PHYSIOLOGY of the CLADOCERA Mud-Living Ilyocryptus Colored Red by Hemoglobin

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PHYSIOLOGY OF THE CLADOCERA Mud-living Ilyocryptus colored red by hemoglobin. Photographed by A.A. Kotov. PHYSIOLOGY OF THE CLADOCERA

NIKOLAI N. SMIRNOV D.SC. Institute of Ecology, Moscow, Russia

WITH ADDITIONAL CONTRIBUTIONS

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Over 700 species of Cladocera are known, the role of iron are also discussed. Studies on and representatives of this group are often the impact of xenobiotics on respiration are dominant in the freshwater fauna and some- reviewed and the blood flows and separating times occur in enormous quantities. They live membranes are described. Heart rate and its in both small and large water bodies from arctic controlling factors are also reviewed. Occasional to tropical latitudes, in open water, on the bot- heart arrest and adhesion of blood cells are tom, in mud, among inshore vegetation, in acid reported. Studies on phagocytosis are pointed pools on bogs, in small accumulations of water out. The data related to excretion are reviewed in epiphytic plants, in narrow aquatic spaces and the distribution of xenobiotics in the body, between moist sand grains. A few species even their bioaccumulation and the transformation left the water and live in moist moss-like of xenobiotics are described. Studies on routes growth on tree trunks in tropical cloud forests. of elimination of xenobiotics and on detoxifica- Some species are specialized for life in saline tion are indicated. lakes and in the sea. Ion exchange is realized via the neck organ The data on organic and inorganic constitu- and limb epipodites. The principal types of ents of cladocerans are reviewed. They may osmotic regulation are: hyperosmotic regulation accumulate physiologically important sub- (in freshwater species), hypoosmotic regulation stances, those of no such importance, or toxi- (in marine Penilia), and amphiosmotic cants. The section on nutrition of Cladocera regulation. The effect of xenobiotics on osmotic comprises a brief review of their anatomical regulation is discussed. Numerous enzymes and environmental backgrounds, with special have been determined in Cladocera and studies consideration of food quality (algae, algal on their role are reviewed. The impact of xeno- lipids, bacteria, and organic debris). Feeding biotics on metabolic links is described, as well and digestion are discussed for both littoral as detoxification in the cladoceran body. and pelagic species and the digestion of pro- Cladocera live for a week up to several tein, carbohydrate, and lipids is reviewed. months, depending on the species and the Cladocera modify only a small amount of the environmental conditions. Body length incre- lipids consumed with algae, and then transfer ments occur between molts. Mechanical dam- them to fish and thus further, to man. The fate age is repaired by regeneration. Effects of of ingested chlorophyll, starvation and the environmental factors and xenobiotics on mor- impact of xenobiotics on feeding and digestion phology and cytology are described and data are also commented on. on the impact of xenobiotics on molting and life Oxygen consumption and the production duration are reviewed. The state of investiga- of CO2 are considered in littoral and pelagic tions on senescence and mortality is also com- Cladocera. Littoral and bottom-dwelling spe- mented on. cies often live under conditions of hypoxia Cladocera are remarkable by their partheno- and anoxia. The dynamics of hemoglobin and genetic reproduction, which is sometimes

ix x PREFACE interrupted by bisexual (gamogenetic) reproduc- xenobiotics on behavior are also reviewed. tion. Physiological aspects of parthenogenetic Ecological aspects discussed comprise consid- and gamogenetic reproduction are discussed. eration of physiological background and the Differences of movement and trajectories of litto- limits of physiological factors, synergism and ral and pelagic species are indicated and data antagonism of factors, pathway of lipids from on the physiology of muscles, immobilization, algae via Cladocera to fish, conditioning of the fatigue, stress, and impact of xenobiotics are also environment by Cladocera, as well as the use reviewed. There is information on neurosecretion, of Cladocera in water quality testing. vision, ability to discern polarized and colored Within certain limits, Cladocera can support light, photoperiod, chemoreception, mechanore- the body’s homeostasis in a dynamic environ- ception, ability for orientation in space, endoge- ment, including the homeostatic level of their nous rhythms, impact of xenobiotics. chemical constituents and of osmotic pressure. Cladocera possess complicated behavior Along with special discussions, introductory patterns, differing in particular species. These remarks are made whenever it seemed to be include migration, swarming, akinesis and necessary to make the matter useful both for escape behavior. The data on the impact of specialists and for non-specialists. Contributors

Stuart K. J. R. Auld School of Natural Sciences, University of Notre Dame, Notre Dame, IN, University of Stirling, Stirling, Scotland, UK USA Margaret J. Beaton Biology Department, Mount Joseph R. Shaw School of Public and Allison University, Sackville, NB, USA Environmental Affairs, Indiana University, Do¨rthe Becker Environmental Genomics Group, Bloomington, IN, USA School of Biosciences, University of Birmingham, Nikolai N. Smirnov Institute of Ecology, Leninski Birmingham, UK Prospect 33, Moscow, Russia John K. Colbourne Environmental Genomics Group, Elizabeth Turner School of Public and School of Biosciences, University of Birmingham, Environmental Affairs, Indiana University, Birmingham, UK Bloomington, IN, USA Carli M. Peters Biology Department, Mount Allison Kay Van Damme Environmental Genomics Group, University, Sackville, NB, USA School of Biosciences, University of Birmingham, Michael E. Pfrender Department of Biological Birmingham, UK Sciences, Environmental Change Initiative,

xi Acknowledgments

The present volume is an attempt at making Many librarians, mostly personally unknown a summary of work of many experts through- to the author, retrieved numerous publications out the world in various fields using special from different countries and time periods. Their methods. A substantial contribution to physiol- care and labor are appreciated, including those ogy of the Cladocera is made recently by of the Biological Department Library of Russian toxicologists. Academy of Sciences, including Ms N.I. Special thanks are due to Dr M.J. Burgis (UK) Gotovskaya and Ms E.V. Morozova. who generously used her time and experience to The present study is supported in part by improve the manuscript. grants on Cladocera projects from the Russian The present review is motivated by the Foundation for Basic Rssearch (12-04-00207, etc.) author’s observations on living, mostly littoral, and from the Program “Living Nature”. cladocerans. Some recent observations are made My wife L.A. Smirnova, Ph.D. (cited here as at the Hydrobiