reynolds numbers”. Reynolds number (Re) is a dimensionless quantity that expresses the ratio between inertial and viscous forces. It is equal to (lρv)/η, where l, ρ, v, and η are a “characteristic length of the system” (in our case length of the ciliate), specific density of the medium, velocity, and viscosity, respectively. Viscosity is a measure of the internal friction in a liquid, e.g. the friction between parallel layers of liquid within a velocity gradient. The viscosity of water (20  C) is about 0.01 dyn cm
2 sec (poise). If Re < 1, viscous forces predominate and if Re > 1, inertial forces are most important. For a swimming bacterium (l ¼ 1 μm, v ¼ 30 μm s
1) Re ¼ 3  10
5. For a swimming Paramecium (l ¼ 150 μm, v ¼ 500 μm s
1), Re ¼ 7.5  10
2. In contrast, for a swimming person, Re is about 106. If a person should perform as a realistic model of the swimming ciliate it would be necessary to swim in a substrate like glycerol (viscosity about: 15 poise) and movement should not exceed about 4 mm per minute. For a general (and entertaining) discussion on “life at low Reynolds number”, see Purcell (1977). Flagella and cilia constitute the most important swimming organelles in protozoa. Both structurally and with regard to the function at the molecular level they are similar. The “sliding microtubule model” suggested and documented by Peter Satir in the 1960s is now generally accepted (see Sleigh 1974; King and Sale 2018). This principle is based on the relative sliding motion of the peripheral microtubules in the flagella mediated by molecules of the protein dynein in the presence of ATP and magnesium ions. From a hydrodynamic point of view, however, flagella and cilia differ somewhat with respect to function. There are usually only one or two flagella per cell whereas a ciliated cell has a large number of cilia. The motion of most flagella is characterized by waves (mostly in one plane) that originates at the base of the flagellum. In contrast, a cilium has only one bend at any one time. Its movement is characterized by an effective stroke in which the cilium bends at its base while the remainder of the cilium is rather straight, and a recovery stroke during which the cilium is drawn back to the initial position while the cilium is kept close and parallel to the cell surface. Typically beat frequencies of cilia and flagella are around 50 Hz, but much slower frequencies can be observed. In all cases, the motility of cilia and flagella serves to propel the organisms through the water or to generate water currents from which food particles can be strained. The flagella of flagellates come in two versions—smooth ones and “hispid” (hairy) ones. The latter type has rows of flagellar hairs, “mastigonemes”, oriented approximately perpendicular to the flagellum and this alters the hydrodynamic properties (Fig. 2.1a). Flagella and cilia cause movement because the drag on a cylinder drawn through a viscous fluid, differ according to whether it is oriented parallel to the direction of movement or perpendicular to it. In the case of a smooth cylinder, the drag perpendicular to the direction of motion is about twice that of the

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Motility: Life in Syrup

17

Fig. 2.1 (a) The freshwater Paraphysomonas butcheri as seen with the Transmission Electron Microscope, negative staining. Cell size: ~5 μm. Photo courtesy of Ken J. Clarke. (b) Prorodon ovum as seen after silver impregnation to reveal the pattern of the ciliary rows. The mouth is located at the top of the cell (dark circle). Note the meridian rows of kinetosomes, one kinetosome per cilium. Cell size: ~200 μm

parallel drag. In a smooth flagellum, therefore, the water will be propelled in the same direction as the wave propagating along the flagellum and the thrust on the flagellum from viscous forces of the water will act in the opposite direction. Choanoflagellates represent an example of flagellates with a smooth flagellum; water is propelled away from the anterior (flagellated) pole of the cell if it is attached to a substrate, while unattached cells will move through the water with the flagellum trailing after them (see Fig. 10.4). In hairy flagella, however, the perpendicular drag of the mastigonemes will exceed that of the drag parallel to the flagellum. Consequently, the generated water flow changes direction and moves in the opposite to that of the flagellar waves. Flagellates with hairy flagella, if unattached, swim with the cell trailing after the flagellum. Hairy flagella are found in many flagellate groups including chrysomonads (see Fig. 2.1a Paraphysomonas), helioflagellates, bodonids, dinoflagellates, and euglenids. Representatives from some of the mentioned groups also possess a sometimes shorter smooth flagellum which may or may not play a role in the propulsion of the organisms. In many flagellate types in which the flagellar beat lies within one plane, hydrodynamics can account for the generation of water currents and the velocity of swimming cells (Holwill 1974; Lighthill 1976). In

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dinoflagellates, there are two flagella, a trailing smooth flagellum and a hispid flagellum situated in an equatorial groove causing a rotational motion of the cell (see following section). Ciliated cells are usually densely covered with cilia arranged in rows. In some ciliates, these rows are arranged in regular meridians (Fig. 2.1b), but in many forms, they are twisted in some way, or parts of the cell may not be ciliated. One of the most obvious features of a swimming ciliate is the metachronal wave generated by ciliary motion (see Chap. 10, Fig. 10.2b). These waves occur in different patterns according to species and swimming mode. The explanation of this phenomenon has been subject to discussion for a long time and various neural or other control mechanisms have been suggested. It is now clear that it is a purely hydrodynamic phenomenon. At the low Reynolds number, each cilium is surrounded by a coat of water which follows its motion. When the cilia are sufficiently close together, neighbouring cilia will share these zones of attached water so that their beat cycles become partly synchronized. The metachronal patterns which arise are therefore solely a function of the geometry of the surface, the position of the cilia, their beat frequency, and the viscosity of the medium, but it does not imply any sort of “nervous” control on the part of the cell (Sleigh 1984). Hydrodynamic models of the motility of ciliates are less complete for ciliates than for flagellar motion. Attempts to model the motion of the cells have been made by describing the action of individual cilia, but also by considering the tips of the cilia as constituting a smooth undulating surface (“envelop models”). Both have had some success in predicting swimming speed, but cannot yet account for the great variety in geometric design of ciliates (see Holwill 1974; Roberts 1981; Lisicki et al. 2019). A special type of ciliary propulsion is provided by the ciliary membranes of some ciliates. Membranelles are rows (usually three, but from two to ten or more can occur) of very densely arranged cilia. These are not attached to each other, but due to their close proximity, hydrodynamic coupling forces them to beat fairly synchronously. Membranelle zones consist of rows of parallel membranelles. Membranelle zones are associated with the left side of the mouth in many ciliates. In hymenostome ciliates there are usually three membranelles in the oral zone; in heterotrich, oligotrich, and hypotrich ciliates the zones contain a larger number of membranelles. The main function of these membranelles is to propel and strain water for food particles, but in oligotrich and hypotrich ciliates they also serve for swimming. Although the plane of the beat of the individual cilia is perpendicular to each membranelle, water is propelled parallel to the membranelles and thus perpendicular to the zone (Fenchel 1980). An explanation of the action of membranelle zones based on the hypotrich Euplotes is offered in Fig. 2.2. In this ciliate, the membranelles beat with a frequency of about 50 Hz, and each membranelle is about one-seventh out-of-phase relative to its neighbour so that a metachronal wave moves forward along the zone away from the cytostome—the area at which food particles are phagocytized. Within each membranelle cilia are also slightly out of phase, the innermost ones being one-seventh of a beat cycle ahead of the peripheral ones. The result is that metachronal waves continuously move from the peristomal cavity outwards driving water out of the cavity rather like a number of parallel peristaltic pumps. Since each membranelle is about 10 μm wide, it is easy to

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Motility: Life in Syrup

19

Fig. 2.2 (a) Water currents generated by the membranelles of the ciliate Euplotes. (b) The mechanism for the generation of water currents. Double lines indicate the basis of the membranelles, single lines their ciliary tips. Individual cilia move in a direction parallel to the membranelle zone, but they are slightly out of phase so that waves move along each membranelle from the peristome side and outward, thus generating water currents

calculate that the metachronal waves move along them with a velocity of about 3.5 mm s
1 which is fairly consistent with the fact that the water velocity through the membranelle zone reaches about 1 mm s
1. It appears that the swimming speed is rather invariant among ciliates and flagellates, respectively: ciliates may swim with velocities up to around 1 mm s
1 irrespective of cell size and for flagellates swimming velocities are around 0.2 mm s
1 (Sleigh and Blake 1977), but in either case, the cells may modulate their swimming speed. That these organisms should be able to swim at about the same velocity irrespective of cell size will seem counterintuitive for many because small protozoa are usually observed at higher magnification than large ones, and because one tends to evaluate the speed of a moving object as a function of its size rather than according to an absolute scale. There are a number of less common swimming mechanisms in protozoa that do not rely on flagella or cilia. Some dinoflagellates are equipped with a tentacle that contracts periodically, rather like a piston (e.g. Erythropsidinium in Fig. 1.2b), and

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the dinoflagellate Noctiluca (Fig. 4.7b) propels itself by deformations of the entire cell brought about by contractions of “myonemes” that are contractile fibrillae. In the peculiar oceanic heliozoan Sticholonche the spicules are moved like oars to produce swimming (see Febvre-Chevalier and Febvre 1982 and references therein; Bray 2001). A species of Vorticella uses the contractile stalk for swimming rather than for permanent attachment, and the pelagic oligotrich ciliate, Tontonia, has a long appendage for swimming. It has often been observed that many protozoa do not swim in straight paths, but rather that the swimming path describes a helix. This is because swimming is based by some sort of deformation of the cells. Such deformation will lead to a translation in the water, but also often to a rotation of the cell. If the axis of rotation is not aligned with the direction of translation the result is that the swimming path becomes a helix. That swimming paths often form a helix rather than a straight line has been noted many times in the literature. Since many protozoa exploit this fact for orientation in chemical gradients, we will treat this phenomenon in more detail in the following chapter. Many protozoa creep or slide along on solid surfaces rather than moving by swimming. This also applies to some organisms that use flagella or cilia for locomotion. In some bodonid and euglenid flagellates, a smooth “trailing” flagellum remains in intimate contact with the substratum, while the hispid, anterior flagellum pulls forward in a manner similar to what is seen in freely swimming relatives. These sliding flagellates can also free themselves from the substratum and swim away. The forces that hold the trailing flagellum to the surface are not known, but it is reasonable to assume that van der Waals forces play a role. Many ciliates slide (e.g. cyrtophorids and loxodids) or walk (hypotrich ciliates) on solid surfaces using cilia, or in the latter case “cirri” (which are bundles of cilia—see Chap. 1, Fig. 1.1). In addition, some forms can also attach temporarily to solid surfaces using “thigmotactic cilia”. This latter term does not constitute an explanation, but again the phenomenon probably involves van der Waals forces. The amoebae, heliozoans, and a few organisms classified as flagellates that have an amoeboid phase in their life cycle use pseudopodia for locomotion. These are transitory extensions of the cells. The motility mechanism involves actin filaments which can slide relative to one another, mediated by myosin molecules and the presence of calcium ions and ATP. The generated force may act on the cell membrane or on microtubules, but the mechanism of pseudopodia formation is still not completely understood. There are several types of pseudopodia: Lobopodia, characteristic of amoebae (see Chap. 10, Figs. 10.1b, c), are thick structures and there is usually one or a few per cell at any time. Filopodia are very thin and numerous; they occur in organisms like the testate amoeba Euglypha. Reticulopodia are anastomosing networks of very slender pseudopodia supported by microtubules; they are characteristic of foraminifera and other amoeboid protozoa (Fig. 2.3a). Finally, axopodia are straight and somewhat stiff structures supported by bundles of microtubules. They occur in heliozoans (Fig. 4.7a), radiolarians, and achantarians. All types of pseudopodia also play a role in catching food particles, and axopodia are not always involved in locomotion. Some heliozoans roll along the substratum by

References

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Fig. 2.3 (a) Network of reticulopodia in the testate amoeba Leptogromia operculata. Photograph by Benedikt Pleyer. (b) An electron microscope image (negative staining) of an undescribed heliozoon (from grassland in Scotland, UK) showing the plate and radial scales. Photograph by Ken J. Clarke

alternatively shortening and extending their axopods. In other types of pseudopodia, locomotion takes place by the extension of a pseudopod, attachment to the substratum, and eventually contraction of the pseudopod. Amoeboid motility is much slower than that of ciliary swimming. Velocities of 5–20 μm s
1 are typical. Actomyosin microfilaments and in some cases pseudopodia-like structures are also involved in phagocytosis and pinocytosis, and this motile principle is also responsible for the movements of organelles within the cells. Contraction requires calcium ions and ATP. Myonemes are bundles of filaments that allow for the contraction of the cells; they are typical of heterotrich ciliates, euglenid flagellates, and some other forms. In other cases, motility occurs in which neither microtubule sliding nor actomyosin apply. In the stalk of sessile peritrich ciliates (see Fig. 10.3c), the contractile filaments, spasmoneme, consist of a protein called spasmin which coils up in the presence of Ca++. The coiling does not require ATP whereas the subsequent stretching requires energy (Amos 1975). One important aspect of motility is, of course, its control. The controlling factor seems in all cases the internal concentration of Ca++ and fluxes of this divalent metal are related to membrane potential. In motile systems based on microtubule sliding, such as in cilia and flagella, increased levels of Ca++ slow down or reverse the beat cycle, whereas hyperpolarization (and the associated efflux of Ca++) leads to an increase in beat frequency. In actomyosin microfilament-based motile systems, the influx of Ca++ leads to contraction, and the presence of the ion in the medium is necessary for the induction of food vacuole formation and for the production of pseudopodia (Cappuccinelli 1980; Naitoh and Eckert 1974; Naitoh and Sugino 1984).

References Amos WB (1975) Contraction and calcium binding in vonicellid ciliates. In: Stephens RE, Inoue S (eds) Molecules and cell movements. Raven Press, New York, pp 411–436 Berg HC (1983) Random walks in biology. Princeton University Press, Princeton

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Bray D (2001) From molecules to motility, 2nd edn. Garland Publishing, Talyor & Francis Group Cappuccinelli F (1980) Motility of living cells. Chapman and Hall Ltd, New York Febvre-Chevalier C, Febvre J (1982) Locomotion processes in some pelagic and benthic protozoa. Ann Inst Océanogr Paris 58:137–142 Fenchel T (1980) Suspension feeding in ciliated protozoa: structure and function of feeding organelles. Arch Protistenkd 123:239–260 Holwill MEJ (1974) Hydrodynamic aspects of ciliary and flagellar movement. In: Sleigh MA (ed) Cilia and Flagella. Academic Press, London, pp 143–175 King SM, Sale WS (2018) Fifty years of microtubule sliding in cilia. Mol Biol Cell 29:698–701 Lighthill J (1976) Flagellar hydrodynamics. SIAM Rev 18:161–230 Lisicki M, Velho Rodrigues MF, Goldstein RE, Lauga E (2019) Swimming eukaryotic microorganisms exhibit a universal speed distribution. Elife 8:e44907 Naitoh Y, Eckert R (1974) The control of ciliary activity in Protozoa. In: Sleigh MA (ed) Cilia and flagella. Academic Press, London, pp 305–352 Naitoh Y, Sugino K (1984) Ciliary movement and its control in Paramecium. J Protozool 31:31–40 Purcell E (1977) Life at low Reynolds number. Am J Phys 45:3–11 Roberts AM (1981) Hydrodynamic in protozoan swimming. In: Levandowsky M, Hutner SH (eds) Biochemistry and Physiology of Protozoa, 2nd edn. Academic Press, New York, pp 6–66 Sleigh MA (ed) (1974) Cilia and flagella. Academic Press, London Sleigh MA (1984) The integrated activity of cilia: function and coordination. J Protozool 31:16–21 Sleigh MAM, Blake JB (1977) Methods of ciliary propulsion and their size limitations. J Protozool 31:16–21

3

Orientation in the Environment

3.1

Mechanisms for Orientation in the Environment

It has long been known that many microorganisms—prokaryote as well eukaryotes—are capable of orientation in the environment and respond to attractants or repellents by motile sensory behaviour. In the case of protozoa, this was first studied by Jennings (1906). Among bacteriologists in particular, the term “taxis” is used for all kind of motile sensory behaviour irrespective of the involved mechanisms, with terms such as “chemotaxis”, “phototaxis”, etc. The tradition among zoologists, on the other hand, is that a taxis means a directed response so that the organisms directly sense the direction of a gradient, whereas “kinesis” refers to other mechanisms, but does not imply that the organisms can at any point detect the direction of a chemical gradient. In this case, taxis is impossible for a microorganism since this would require that the cells could sense the concentration difference between their anterior and their posterior ends, but due to the small number of molecules in the immediate vicinity of the cell and statistical noise, this is impossible. Likewise, microorganisms cannot sense the direction of water flow and thus be able to swim upstream when sensing an attractive compound in the way that many animals move against the direction of wind or water currents when sensing the smell of prey or of a potential mate. Among protozoans, the only examples of a real taxis include phototaxis and geotaxis, both of which occur only in a relatively few species and special organelles are required (see Chap. 1, Fig. 1.2a). Other types of sensory motile behaviour that make microorganisms accumulate in attractive places and avoid deterrents are referred to as “kinesis” (Dunn 1990; Van Houten et al. 1981). A general type of motile behaviour in a homogenous environment is based on “runs” and “tumbles”. During runs, the cells move along an approximately linear path. But at intervals, the runs are more or less regularly being interrupted by tumbles; that is, the cell stops and then continues in another direction that may be random or in some cases opposite to the original direction. The result is a kind of diffusion process so that the dispersal of cells is proportional to v/τ where v is the swimming velocity and τ is the mean time between tumbles (Berg 1983). # Springer Nature Switzerland AG 2020 G. F. Esteban, T. M. Fenchel, Ecology of Protozoa, https://doi.org/10.1007/978-3-030-59979-9_3

23

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3 Orientation in the Environment

It can be shown that in a patchy environment, cells will tend to accumulate where their swimming velocity is lowest—and when a steady state is reached the cell density will everywhere be inversely proportional to the local swimming velocity (Schnitzer et al. 1990). This is an important and widely occurring mechanism: if you are in a good place do not move around too much. Another important mechanism for guiding protozoa is referred to as “transient responses”, “kinesis with memory”, or “run and tumble reaction”. A protozoan cannot sense the direction of a gradient of an attractant or of a repellent at any point, but it can swim for a distance and if, it over a run, it senses an increase in the concentration of a repellent this induces tumbling or in some cases a 180o turn—an “avoidance reaction”. And conversely, if the cells experience an increase in the concentration of an attractant, tumbling is suppressed so that the cells continue in “the right direction”. This implies a sort of “memory” as they move along a gradient. This mechanism can be referred to as a “biased diffusion process”. An example is the ciliate Euplotes that walk on surfaces—as they approach the source of an attractant they keep walking without changing direction, and while moving away from the source they frequently change direction (Fenchel 2004). A variation of “kinesis with memory” is based on the fact that the swimming path of many protozoa is helical. The basic reason for this is that motion of the cells results not only in translation through the water, but also a rotation of the cell. If the axis of rotation is not parallel to the direction of the translation of the cell through the water, the swimming path of the cell forms a helix. This is very common among ciliates and dinoflagellates, and this is exploited for orientation in environmental gradients. The mechanism was first studied by Crenshaw (1993) and Crenshaw and Edelstein-Keshet (1993) and was termed “helical klinotaxis”. The underlying mechanism is that as the protozoan swims in a helical path and passes a gradient of an attractant it will alternatively sense a higher and lower concentration of the attractant as it swims along. As the cell senses an increasing concentration it can approach the source of the attractant by changing the angular velocity of one of the components of the cell’s rotation and this again results in bending the axis of the helical path towards the attractant source, as explained in the case of a dinoflagellate in Fig. 3.1. If the transversal flagellum of the dinoflagellate is inactive the cell would swim in a circle with a translation velocity of v and an angular velocity ω1. When the transversal flagellum is active this will lead a rotation with the angular velocity ω2. These rotations can be described as vectors indicating the axis of rotation and where the length of the vector is proportional to the angular velocity of the swimming path. The resultant of these rotations is the vector sum which again is parallel to the axis of the resulting helical swimming path which is completely described by the mentioned parameters (including the radius and pitch of the helix). But the essential here is that the cell by modulating ω2 can bend the axis of the helical swimming path; in Fig. 3.1 an increase in ω2 would lead to the axis of the helix bend towards the right in the figure. Figure 3.2 shows how the ciliate Strombidium approaching a drop of a bacterial suspension is applying helical klinotaxis. It can be seen that the parameters describing the helical path changes as the axis of the helix bends in some direction.

3.1 Mechanisms for Orientation in the Environment

25

Fig. 3.1 Helical swimming in a dinoflagellate. For further explanation, see text. After Fenchel (2002)

Fig. 3.2 Tracks of Strombidium sulcatum cells as they enter a cluster of bacteria (the visible part is indicated by shading, but the bacteria probably extended somewhat further). Period between dots: 0.04 s. (After Fenchel and Blackburn 1999)

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Fig. 3.3 The ciliate Uronema nigricans in an oxygen gradient. At the meniscus pO2 is 100% atm. sat., to the left of the band formed the water is anoxic. In the middle of the band (measured by inserting an oxygen microelectrode) the oxygen tension is about 10% atm. sat. Scale bar: 100 μm

Similarly, the cells can bend the axis away from a source of deterrent by changing the angular velocity with respect to one component axis. Helical klinotaxis is widespread among ciliates in order to approach sources of attractants or orientation in oxygen gradients (Fenchel and Bernard 1996, Fenchel and Blackburn 1999). Perhaps all free-living protozoa respond to oxygen tension and many prefer a pO2 within the range 0–10% atm. sat. (Fenchel and Bernard 1996, and Fig. 3.3). Oxygen tension is the major factor that determines the vertical distribution of different species in sediments and in stratified water columns (Chaps. 9 and 10). Several examples of sensory motile responses to attractants are given in Fenchel and Blackburn (1999) and modelling of the different mechanisms is treated in Blackburn and Fenchel (1999).

3.2

Geotaxis and Responses to Light

Many protozoa occur at particular depths in the water column or in sediments. In most cases, this is a response to the ambient oxygen tension in sediments or in the stratified water column, or sometimes light intensity may play a role. True geotaxis—in the meaning that the cells can sense what is up and down, has so far been demonstrated only for the freshwater ciliate Loxodes spp. and in their marine relatives, Remanella spp. (Fig. 3.4; Fenchel and Finlay 1984, Finlay et al. 1986; see also Chap. 1). Many ciliates can control their vertical position in the water column without response to light or oxygen tension, to directly detect what is up and down. Roberts (1970) modelled how ciliates may regulate their vertical position in the water column inspired by the ability of Paramecium cells to regulate its vertical position by sinking. The specific gravity (relative density) of the cells exceeds unity (like most other protozoa) and so without net upwards orientation, they will sink in the water column due to gravity. The shape of the Paramecium cells is characterized by a thicker posterior end and a slimmer anterior end. Consequently, when they

3.2 Geotaxis and Responses to Light

27

Fig. 3.4 Positive geotaxis in Loxodes striatus induced by increased light intensity in a photometer cell and recorded with dark field illumination. The cells had been kept in the dark at 5% atm. Oxygen tension (a). Subsequent recordings were made 0–5, 30–35, 120–125, and 30–305 sec after exposure to an illumination of 10 klx. And it can be seen that the cells become aligned in a vertical position and swim downwards. Scale bar: 5 mm. Below: A kinetic response towards increased light intensity in a horizontal preparation. It can be seen that the cells increase their swimming speed after being exposed to light (10 klx). Scale bar: 5 mm. (After Fenchel and Finlay 1986)

passively sink down, the cells become re-orientated so that their anterior end points upwards, and they will therefore swim upwards. However, if they make frequent tumbles so that the orientation of the cells becomes random the effect of gravity overrides the tendency to orient the anterior end upward and the cells will tend to sink. But if they sustain from tumbling their anterior end will tend to point upwards and so they will swim upwards. Thus, the cells can regulate whether they move upwards or downwards in the water column by regulating tumbling rates but it is not a true geotaxis in the sense that they can detect the direction of gravity. A similar mechanism has been found in the marine plankton ciliate Mesodinium rubrum, a ciliate that performs diurnal vertical migrations (Fenchel and Hansen 2006). The only experimentally established case of a true geotaxis is that of the freshwater ciliate Loxodes. A complex mechanoreceptor is present, the Müller vesicles (see Chap. 1, Fig. 1.2a), which can account for the behaviour (Fenchel and Finlay 1986; Finlay and Fenchel 1986; Finlay et al. 1986), but similar organelles have not yet been found in other protozoa other than their close relative Remanella spp. Loxodes is sensitive to high oxygen tensions especially when combined with light exposure, and at sufficiently high light intensities they prefer complete anoxia (Finlay et al. 1986). Cells surrounded by anoxic water remain insensitive, but cells

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in contact with oxygen are sensitive to light. Figure 3.4 shows the effect of increasing light intensity in cells suspended in water with a pO2 of 5% initially exposed to dim light. After increasing the light intensity the cells immediately align in a vertical position and swim downwards. The figure also shows a kinetic response in a horizontal preparation as an effect of increased light intensity. That phototrophic protists and protozoa with phototrophic endosymbionts are attracted by light by some mechanisms is not surprising (see Chap. 8). Paramecium bursaria normally contains symbiotic Chlorella cells (see Chap. 8). These ciliates are attracted to light, but if the endosymbionts are removed they lose this behaviour. Their light responses include both kinetic and transient ones, and they are mediated, at least in part, by oxygen produced by the symbionts in light (Cronkite and Van den Brink 1981; Esteban et al. 2010). But many ciliates without symbionts show a negative response to light in addition to the mentioned example of Loxodes. This seems to apply particularly to pigmented forms, and (besides of Loxodes) include Blepharisma and Stentor species (Fabczak 2000, Matsuoka 1983; Pill-Soon and Walker 1981). The pigments are flavins or hypericins. It is likely that—as is the case of Loxodes spp.—it is a question of oxygen toxicity. That is, when exposed to light in the presence of oxygen the pigments generate toxic oxygen radicals (superoxide and peroxide). Pigments in some ciliates act as a protection against predators—just like some plants produce hypericins for that purpose. Blepharisma japonicum is a ciliate with pink granules that give the ciliate its rose hue. The granules have five blepharismin (a type of hypericin) pigments in its extrusive pigment granules that provide both light perception and chemical defence against predators (Buonanno et al. 2017). The defensive function of the granules has also been studied by comparing “red” and albino-mutant cells of Blepharisma, respectively, as prey for the ciliate Dileptus (Miyake et al. 1990). Albino cells were eaten more readily than red cells. Furthermore, under certain conditions, Dileptus was killed by the cell-free pink fluid (blepharismins). Similar findings have been recorded for the blue Stentor coeruleus (Miyake et al. 2001).

3.3

Jumping Ciliates

Tamar (1979) brought attention to what many have observed when watching a water sample under the microscope: some ciliates, apparently spontaneously, make sudden—often backward—leaps for a short distance with a velocity that exceeds the normal swimming speed. At least in some cases, it is an avoidance reaction against predators. This has been shown for several marine plankton ciliates (Fenchel and Hansen 2006; Jakobsen 2001), and it is characteristic of the freshwater planktonic ciliate Halteria (Gilbert 1994). In these cases the escape jump response is brought about by the cells sensing shear in the surrounding water; the ciliate leaps backward even if the predator has not established contact with it. Plankton consumers, such as copepods, draw water through the bristles of the oral limbs and the resulting water currents have steep velocity gradients. Jumping behaviour can be demonstrated by

3.4 Dispersal

29

Fig. 3.5 Swimming tracts of a population of the ciliate Pseudocohnilembus pusillus in marine sediment. The cells swim slowly when feeding, with frequent changes in swimming direction, and so they are not likely to stray away. Deprived of food, the ciliates’ swimming velocity increases, and the frequency of changing direction of the swimming path drops (after Fenchel 1990)

drawing a water current into a capillary glass tube in the vicinity of the ciliates thus avoiding to be drawn into the tube.

3.4

Dispersal

We have so far mainly considered motile response to attractants or repellents. But under certain adverse conditions, it is adaptive simply to disperse. The world is patchy and if conditions become unfavourable in a particular spot it may be favourable to look for a better place. Suppose that a population of ciliates feeding on bacteria have exploited a patch for food particles. Under this circumstance, it is adaptive for the population to disperse to increase the probability that some cells will find another patch with food. It is a sort of lottery—but the further the cells can disperse the higher is the probability that some of the cells may find a favourable patch. Figure 3.5 shows the swimming tracts of a population of the bacterivorous

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ciliate Pseudocohnilembus pusillus that naturally occurs in or on marine sediments. While feeding, the cells swim slowly and with frequent changes in swimming direction, and so they are not likely to stray away. But a few minutes after the cells have been deprived of food swimming velocity increases and the frequency of changing the direction of the swimming path drops. Calculations based on these data show that their tendency to disperse (“diffusion coefficient”) increases by a factor of about 50 as an immediate response to the disappearance of food particles.

References Berg HC (1983) Random walks in biology. Princeton University Press, Princeton, NJ Blackburn N, Fenchel N (1999) Modelling of microbial patch encounter by chemotactic protozoa. Protist 150:337–343 Buonanno F, Anesi A, Guella G, Ortenzi C (2017) Blepharismins used for chemical defense in two ciliate species of the genus Blepharisma, B. stoltei and B. undulans (Ciliophora: Heterotrichida). Eur Zool J 84:402–409 Crenshaw HC (1993) Orientation by helical motion. Microorganisms can orient to stimuli by changing the direction of their rotational velocity. Bull Math Biol 55:231–255 Crenshaw HC, Edelstein-Keshet L (1993) Orientation by helical motion. 2. Bull Math Biol 55:212–230 Cronkite D, Van den Brink S (1981) The role of oxygen and light in guiding “photoaccummulation” in the Paramecium bursaria-Chlorella symbiosis. J Exp Zool 217:171–177 Dunn GA (1990) Conceptual problems with kinesis and taxis. In: Armitage JP, Lackie JM (eds) In Biology of the chemotactic response. Cambridge University Press, Cambridge Esteban GF, Fenchel T, Finlay BJ (2010) Mixotrophy in ciliates. Protist 161:621–641 Fabczak H (2000) Protozoa as model system for studies of sensory light transduction: photophobic response in the ciliate Stentor and Blepharisma. Acta Protozool 39:171–181 Fenchel T (1990) Adaptive significance of polymorphic life cycles in Protozoa: responses to starvation and refeeding in two species of marine ciliates. J Exp Mar Biol Ecol 136:159–177 Fenchel T (2002) How dinoflagellates swim. Protist 152:329–338 Fenchel T (2004) Orientation in two dimensions: chemosensory motile behaviour of Euplotes vannus. Eur J Protistol 40:49–55 Fenchel T, Bernard C (1996) Behavioural responses in oxygen gradients of ciliates from microbial mats. Eur J Protistol 32:55–63 Fenchel T, Blackburn N (1999) Motile Chemosensory behavior of phagotrophic protists: mechanisms for and efficiency in congregating at food patches. Protist 150:325–336 Fenchel T, Finlay BJ (1984) Geotaxis in the ciliated protozoon Loxodes. J Exp Biol 110:17–33 Fenchel T, Finlay BJ (1986) Photobehaviour of the ciliated Protozoon Loxodes: taxic, transient and kinetic responses to the presence and absence of oxygen. J Protozool 33:139–145 Fenchel T, Hansen PJ (2006) Motile behavior of the bloom-forming ciliate Mesodinium rubrum. Mar Biol Res 2:33–40 Finlay BJ, Fenchel T (1986) Photosensitivity in the ciliate Loxodes. pigment granules, absorption and action Spectra, blue light perception, and ecological significance. J Protozool 331:534–542 Finlay BJ, Fenchel T, Gardner S (1986) Oxygen perception and 02 toxicity in the freshwater ciliated protozoon Loxodes. J Protozool 33:157–165 Gilbert JJ (1994) Jumping behavior in the Oligotrich Ciliates Strobilidium velox and Halteria grandinella, and its significance as a defense against rotifer predators. Microb Ecol 27:189–200 Jakobsen HH (2001) Escape response of planktonic protists to fluid mechanical signals. Mar Ecol Prog Ser 214:67–78 Jennings HS (1906) Behaviour of Lower Organisms. Columbia University Press, New York

References

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Matsuoka T (1983) Negative phototaxis in Blepharisma japonicum. J Protozool 30:409–414 Miyake A, Harumoto T, Salvi B, Rivola V (1990) Defensive function of pigment granules in Blepharisma japonicum. Eur J Protistol 25:310–315 Miyake A, Harumoto T, Lio H (2001) Defence function of pigment granules in Stentor coeruleus. Eur J Protistol 37:77–88 Pill-Soon S, Walker EB (1981) Molecular aspects of photoreceptors in protozoa and other microorganisms. In: Levandowsky M, Hutner SH (eds) Biochemistry and Physiology of Protozoa, vol 4, 2nd edn. Academic Press, New York, pp 199–233 Roberts AM (1970) Geotaxis in motile microorganisms. J Exp Biol 53:687–699 Schnitzer MJ, Block SM, Berg HC, Purcell EM (1990) Strategies for chemotaxis. In: Armitage JP, Lackie JM (eds) Biology of the chemotactic response. Cambridge University Press, Cambridge, pp 15–34 Tamar H (1979) The movement of jumping ciliates. Arch Protistenkd 122:290–327 Van Houten J, Hauser DCR, Levandowsky M (1981) Chemosensory behavior in protozoa. In: Levandowsky M, Hutner SH (eds) Biochemistry and physiology of Protozoa, vol 4, 2nd edn. Academic Press, New York, pp 67–124

4

Feeding

4.1

General Considerations

Phagocytosis—the uptake of particulate food particles—is an essential feature of protozoan feeding. Some protozoa obtain additional energy and materials for growth by other mechanisms, such as the uptake of dissolved materials, which will be discussed at the end of this chapter, while symbiotic relations with photosynthetic organisms is discussed in Chap. 8. Feeding consists of two processes, each of which may limit the actual rate of feeding. The first one of these is the process of phagocytosis, that is, the enclosure of a food particle in a membrane-covered vacuole in which digestion takes place. In ciliates and in most phagotrophic flagellates this occurs at a special site on the cell surface, the “cytostome” which is covered by a single unit membrane from which food vacuoles are formed. The cytostome is often associated with various organelles in the cytoplasm, in particular bundles of microtubules, which play a role in the transport of the captured particles. On the surface, surrounding the cytostome, a variety of ciliary and other organelles serve to concentrate or retain food particles. The entire area is usually referred to as the “mouth”. For ciliates, a rather specialized terminology of mouth morphology has developed due to its taxonomic significance (see Corliss 1979; Lynn 2008). However, in, e.g. amoebae phagocytosis seems to take place anywhere on the cell surface. The process of phagocytosis has been extensively studied in ciliates (Allen 1984; Nilsson 1979, and references therein) but only more rarely in other types of protozoa (e.g. Linnenbach et al. 1983). The induction of phagocytosis is, at least in part, due to mechanical stimulus by food particles. Food vacuole formation does not take place in particle-free water, while filter-feeding protozoa ingest inert particles such as latex beads and suspended carbon particles at a rate similar to that of similarly sized food particles (Fenchel 1986; Mueller et al. 1965). The newly formed food vacuole may be subject to some water absorption, after which it fuses with lysosomes to form secondary vacuoles in which hydrolytic degradation of the food particles takes place. The dissolved nutrients are removed from the vacuole through pinocytotic vesicles. # Springer Nature Switzerland AG 2020 G. F. Esteban, T. M. Fenchel, Ecology of Protozoa, https://doi.org/10.1007/978-3-030-59979-9_4

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Feeding

Indigestible remains are eventually removed from the cell through a fusion of the vacuole with the surface membrane; in ciliates this takes place at a special site, the “cytoproct”. The last part of this cycle is the transport of membrane material in the form of small vesicles back to the cytostome area to form new food vacuoles. Thus, during the lifetime of a food vacuole, it is moved around in the cell. Individual food vacuoles are easy to follow when labelled with inert, light-refringent or coloured particles. It seems, at least in ciliates, that the elimination of food vacuoles is random so that the lifetime of vacuoles has an exponential distribution. The expected lifetime of a vacuole decreases with increasing rate of feeding, so that if the cells are starved the elimination rate is much lower. The average lifetime for a food vacuole in a feeding ciliate is about 20 minutes (Berger and Pollock 1981). The maximum rate at which phagocytosis can take place (which may be limited by the rate at which membrane material can be recycled within the cell) sets an upper limit to the feeding rate of a protozoon. The maximum volume ingested by a small ciliate or flagellate is about 100% of the cell volume per hour, which is consistent with minimum doubling times of such organisms of about 3 hours. In larger protozoa this figure is lower; about 50% of the body volume per hour (Fenchel 1980c, 1982b). The other process involved in feeding is that of concentrating food particles from the environment. It is possible that the first Precambrian protozoan simply engulfed portions of the surrounding water and ingested whatever bacteria were present, but extant forms have developed various mechanisms by which the dilute food particles of the environment can be concentrated prior to phagocytosis. The variety of these adaptations contributes to the diversity of protozoan forms; this will be discussed in the following section, while we devote this section to some general aspects of particle capture. The first thing to consider is how to quantify the rate at which the organisms concentrate particles. A reasonable measure is “clearance”, F, by which is meant the volume of water cleared of food particles per unit time. In discussing clearance, we will, for the moment, consider only organisms which feed on suspended particles, although the principles discussed below can easily be adapted to organisms which feed on particles associated with solid surfaces. Clearance is then equal to the particle uptake per unit time, U, divided by particle concentration in the surrounding medium, x, or: F ¼ U (x)/x. The rate of food uptake, however, is not likely to increase linearly with particle concentration, since as x increases, the rate of phagocytosis becomes limiting. Assuming that it takes a finite time, t’, to phagocytize one unit of food particles, during which additional phagocytosis cannot take place, the rate of food uptake as a function of food particle concentration (the “functional response”) then becomes: U (x) ¼ xFm (1  t’U), where Fm is a maximum value of clearance realized for very low values of x. Rearranging the equation we get: U ¼ xUm/ (x + Um/Fm), where Um (¼ 1/t’) is the maximum rate of phagocytosis. This equation is a hyperbolic function in which, as x becomes very large, U approaches Um and the slope at the origin is Fm (Fig. 4.1). This is analogous to the Michaelis–Menten equation for describing enzyme kinetics, which also describes, for example, the rate of uptake of a dissolved nutrient by a bacterial cell as a function of concentration. This way of deriving the equation shows that the

4.1 General Considerations

35

Fig. 4.1 The uptake of 2 μm latex beads by the oligitrich ciliate Halteria grandinella as a function of particle concentration. The data are fitted to a hyperbolic function. The slope at the origin is the rate of maximum clearance (Fm) which is 6.7  104 ml/ h. The volume of the ciliate is about 8  103 μm3 so it clears about 8  104 times its own cell volume per hour. The maximum uptake rate is about 190 beads per hour. (Redrawn from Fenchel 1986)

“half-saturation constant” Um/Fm (or Km) is an ad hoc parameter which has no obvious biological significance. This half-saturation constant can be interpreted as the ratio between the capacity to ingest particles and the efficiency with which particles are concentrated from the environment. This is an analogy to the uptake of dissolved molecules by a bacterial cell. Here the half-saturation or Monod constant measures the ratio between the transport capacity of the cell membrane and the uptake rate when only limited by the substrates diffusivity outside the cell. Also, in this case, it is the latter measure rather than the not half-saturation constant which gives information on the competitive ability at low substrate concentrations (Koch 1971). The significant parameters are Um and Fm; (and not the ratio Um/Fm) are direct measures of competitive ability for resources. This has been widely misunderstood in the literature and has led to incorrect conclusions (as discussed below) about the role of dissolved organic materials as food for protozoa. The actual values of Um and Fm may be determined in various ways (Fenchel 1980b, 1986). One is to measure the uptake of either real food particles or inert particles (e.g. latex beads) as a function of concentration. It is also possible to calculate the values from culture experiments since the exponential growth rate constant is proportional to the food consumption rate (the growth yield being the proportionality constant). For any one species, Fm and Um are functions of particle quality. In particular, particle size affects both the efficiency of retention and the rate of phagocytosis. The “volume-specific clearance”, that is, clearance divided by cell volume, is often a useful number, since it allows a comparison of clearance values between different-sized organisms. If the clearance of an organism has been determined and the particle concentration in the natural environment is known, it is possible to estimate the consumption rate in nature. Values of maximum volume-specific clearance for protozoa feeding on suspensions of particles are usually of the order of 105 per hour; that is, the protozoa can, in an hour, clear food particles from a

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Feeding

volume of water which is 105 times their own cell volume. Species which filter very small particles or those which live in interstitial environments with narrow crevices have values of clearance which are as much as ten times lower. Which mechanisms do protozoa utilize for concentrating food particles? At the dimensions and velocities characteristic of protozoa, any mechanism based on inertial forces can be ruled out; some quantitative calculations demonstrating this are given in Fenchel (1980a). For organisms feeding on suspended particles, there are three possibilities; these mechanisms can be termed “filter feeding”, “direct interception”, and “diffusion feeding”, respectively (Fenchel 1984, 1986). These mechanisms are analogous to methods for catching fish, namely, trawling, spear fishing, and the use of fish traps. Filter feeding depends on the transport of water through a sieve formed by cilia or pseudopodial tentacles that strain food particles from the water. In direct interception or “raptorial feeding”, food particles carried along the flow lines are directly intercepted by the protozoan surface which is “sticky”, thus retaining the particle in some way until it is phagocytized. These two mechanisms depend on the motility of the protozoon. The third mechanism, diffusion feeding, requires motility of the prey, which is intercepted by a typically motionless consumer. In order to get a feeling for the relative efficiency and importance of these mechanisms, it is useful to consider very simplified models of suspension-feeding protozoa. In the case of a filter-feeding protozoon, the capture rate is proportional to the concentration of food particles, to the area of the filter, and to the velocity of the water current which the cell generates: Thus, the capture rate is xR2v; where x is the particle concentration in the environment; R is a length measure of the cell; and v, water velocity. Volume-specific clearance is then found by dividing with x and with cell volume, which is proportional to R3, to give the expression R1v. (Any realistic model which is to predict the clearance rate of a real protozoon must, of course, take into account details of the geometry of the cell as well as hydrodynamic considerations. However, the above expression yields results of the correct order of magnitude: If v ¼ 300 μm/sec and R ¼ 10 μm, then volume-specific clearance is about 105 per hour.) For a spherical protozoon (radius: R) catching particles (radius: r) by the second mechanism of direct interception, there exist “critical flow lines” within which particles will be intercepted. The transectional area of the flow past the cell within the critical flow lines is 2πRr (if R >> r, and if it is assumed that particles can be intercepted along the equator of the cell), so the volume-specific clearance will be proportional to R2rv (based on arguments quite similar to those applied for the filter feeder above). The efficiency of a raptorial feeder, therefore, depends on the size of the prey. To compare the efficiencies of a filter feeder and a raptorial feeder of equal size and capable of generating a similar water velocity, we can divide the expression for clearance of the latter by that for the former to arrive at the expression, r/R. This informs us that the efficiency of removal of particles is a function of the ratio between predator and prey size. If the food particles are sufficiently small, filterfeeding is the most efficient mechanism. Empirical evidence for ciliates shows that if the prey:predator size ratio exceeds about 0.1, raptorial feeding predominates, but if

4.1 General Considerations

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Fig. 4.2 Average food particle size as a function of cell size for a number of filterfeeding (filled circles) and raptorial (open circles) protozoa. The line corresponds to a food particle: cell size ratio of 1:10. (After Fenchel 1986)

the food particles are smaller, filter-feeding is found (Fig. 4.2, Fenchel 1986). Thus, many smaller, predatory ciliates (e.g. Litonotus, Didinium) feed on other ciliates such as Colpidium and Paramecium by direct interception, while the giant ciliate Bursaria (which measures up to 1 mm) feeds on similar prey organisms by filter feeding. The last mechanism is feeding by diffusion. It is related to the mechanism by which a bacterium takes up organic molecules from a solution. It can be shown (Koch 1971; Roberts 1981) that at low particle concentrations (the diffusion-limited case in which the particle concentration at the surface of the organism is zero) the uptake rate by a spherical collector is 4πrRDx, where D is the motility (“diffusion coefficient”) of the particles (or molecules): Hence the volume-specific clearance is proportional to R2D. The efficiency of the mechanism depends on the motility of the prey. For small bacterivorous heliozoa, Fenchel (1984) calculated that in the case of non-motile bacteria, Brownian motion alone will yield a clearance which is two to three orders of magnitude too low to make these protozoa competitive with other bacterivorous forms. If motile bacteria are considered, however, the efficiency of the mechanism is comparable to that of protozoa utilizing direct interception (e.g. chrysomonad flagellates) or filter-feeding (e.g. choanoflagellates). We have so far considered only suspension-feeding forms. For forms that feed on food particles associated with solid surfaces, filter-feeding does not occur. However, it is still possible to distinguish between forms which slide along the surface in order to intercept prey and those which spread their pseudopodia over a large area to trap motile prey.

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There are a number of mechanisms that may enhance the capture of particles. One of these is to attract the prey (or to return to the fishing analogy, to use bait). Apparently, some foraminifera and heliozoa excrete substances that attract flagellates, which then can be captured by the predators (Lee 1980, and references therein). Unfortunately, the phenomenon has not been studied in detail. Certain physical principles could possibly act to increase the probability of capture in protozoa where food particles stick to the collector surface, i.e. electrostatic attraction between food particles and the predator surface. Another such mechanism is based on the observation that particles of a finite size following a viscous flow close to a solid surface will occupy a part of a velocity gradient. This will lead to a rotation of the particle which will tend to migrate across the flow lines and towards the surface (Spielman 1977). The significance of such effects for suspension-feeding organisms has not yet been investigated. In the case of filter-feeding protozoa, however, they can be ruled out since food particles are not trapped by mucus, extrusomes, or other mechanisms prior to phagocytosis (food particles show Brownian motion in the food vacuoles); so in this case, a purely mechanical sieving effect accounts for particle capture. The simple models discussed above ignore certain hydrodynamic constraints that apply in particular to filter feeders. These constraints are related to the fact that when a viscous fluid flows past a solid surface, the velocity is zero at the surface (the “noslip condition”), and a steep velocity gradient will be present immediately above the surface. One consequence of this (which is well known from the practical application of filters in the laboratory) is the pressure drop across a filter necessary to overcome viscous forces. Ciliary propulsion mechanisms can only sustain a very low hydrostatic pressure. The pressure drop across a filter is proportional to fluid velocity, viscosity, and the thickness of the filter, and it is also a rather complex function of the geometry of the filter elements; in general, it increases greatly when the size ratio between the filter elements and the pores becomes large (> 1). The pressure drop of some protozoan filters (those consisting of parallel arrays of cilia or tentacles) can be estimated. This pressure drop is always about 10 dyn/cm2 (¼ 0.1 mm H20). Since the diameter of the filter elements is fixed at 0.2 μm (cilia) or down to 0.1 μm in some types of tentacle filters, it means that the flow rate through the filters (and thus clearance) is much reduced in filter-feeding species which specialize in feeding on very small food particles. For example, choanoflagellates, which filter the smallest prokaryotic cells (the distance between neighbouring pseudopodial tentacles is only 0.1–0.3 μm) have flow rates through the filter of only 10–20 μm/sec; whereas in the helioflagellate, Pteridomonas, the free distance between the tentacles is 1–3 μm and the flow velocity through the filter is nearly 100 μm/sec. The specialization to catch very small food particles is therefore correlated with a decrease in clearance (Fenchel 1986). The flow past a filter feeder is larger when the organism is attached to a surface than when it is swimming. This is because the thrust of the flagellum or cilia of a swimming organism must balance the viscous drag of the cell as it moves through the water. In accordance with this, most filter-feeding protozoa tend to attach to a solid object when feeding. Some forms are permanently attached (e.g., peritrich

4.1 General Considerations

39

Fig. 4.3 Ratio between the distance of a filter from the solid substrate and the radius of the filter in three filter-feeding protozoa which generate water currents perpendicular to the substrate (a–c) and two species (d, e) which generate water currents parallel to the substrate. In the former case, this ratio is around 8 and in the latter case 3–4. (a) the choanoflagellate Salpingoeca, (b) Vorticella, (c) Stentor, (d) Cyclidium, and (e) Euplotes. F shows the small mouth of filter-feeding living in narrow crevices (Colpoda). Scale bars: a: 10 μm, b: 100 μm, c: 1 mm, e and f: 50 μm

ciliates and some choanoflagellates) and others attach temporarily while feeding (e.g. many ciliates such as Cyclidium or Euplotes). This, however, poses another problem for the protozoan, namely, the viscous drag due to the proximity of a solid surface, an effect which could slow the water flow considerably (ChristensenDalsgaard and Fenchel 2003; Fenchel 1986). It can be shown (Fig. 4.3) that for a given distance from the substrate, the effect is considerably larger if the feeding current is perpendicular to a solid surface than if it is parallel. Thus, choanoflagellates, and the ciliates Vorticella and Stentor, which propel water perpendicular to the surface, have stalks, while organisms such as Euplotes and Cyclidium, which propel water parallel to the surface, only need cilia to rise sufficiently far above the surface. The effect of a solid surface is a function of the ratio of the distance to the surface and the radius of the filter. When, in the case of a perpendicular flow, this ratio exceeds eight, the effect of a solid surface becomes negligible, and this is the ratio of stalk lengths to filter sizes found in filter-feeding protozoa, ranging from 10 to 20 μm-tall choanoflagellates to nearly 2-mm-high stentors (Fig. 4.3). The discussion of the role of dissolved organics in the nutrition of aquatic invertebrates, and by implication protozoa, has now persisted for about 75 years; this literature has been reviewed by Jørgensen (1976) and Steinberg (2003). Much of the work attempting to show the significance of dissolved organics in the nutrition of animals is uncritical; in part, this is due to a misunderstanding of the significance of the half-saturation constant of the Michaelis–Menten equation as discussed above.

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It is a fact that protozoa (and the epithelial cells of many invertebrates) show both an active uptake of low-molecular-weight organic compounds and an uptake of dissolved macromolecules through “pinocytosis”. Some smaller protozoa can be grown axenically in solutions of substrates like peptone or even in chemically defined media. The mouthless mutants of Tetrahymena studied by Rasmussen and Orias (1975) constitute a very convincing proof of this. Nevertheless, a number of arguments render it very improbable that dissolved nutrients play any role for freeliving protozoa in nature. The most important argument in favour of this view is, perhaps, that no protozoan groups have evolved free-living (non-photosynthetic) forms which do not ingest particulate material. A few parasitic forms (e.g. the astome ciliates living in the intestine of annelid worms) must depend on dissolved nutrients, but even many parasitic protozoa (e.g. Entamoeba hystolitica) are phagotrophs. A few other protists (e.g. apochlorotic chlamydomonad flagellates and diatoms) which live in special environments with high concentrations of dissolved organic material exist, but this is also very rare. The reason for this is probably that the relatively larger protozoa cannot compete with bacteria for dissolved nutrients. The fact that the specific clearance of a diffusion feeder is inversely proportional to the square of its length suggests that large cells will be less efficient than small cells in competing for dissolved nutrients. In order to grow Tetrahymena cells axenically, peptone solutions of 2–5 g per litre of medium are necessary (see Rasmussen and Orias 1975, and Cassidy-Hanley 2012). These ciliates will, however, grow at approximately the same rate in a suspension of about 5  106 bacteria/ml which is only about 5  104 times the concentration of organic material needed for growth based on dissolved nutrients. Comparisons of the volume-specific clearance for amino acids by Escherichia coli reported by Koch (1971) are about a thousand-fold higher than similar values for amino acid uptake in Tetrahymena, as calculated from the data in Jørgensen (1976). In this context the observation that the half-saturation constants (Km) for amino acid uptake are comparable (within the range of 1–8 μM for the bacterium, and within the range 8–94 μM in the case of the ciliate) is misleading. Since Km is proportional to the maximum rate of uptake, Vm, this observation only reflects the fact that the Vm per unit volume of ciliates is very low as compared to similar values for bacteria. It is therefore unlikely that the uptake of dissolved organic matter or that phenomena such as pinocytosis in large amoebae (Chapman-Andresen 1967) play any significant nutritive role in free-living protozoa. The real function of these processes in protozoa (and in epithelial cells of invertebrates) is not well understood. It may be possible that their primary function is related to the maintenance of the steep chemical gradients of dissolved organic compounds which occur across the cell membrane.

4.2 Feeding in Real Protozoa

4.2

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Feeding in Real Protozoa

In spite of the diversity of feeding mechanisms, it is still possible to classify protozoa according to three categories, namely, filter feeders, raptorial feeders, and diffusion feeders. This classification will be used as a framework for the following discussion. Filter feeding is an adaptation for feeding on small, suspended food particles. In filter-feeding flagellates, a flagellum propels water through a collar of nearly straight and seemingly rigid tentacles or pseudopodia which act as a filter. In the choanoflagellates, the collar typically consists of 20–50 tentacles, each with a diameter of about 0.1 μm. The free distance between neighbouring tentacles is only 0.1–0.3 μm. In the centre of the base of the collar one smooth flagellum is situated; it drives water away from the cell so that suspended particles are collected on the outside of the collar. The food particles are then phagocytized by pseudopodia arising from the periphery of the collar (Fenchel 1982a; Laval 1971; Leadbeater and Morton 1974; Leadbeater 2015—see also Figs. 4.4, 9.2, and 10.4). The choanoflagellates are specialized for feeding on the smallest prokaryotic cells so they play an important role in planktonic environments. The low porosity of the filter explains the slow water currents through it (10–20 μm/sec), but due to the small size of choanoflagellates and thus their large surface to volume ratio, they filter about 105 times their cell volume of water per hour, equivalent to 1 to 4  106 ml/h (Fenchel 1986). Certain chrysomonads and helioflagellates, although not related to the choanoflagellates, apply a similar system for catching bacteria. However, these forms have hairy flagella (see Fig. 2.1), and so in this case water current is directed against the cell and the food particles are intercepted along the inside of the tentacle collar. These forms have a much coarser filter and generate much faster water currents than do the choanoflagellates. Due to the simple geometry of filter-feeding flagellates, and in particular their radial symmetry, it has been possible to make rather detailed hydrodynamic models of the feeding currents and to compare them with the flow fields and filtration rates of the real organisms (Fenchel 1986; Lighthill 1976). It is among the ciliates that we find the greatest diversity and specialization for filter feeding. Filter feeding is often found among the oligohymenophorans (hymenostomes such as Tetrahymena, Colpidium, and Paramecium, scuticociliates such as Cyclidium, and the peritrichs), among the colpodids, and among the polyhymenophorans (hypotrichs such as Euplotes, heterotrichs such as Stentor and Blepharisma, and oligotrichs such as Halteria and the planktonic tintinnids). In filter-feeding ciliates, the water currents are always generated by membranelle zones which are situated on the left side of the mouth (Chap. 2). However, there are two different principles of filtration. In some forms, the membranelle zone not only generates the water currents but also functions as a filter. The membranelles pump water out of the mouth, while particles that are too large to pass between neighbouring membranelles are retained along the inside of the zone and eventually accumulate at the cytostome. Such “upstream” filtration (Nielsen and Riisgård 1998) is typical of all the polyhymenophoran ciliates (Figs. 2.2 and 4.4) and the colpodids;

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Fig. 4.4 Flow lines generated by protozoan filter feeders. (a) The choanoflagellate Diaphanoeca; (b) The oligotrich ciliate Halteria, food particles are intercepted on the inside of the membranelle zone. (c, d) The scuticociliate Cyclidium: food particles are intercepted on the paroral membrane which consists of a row of immobile parallel cilia. In d the position of a latex bead was recorded every 20 msec on a video recorder; it shows how water is accelerated by the oral membranelles (not shown) to reach velocities around 300 μm per second and how the particle is eventually intercepted by the ciliary membrane. Al scale bars are 10 μm. (Redrawn from Fenchel 1986)

it is also found in a few oligohymenopheran ciliates. High water velocities (0.5–1 mm/sec) are characteristic for this type of particle retention. The lower part of the size spectrum of retained particles is also ill-defined (Fig. 4.5). This is because the distance between neighbouring membranelles varies during the beat cycle. The mechanism does not allow the retention of very small particles; the species with the tightest arrangement of membranelles will retain 100% of particles exceeding 1–2 μm.

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Fig. 4.5 The retention efficiencies for the lower size range of intercepted particles in seven filterfeeding ciliates. Open circles: interception with a paroral membrane; filled circles with a membranelle zone. (a) Cyclidium citrulllus, (b) Colpidium colpoda, (c) Uronema marinum, (d) Halteria grandinella, (e) Euplotes moebiusi (f) Blepharisma japonicum, (g) Bursaria truncatella. (After Fenchel 1986)

In most oligohymenophoran ciliates the water currents generated by the membranelles are driven through an array of parallel and nearly motionless cilia on the right side of the mouth, the “paroral” or “undulating membrane”, which acts as a sieve. A characteristic example is Cyclidium (Fig. 4.4). In peritrich ciliates, one membranelle and the paroral membrane run counter-clockwise all the way around the pole of the cell and then enter the funnel-shaped “infundibulum” leading to the cytosome. The undulating motion of the membranelle drives water in between it and the paroral membrane and towards the infundibulum. As the water moves through this passage, most of the water seeps out between the cilia of the membrane, so a concentrated suspension of particles enters the infundibulum. The organisms which use this form of filtration are specialized to feed on relatively small particles (typically as small as 0.3 μm). The size spectra of retained particles show a sharply defined lower-size range in accordance with the fact that the cilia of the undulating membrane are held parallel and nearly motionless during the filtration. The water currents generated by the membranelles have velocities comparable to those of upstream filterers. However, the water current approaches the paroral membrane at an acute angle so that the flow velocity through the membrane is correspondingly lower, typically around 20 μm/sec. Consequently, in many of these forms, the filter area is huge (e.g. Cyclidium and the peritrichs). Some filter-feeding ciliates, however, have quite a small mouth and so have a low clearance. This applies to soil ciliates, such as Colpoda, and also to tetrahymenine ciliates, such as Colpidium and Glaucoma. This is probably an adaptation to life in small crevices (e.g. soil, detritus, and carrion). As discussed in the preceding section, the hydrodynamic resistance to flow due to the proximity of solid surfaces is a function of the transectional area of the water flow that is filtered. When a ciliate is sufficiently close to a surface, there is no gain in clearance by increasing the filter

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area because this would be counteracted by a corresponding decrease in the average flow velocity through it (Fenchel 1986).

4.3

Raptorial Feeding

Raptorial feeding is found in many small flagellates. These and the small amoebae are the only protozoa which are sufficiently small to feed on bacteria in this way. Chrysomonads and the possibly related bicoecids drive water currents against the cell with their anterior, hairy flagellum. Particles which touch a lip-like structure supported by bundles of microtubules surrounding the cytostome are phagocytized (Fig. 4.6a). The free-living kinetoplastid flagellates are also raptorial feeders. Some forms, such as Pleuromonas jaculans, feed on suspended bacteria much as the chrysomonads do. Most kinetoplastids, however, are specialized on bacteria associated with solid surfaces. These flagellates slide on their trailing flagellum and ingest particles that come in contact with the cytostome. The characteristic structure of phagotrophic kinetoplastids are microtubular rods, which form a “cytopharynx” and that must play a role in the ingestion of the food particles (Fig. 4.6; Fenchel 1982a). Similar rods are also found in the phagotrophic euglenids Peranema and Entosiphon, which also find their food particles along surfaces. Many dinoflagellates are obligatory or facultative predators of other organisms. Phagocytosis takes place in the “sulcus” close to the place where the flagella are attached. In

Fig. 4.6 (a) The chrysomonad flagellate Ochromonas (fixed with a mixture of OsO4 and HgCl solutions) immediately after having ingested a bacterium (the bulge above the closing cytostome). The short and the long flagellum are also seen. (b) The kinetoplastid flagellate, Pleuromonas jaculans showing the cytostome enforced by microtubules as well as the two flagella arising in a flagellar pocket. (c) A Transmission Electron micrograph of Pleuromonas showing the microtubules of the cytostome which plays a role in the transport of captured bacteria into food vacuoles (also seen to the right is the swollen DNA-containing part of the mitochondrion—the “kinetoplast”). Scale bars: a, b: 1 μm; c: 0.5 μm

4.3 Raptorial Feeding

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Fig. 4.7 (a) Heliozoon (size: 500 μm) ingesting a rotifer. (b) The giant bioluminescent marine dinoflagellate Noctiluca (cell size 500 μm). (Both images photographed by Benedikt Pleyer)

some dinoflagellates (e.g. Gymnodinium) an extensible structure, the peduncle, which protrudes from the sulcus area, is used for the capture of prey cells and for the ingestion of their cytoplasmic contents (Spero 1982; Lee et al. 2014). Other dinoflagellates, like Gyrodinium and some of its relatives, exploding trichocysts immobilize the prey organism prior to phagocytosis (Biecheler 1952). Among the heterotrophic flagellates, Oxyrrhis and Noctiluca have been especially well studied (Droop 1966; Prasad 1958). The latter (Fig. 4.7b), a giant marine dinoflagellate, feeds on algal cells, other protozoa, and even crustacean larvae and other small invertebrates. Prey organisms stick to the motile tentacle of the flagellate and are then brought to the cell surface and phagocytized. Filter feeders retain and ingest all properly sized particles. Raptorial protozoa, on the other hand, which capture each particle individually rather than in bulk, may show a considerable degree of discrimination on the basis of qualities other than size. Among the ciliates, there are several examples of this. It is especially the karyorelectids, the prostomatids, and the pleurostomatids which feed this way. The most common prey item among karyorelectids (e.g. Loxodes) seems to be microalgae and flagellates, but other ciliates and even testate amoebae are also ingested (Fig. 4.8b) (Fenchel 1968; Hines et al. 2016). Prey size plays a considerable

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Fig. 4.8 (a) A suctorian ciliate showing the bundles of tentacles (cell size: 90 μm). (b) The giant ciliate Loxodes rex (cell size: 1000 μm) with an ingested testate amoeba. (c) Frontonia (cell size: 300 μm) with ingested diatoms. (b, c: Photographed by Hunter N. Hines)

role in determining whether a particle is ingested or not and there is a close correlation between protozoan cell size and the preferred particle size (Fenchel 1968; Finlay and Berninger 1984; see also Fig. 7.1). Some marine interstitial ciliates (Chap. 9) are commonly found with ingested sand grains (Fig. 4.9). This maybe is due to the fact that microorganisms like bacteria, diatoms, cyanobacteria and others live attached to the sand grains. By ingesting sand grains ciliates are probably able to use these attached microorganisms as food, but the reasons are not really known. The phenomenon was already noted by Meadows and Anderson (1966), and Fenchel (1968). The prostomatids comprise some of the best-known protozoan predators. The classical studies of Gause (1934) on the kinetics of a prey–predator system were based on Didinium nasutum, which specializes in the use of Paramecium as food. This system has been re-examined many times (see Luckinbill 1973, 1974; Salt 1979, and also Kozlova et al. 2002 and references therein), mainly with emphasis on the functional response of this predator and the stability properties of the system. The prey organism has a length comparable to a Didinium cell and the ingestion process, after the predator has immobilized its prey is beautifully illustrated in Wessenberg and Antipa (1970). Like other predatory prostomatids and pleurostomatids, Didinium has “toxicysts”, extrusomes that explode when they are touched by the prey cell and which subsequently immobilize it. Other common predators of ciliates include Lacrymaria, Homalozoon, Dileptus (which may prey on small metazoa as well), Litonotus, and Loxophyllum (Dragesco 1962, 1963; Fenchel 1968; Kuhlmann et al. 1980; see also Fig. 4.10). Some of these forms feed predominantly on colonial, peritrich ciliates (Canella 1951).

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Fig. 4.9 The marine interstitial ciliate Geleia sp. photographed in the process of feeding. Same specimen seen from different angles. The ciliate significantly widens the oral area, with expansion of the oral opening. This cell seems to have ingested a sand grain—a common phenomenon observed in sand-dwelling marine ciliates. Scale bars: 100 μm

Predators also are found among other ciliate groups. Some hymenostomes, such as Lembadion (Fig. 10.5d) have secondarily evolved raptorial feeding, as has the hypotrich Uronychia. Frontonia, which is a close relative to Paramecium, lacks the filter-feeding habits of its relatives and feeds predaciously on dinoflagellates, large diatoms, and cyanobacteria (Fig. 4.8c). The hypostome ciliates are characterized by a cytopharyngeal basket of microtubular rods which assists in the ingestion of filamentous organisms (Tucker 1968). In Nassula and its relatives, this organelle is used for feeding on filamentous cyanobacteria on which they have specialized (Fig. 4.11; Dragesco 1962; Tucker 1968). The chlamydodontids and the dysteriids feed primarily on bacteria and filamentous microorganisms associated with surfaces (see Chap. 9). The diversity of the structure of the pharyngeal rods found among these forms suggests a high degree of specialization for specific foods (Deroux 1976; Peck 1985; see also Fig. 4.10). Histophagy is a specialized type of raptorial feeding which has developed independently in several different ciliate taxa. Histophagous ciliates attack damaged, but live, animals such as annelid worms and small crustaceans. The ciliates enter wounds, to which they are attracted by a chemosensory mechanism, and ingest animal tissue. If many ciliates attack simultaneously, which is often the case,

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Fig. 4.10 (a) Didinium nasutum ingesting a Paramecium cell. (b) The anterior end of Dileptus anser ingesting a Colpidium cell. Toxicysts situated in the trunk lyse the prey cell which is then ingested. (c) Pseudomicrothorax dubius using its pharyngeal (microtubular) rods to ingest an Oscillatoria filament (scale bar: 110 μm). (d) The pharyngial baskets of rods of some dysteriid ciliates (1 and 2 redrawn from Dragesco 1962; 3 redrawn from Peck 1985; 4 redrawn from Deroux 1976)

everything but the cuticle may be devoured within 1 hour. The most specialized histophages have a polymorphic life cycle including a swarmer stage, a feeding stage, and a cyst stage. In these forms, the trophic stage is short lived (30–60 minutes) and during this period the ciliates may ingest ten times their own volume. Histophagy occurs among some prostomatids (e.g. Coleps and Prorodon) where it has evolved as a result of the carnivorous habits of these ciliates. In other groups, histophagous species evolved from forms which feed on bacteria in carrion (e.g. some species of Tetrahymena and some philasterid ciliates). The most specialized histophages are found in the genus Ophryoglena (Fig. 4.12). Some scuticociliates can also have devastating impact—known as “scuticociliatosis”— on fisheries and shellfish aquacultures (Song et al. 2009; Stidworthy et al. 2013). The biology of histophagous ciliates has been the subject of several studies (e.g. Canella and Rocchi-Canella 1976; Corliss 1973; Fenchel 1968; Jee et al. 2001; Ofelio et al. 2014; Savoie 1968).

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Fig. 4.11 (a) The freshwater ciliate Nassula tumida feeding on a filament of a cyanobacterium. (b) The marine amoeba Cochliopodium bilimbosum catching a small flagellate. Note the layer of microscales surrounding the amoeban cell

Fig. 4.12 The marine histophagous ciliate Ophryoglena macrostomum. (a) Tomites entering cut in an oligochaete worm. (b) Scanning Electron Microscope image of young trophonts feeding on a cut oligochete. Scale bars: 100 μm

Among the sarcodines, raptorial feeding is especially common in lobose amoebae—see Chap. 10, Fig. 10.1). The feeding of large amoebae on ciliates and the method by which amoebae surround the ciliates and other prey using pseudopodia and enclose them in a “food cup” prior to food vacuole formation is well documented and has probably been observed by most biologists (Lindberg and Bovee 1976). The numerous species of small marine, freshwater, and soil amoebae depend primarily on bacteria and also on microalgae for food (Fig. 4.11; Page 1976, 1983; Schuster 1979). This also applies to most lobose and filose testaceans (Anderson 2017; Heal 1961; Ogden and Hedley 1980; Stump 1935).

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4.4

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Feeding

Diffusion Feeding

Diffusion feeding is found among many types of sarcodines. In heliozoa, axopods radiate out in all directions. Prey cells which happen to collide with the axopods are held by mechanisms which are not fully understood but which involve special extrusomes. The prey may then be brought closer to the cell through the bending of the axopods or through cytoplasmic streaming on the surface of the axopods. Eventually, a pseudopodium arising from a cell surface engulfs the prey and forms a food vacuole. The largest heliozoan (Actinosphaerium) feed on large ciliates and on small metazoa, such as rotifers (Fig. 4.7) and crustaceans, while the smallest forms depend on motile bacteria and small flagellates. Heliozoan feeding has been described in detail by Dragesco (1964) and by Hausmann and Patterson (1982). Feeding in planktonic foraminifera was studied by Anderson and Be (1976) and Anderson et al. (1979). They examined the process of feeding of the large (1 mm) pelagic Hastigerina on larvae of the crustacean Artemia. In this case, the larval prey stick to the reticulopodial network, which eventually envelopes it and then penetrates it. The prey is then drawn into the ectoplasmic “bubble layer” where digestion takes place in a food vacuole. In radiolaria, axopodia or reticulopodia hold on to any prey organisms which collide with them. Some radiolaria are also surrounded by a “sticky jelly” which helps retain prey (Fig. 9.5), while in small species the spines may assist mechanically as the reticulopodia are anchored to them as well as to the struggling prey. Radiolarian species of different sizes prey on small metazoa, ciliates, and algae (Anderson 1983; Matsuoka 2007; Swanberg and Anderson 1985). In contrast to the organisms discussed above, most Granuloreticulosa and thus the majority of the foraminifera are associated with solid surfaces over which they spread a network of often extremely thin reticulopodia (Fig. 4.13—see also Fig. 2.3). Prey organisms, such as algal cells, bacteria, or other small cells, stick to the pseudopodia and are drawn towards the predator by contraction or cytoplasmic streaming. Many foraminifera are surrounded by empty diatom frustules and other

Fig. 4.13 The foraminifer Elphidium williamsoni showing the test (a), and the reticulopodia which trap food particles (b) Scale bars: 100 μm

References

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remains, the contents of which were apparently digested in food vacuoles outside the test; in other cases, particulate material is brought inside the test. Detailed studies on the feeding and food requirements of mainly estuarine species have been carried out by Lee (1980); Lee et al. (1969); Lee and Muller (1973), Muller (1975), and Murray (2006). The study by Christiansen (1971) demonstrates a diversity and inventiveness among some benthic foraminifera in their use of pseudopodial nets for the capture of different kinds of prey. Among the ciliates, the suctorians make use of diffusion feeding and their prey organisms are almost exclusively other ciliates (Fig. 4.8). Mature suctorians are devoid of cilia and in most cases are attached (with or without a stalk) to a substrate. They possess bundles of tentacles which are supported by an internal cylinder of microtubules (Fig. 4.8). Extrusomes on the tip of the tentacles serve to attach and immobilize any ciliates or flagellates which touch them. The tentacles eventually penetrate the cell membrane of the prey, whose contents are then drawn through the tentacle and into the suctorian predator. Bardele and Grell (1967) and Bardele (1972) described the adhesion of the prey and suggested a mechanical model for the movement of prey cytoplasm through the tentacle. Some suctorians have adopted a parasitic life, living intracellularly in ciliates which are much larger than themselves (Esteban et al. 1991). The haptorid ciliates belonging to the genus Actinobolina (Fig. 10.5c) also use tentacles to catch swimming prey organisms such as other ciliates. Their tentacles carry toxicysts that immobilize the prey which is subsequently ingested through the cytostome.

References Allen BD (1984) Paramecium phagosome membrane: from oral region to cytoproct and back again. J Protozool 31:1–6 Anderson OR (1983) Radiolaria. Springer-Verlag, New York Anderson OR (2017) Amoebozoan Lobose Amoebae (Tubulinea, Flabellinea, and Others). In: Archibald JM, Simpson AGB, Slamovits CH, Margulis L, Melkonian M, Chapman DJ (eds) Handbook of the Protists. J.O. Springer, Corliss, pp 1–31 Anderson OR, Be AWH (1976) A cytochemical fine structure of phagotrophy a planktonic foraminifer Hastegerina pelagica (d’Orbigny). Biol Bull 151:437–449 Anderson OR, Spindler M, Be AWH, Hemleben C (1979) Trophic activity of planktonic foraminifera. J Mar Biol Assoc UK 59:791–799 Bardele CF (1972) A microtubule model for ingestion and transporting in the suctorian tentacle. Z Zellforsch Mikrosk Anat 126:116–134 Bardele CF, Grell KG (1967) Elektronenmikroskopische Beobachtungen zur Nahrungsaufnahme bei dem Suktor Acineta tuberosa Ehrenberg. Z Zellforsch Mikrosk Anat 80:108–123 Berger JD, Pollock C (1981) Kinetics of food vacuole accumulation and loss in Paramecium tretraurelis. Trans Am Microsc Soc 100:120–133 Biecheler B (1952) Recherches sur les Peridiniens. Bull Biol Fr Belg (Suppl) 36:1–149 Canella MF (1951) Contribution à la connaissance de gymnostomes des genres Holophrya, Amphileptus et Litonotus prédateurs de Carchesium polypinum et d’autres peritriches sessiles. Ann Univ Ferrara NS 1:1–11 Canella MF, Rocchi-Canella L (1976) Biologie des Ophryoglenina. Ann Univ Ferrara NS 3:1–150

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Cassidy-Hanley DM (2012) Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage. Methods Cell Biol 109:237–276 Chapman-Andresen C (1967) Studies on endocytosis in amoebae. The distribution of pinocytically ingested dyes in relation to food vacuoles in Chaos chaos. I. Light microscopic observations. CR Trav Lab Carlsberg 36:161–187 Christensen-Dalsgaard KK, Fenchel T (2003) Increased filtration efficiency of attached compared to free-swimming flagellates. Aquat Microb Ecol 33:77–86 Christiansen BO (1971) Notes on the biology of foraminifera. Vie Milieu Suppl 22 2: 465 Corliss JO (1973) History, taxonomy, ecology, and evolution of species of Tetrahymena. In: Elliot AM (ed) Biology of Tetrahymena. Dowdon, Hutchinson & Ross, Inc., Stroudsburg, Pennsylvania, pp 1–55 Corliss J0 (1979) The ciliated protozoa: characterization, classification, and guide to the literature. Pergamon Press, Oxford Deroux G (1976) Plan corticale des cyrtophorida III-Les structures differen- ciatrices chez les dysterina. Protistologica 12:505–538 Dragesco J (1962) Capture et ingestion des proies chez les infusoires ciliés. Bull Biol Fr Belg. 46:123–167 Dragesco J (1963) Revision du genre Dileptus Dujardin 1871 (Ciliata Holotricha) (systematiqes, cytology, biologie). Bull Biol Fr Belg 97:103–145 Dragesco J (1964) Capture et ingestion des proies chez Actinosphaerium eicborni (Rhizopoda, Heliozoa). Arch Zool Exp Gen 104:163–175 Droop MR (1966) The role of algae in the nutrition of Heteramoeba clara Droop with notes on Oxyrrhis marina Dujardin and Philodina roseola Ehrenberg. In: Barnes H (ed) Some contemporary studies in marine science. Allen & Unwin, London, pp 269–282 Esteban G, Téllez C, Muñoz A (1991) Infraciliature, morphogenesis and life cycle of Endosphaera terebrans (Suctoria, Tokophridae). J Protozool 38:483–488 Fenchel T (1968) The ecology of marine microbenthos. II. The food of marine benthic ciliates. Ophelia 5:73–121 Fenchel T (1980a) Suspension feeding in ciliated protozoa: structure and function of feeding organelles. Arch Protistenkd 123:239–260 Fenchel T (1980b) Suspension feeding in ciliated protozoa: functional response and particle size selection. Microb Ecol 6:1–11 Fenchel T (1980c) Suspension feeding in ciliated protozoa,: feeding rates and their ecological significance. Microb Ecol 6:13–25 Fenchel T (1982a) Ecology of heterotrophic microflagellates. I. Some important forms and their functional morphology. Mar Ecol Prog Ser 8:211–223 Fenchel T (1982b) Ecology of heterotrophic microflagellates. II. Bioenergetics and growth. Mar Ecol Prog Ser 8:225–231 Fenchel T (1984) Suspended marine bacteria as food source. In: Fashham MJ (ed) Energy and materials in marine ecosystems. Plenum Press, New York, pp 301–315 Fenchel (1986) Protozoan filter-feeding. Prog Protistol 1:65–113 Finlay BJ, Berninger UG (1984) Coexistence of congeneric ciliates (Karyorelectida, Loxodes). J Anim Ecol 53:929–943 Gause GF (1934) The struggle for existence. Williams and Wilkins, Baltimore Hausmann K, Patterson DJ (1982) Pseudopod formation and membrane production during prey capture by a heliozoon (feeding by Actinophrys, II). Cell Motil 2:9–24 Heal GW (1961) The distribution of testate amoebae (Rhizopoda, Testacea) in some ferns and bogs in Northern England. Zool J Linn Soc 44:369–382 Hines HN, McCarthy PJ, Esteban GF (2016) The first record for the Americas of Loxodes rex, a flagship ciliate with an alleged restricted biogeography. Microb Ecol 71:5–8 Jee B-Y, Kim Y-C, Park MS (2001) Morphology and biology of parasite responsible for scuticociliatosis of cultured olive flounder Paralichthys olivaceu. Dis Aquat Org 47:49–55

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Jørgensen CB (1976) August Pütter, August Krogh, and modern ideas on the use of dissolved organic matter in aquatic environments. Biol Rev 51:291–328 Koch AL (1971) The adaptive respponses of Escherichia coli to a feast and fanibe existence. Adv Microb Physiol 6:147–217 Kozlova I, Singh M, Easton A (2002) Predator-prey models with diffusion based on Luckinbill’s experiment with Didinium and Paramecium. Math Comput Model 36:83–102 Kuhlmann S, Patterson DJ, Hausmann K (1980) Untersuchungen zu Nah- rungserwerts und Nahrungsaufname bei Homalozoon vermiculare, Stokes 1887. Protistologica 16:39–55 Laval M (1971) Ultrastructure et mode de nutrition du chaonaflagellate Salpingoeca pelagic asp. Nov. Comparaison avec les choanocytes des spongaires. Protistologica 7:325–336 Leadbeater BSC (2015) The Choanoflagellates: evolution, biology and ecology. Cambridge Unviersity Press. 315pp Leadbeater BSC, Morton C (1974) A microscopical study of a marine species of Codosiga JamesClarck with special reference to the ingestion of bacteria. Biol J Linn Soc 6:337–347 Lee JJ (1980) Nutrition and physiology of foraminifera. In: Levandowsky M, Hutner SH (eds) The biochemistry and physiology of protozoa, vol 3, 2nd edn. Academic Press, New York, pp 43–66 Lee JJ, Muller WA (1973) Trophic dynamics and niches of salt marsh foraminifera. Am Zool 13:215–223 Lee JJ, McEnery ME, Rubin H (1969) Quantitative studies on the growth of Allogromia laticollaris (Foraminifera). J Protozool 16:377–395 Lee KH, Jeong HJ, Jang TY, Lim AS, Kang NS, Kim J-H, Kim KY, Park K-T, Lee K (2014) Feeding by the newly described mixotrophic dinoflagellate Gymnodinium smaydae: feeding mechanism, prey species, and effect of prey concentration. J Exp Mar Biol Ecol 459:114–125 Lighthill J (1976) Fagellar hydrodynamics. SIAM Rev 18:161–230 Lindberg RE, Bovee EC (1976) Chaos caroliensis, induction of phagocytosis and cannibalism. J Protozool 23:333–336 Linnenbach M, Hausmann K, Patterson DJ (1983) Ultrastructural studies on the food vacuole cycle of a helizoan (Feeding by Actibophrys, III). Protoplasma 115:43–51 Luckinbill LS (1973) Coexistence in laboratory populations of Paramecium aurelia and its predator Didinium nasutum. Ecology 54:1320–1327 Luckinbill LS (1974) The effects of space and enrichment on a predator-prey system. Ecology 55:1142–1147 Lynn D (2008) The Ciliated protozoa: characterization, classification, and guide to the literature. Springer. 605pp Matsuoka A (2007) Living radiolarian feeding mechanisms: new light on past marine ecosystems. Swiss J Geosci 100:273–279 Meadows PS, Anderson JG (1966) Micro-organisms attached to marine and freshwater sand grains. Nature 212:1059–1060 Mueller M, Röhlich P, Torö I (1965) Studies on feeding and digestion in protozoa. VII. Ingestion of polystyrene latex particles and its early effect on acid phosphatase in Paramecium mulitimicronucleatumn and Tetrahymena pyriformis. J Protozool 12:27–34 Muller WA (1975) Competition for food and other niche-related studies of three species of saltmarsh foraminifera. Mar Biol 31:339–351 Murray JW (2006) Ecology and applications of benthic foraminifera. Cambridge University Press, p 426 Nielsen C, Riisgård HU (1998) Tentacle structure and filter-feeding in Crisia eburnea and other cyclomatous bryozoans—collecting mechanisms. Mar Ecol Prog Ser 168:163–186 Nilsson JB (1979) Phagotrophy in Tetrahymena. In: Levandowsky M, Hutner SH (eds) Biochemistry and physiology of protozoa, vol 2, 2nd edn. Academic Press, New York, pp 339–379 Ofelio C, Blanco A, Roura Á, Pintado J, Pascual S, Planas M (2014) Isolation and molecular identification of the scuticociliate Porpostoma notata Moebius, 1888 from moribund reared Hippocampus hippocampus (L.) seahorses, by amplification of the SSU rRNA gene sequences. J Fish Dis 37:1061–1065

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Ogden CG, Hedley RH (1980) An Atlas of freshwater testate amoebae. Oxford University Press, Oxford Page FC (1976) An illustrated key to freshwater and soil amebae. Freshwater Biological Association, Ambleside, UK Page FC (1983) Marine gymnamoebae. Institute of Terrestrial Ecology, NERC, Camridge, UK Peck RK (1985) Feeding behavior in the ciliate Pseudomicrothorax dubius in a series of morphologically and physiologically distinct events. J Protozool 32:492–501 Prasad RR (1958) A note on the occurrence and feeding habit of Noctiluca and their effects on the plankton communities and fisheries. Proc Ind Acad Sci B 47:331–337 Rasmussen L, Orias E (1975) Tetrahymena growth without ophagocytosis. Science 190:464–465 Roberts AM (1981) Hydrodynamics in protozoan swimming. In: Levandowsky M, Hutner SH (eds) Biochemistry and physiology of pro-tozoa, vol 4, 2nd edn. Academic Press, New York, pp 6–66 Salt GW (1979) Density, starvation, and swimming rate in Didinium populations. Am Nat 113:135–143 Savoie A (1968) Les cilliés histophages e biologie cellulaire. Ann Univ Ferrara NS 3(6):65–71 Schuster FL (1979) Small amoebas and amoeba flagellates. In: Levandowsky M, Hutner SH (eds) Biochemistry and physiology of protozoa, 2nd edn. Academic Press, New York, pp 215–285 Song JY, Kitamura SI, Oh MJ, Kang HS, Lee JH, Tanaka SJ, Jung SJ (2009) Pathogenicity of Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum (Ciliophora, Scuticociliatida). Dis Aqat Organ 83:133–143 Spero HJ (1982) Phagotrophy in Gymnodinium fungiforme (Pyrrhophyta): the peduncle as an organelle of ingestion. J Phycol 18:356–360 Spielman LA (1977) Particle capture from low speed laminar flows. Ann Rev Fluid Mech 9:297–319 Steinberg C (2003) Ecology of humic substances in freshwaters: determinants from geochemistry to ecological niches. Springer, p 440 Stidworthy MF, Garner MM, Bradway DS, Westfall BD, Joseph B, Repetto S, Guglielmi E, Schmidt-Posthaus H, Thornton SM (2013) Systemic scuticociliatosis (Philasterides dicentrarchi) in sharks. Vet Pathol 51:628–632 Stump AB (1935) Observations on the feeding of Difflugia, Pontigulasia and Lesqereusia. Biol Bull 69:136–142 Swanberg NR, Anderson OR (1985) The nutrition of radiolarians: trophic activity of some solitary Spumellaria. Limnol Oceanogr 30:646–652 Tucker JB (1968) Fine structure and function of the pharyngeal basket in the ciliate Nassula. J Cell Sci 3:493–514 Wessenberg H, Antipa GA (1970) Capture and ingestion of Paramecium by Didinium nasutum. J Protozool 17:250–270

5

Bioenergetics

5.1

Balanced Growth: Efficiency of Conversion and the Relative Importance of Power Generation for Different Functions

In order to grow and divide, protozoa must assimilate building blocks for the synthesis of cell constituents (known as the assimilatory metabolism). They also need free energy for the synthesis of macromolecules, for the maintenance of the integrity of the cell, and for various processes that enhance the survival of the cell including osmotic and electric work and motility. In heterotrophic organisms, this is all provided for by the dissimilatory metabolism. In heterotrophic eukaryotes, the dissimilatory metabolism is based on aerobic or anaerobic degradation of organic molecules and the assimilatory metabolism also depends on organic molecules. In an ecological context, it is of interest to know the efficiency of conversion of food to cell biomass and the rate at which this conversion takes place. In most treatments of ecological bioenergetics, “balanced growth” is implicitly assumed. In a constant environment that meets the cells’ requirements, populations will increase exponentially and irrespective of how we quantify the population, e.g. by cell numbers, total organic C, DNA, or O2-consumption, the measured growth rate constant will remain the same. Balanced growth implies that the “age structure” (that is the relative abundance of different life cycle stages) remains constant over time. Balanced growth is a property of the population, not of individual cells, in which different processes, such as, DNA synthesis, take place during different stages of the cell growth cycle. Balanced growth occurs in a chemostat, and balanced growth can also be attained for a limited time period in a batch culture if the initial inoculum is small, so that the population can grow for several generations without changing the environment much. If balanced growth is realized in nature it occurs only over short periods due to environmental fluctuations or because the growth rate declines over time due to exploitation of the available food particles; starvation results in a rapid decline in respiration rates (see Chap. 6). # Springer Nature Switzerland AG 2020 G. F. Esteban, T. M. Fenchel, Ecology of Protozoa, https://doi.org/10.1007/978-3-030-59979-9_5

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5 Bioenergetics

Fig. 5.1 Left: growth rate constant (20  C) of the phagotrophic flagellate Paraphysomonas vestita as a function of the concentration of bacteria. Right: the eventual yield of flagellates as a function of the initial concentrations in four batch cultures. (Data from Fenchel 1982)

When expressed in units of energy, the ingested material is equal to the sum of respiration + growth + egested and excreted material. Respiration represents the dissimilatory metabolism; in practice it is usually measured as O2-consumption. In aerobic organisms, oxygen consumption is an adequate measure of power generation: about 2
108 erg (¼20 J) per ml O2. Egested and excreted material represents non-digestible parts of the food and loss of dissolved low molecular weight compounds. The “gross growth efficiency” or “yield” is defined as growth divided by ingestion and “net growth efficiency” is growth divided by growth + dissimilated matter. Figure 5.1 shows the growth rate constant and cell yield (final number of consumers divided by the initial number of food particles) in batch cultures of the heterotrophic flagellate Paraphysomonas vestita with different initial concentrations of food bacteria. It can be seen that yield is invariant with the growth rate constant. Basically, this reflects that, in contrast to the case for macroscopic organisms, growing protozoa spend a much larger fraction of the energy generated by the dissimilatory metabolism for growth, that is, for the energetic costs of synthesizing macromolecules (proteins and nucleic acids). This is also evident from the fact that a small aerobic protozoon (at 20  C) is capable of doubling its biomass within 3–4 h— something that is impossible for macroscopic organisms. It is also clear that the respiration rate must be almost linearly proportional to the growth rate within the range of food availability that can maintain balanced growth. The concept of “basal metabolism” as used by zoo-physiologists has therefore little meaning in the case of protozoa. This again reflects that in growing small organisms by far the largest part of the power generation is involved in macromolecular synthesis and thus directly coupled to growth. Conversely, only a relatively smaller fraction of the energy budget is spent on motility and electrical and osmotic work.

5.2 The Rate of Living

57

It is here illuminating to calculate the approximate cost of motility. As an example, consider a spherical flagellate (radius 4 μm) which swims with a velocity of 60 μm/sec. From Stokes’ law, we find that the necessary power is 6πrv2η, where v is velocity and η viscosity (0.01 poise). In the case of the swimming flagellate, this works out to be 2.7
107 erg s1. This estimate agrees with independent estimates for the power consumption of a single flagellum of 2  8
107 erg s1 (Sleigh 1974). A growing cell of this size consumes at 20  C about 4.5
103 nl O2 h1 corresponding to 2.5
104 erg sec1 and so then only about 0.3% of the energy budget of the flagellate is used for swimming. In contrast, mechanical work (swimming, flying, and running) represents a substantial part of the energy budget of large motile animals. There are two reasons for this difference between macroscopic animals and microbes. One is that the weight-specific power generation of protozoa is much higher than that of macroscopic organisms, and the other is that the swimming velocities of protozoa are very low in absolute terms—in the case of the flagellate example it only amounts 22 cm h1. Similar considerations show that the power needed for osmotic and electric work is also modest relative to the power generation of growing protozoa. For bacteria, it has been shown that during balanced growth the generation of one mole of ATP leads to the production of approximately 10 g dry weight of cells (Beauchop and Elsden 1960; Paine 1970). If—as is nearly always the case for eukaryotic cells—the C atoms of the substrate have an oxidation level similar to that of glucose, this corresponds to a net growth efficiency of 67%. In unicellular eukaryotes (and in growing tissue of poikilothermic metazoans) it has also been found that net growth efficiency is around 60% (Calow 1977; Fenchel and Finlay 1983). This value is invariant with the size of the organism and within wide limits, and it probably represents some fundamental limit to growth efficiency. Gross growth efficiency is a more variable parameter. Typical values are 30–50% so that as much as 50% may be egested in an undigested state or is lost as dissolved organic matter, and it varies according to the qualitative nature of food particles.

5.2

The Rate of Living

It has long been recognized that respiration rates of different animals are not proportional to body weight, but can be described by the relation: R ¼ aWb where a and b are constants. It was further found empirically that when organisms of different sizes were compared, the constant b takes the value of 3/4 (e.g. Hemmingsen 1960) and so the weight-specific respiration rate should decrease with a power of 1/4 as a function of size. There are, however, many deviations from this rule, when different taxonomic groups or ontogenetic stages are compared and, e.g. endothermic animals have a higher value of the constant a compared to that of exothermic animals. With respect to protozoa, Hemmingsen (1960) found that the value of the constant, a, is somewhat lower in protozoa as compared to that of invertebrates. Fenchel and Finlay (1983) found that this is due to the fact that many data on respiration rates of protozoa were based on non-growing and starving cells.

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Fig. 5.2 Left: maximum respiration rates (20  C) of different ciliates and flagellates (filled circles) and amoebae (open circles) during exponential growth and as a function of cell volume. The slope of the regression line is 0.75. Right: the maximum attainable growth rate constant (20  C) for some protozoan species: the slope of the regression line is 0.25 (Data from Fenchel and Finlay 1983)

Including only measurements of the respiration of rapidly growing cells, the data points represent an extrapolation of the data for multicellular exothermic animals (Fig. 5.2). There have been many attempts to rationalize the value of 3/4 for the constant b (e.g. Glazier 2010), but they have so far not been quite successful. The maximum growth rate constants of protozoa also scale with cell volume with an exponent of 1/4 (Fenchel 1974). However, among the tiniest protozoa (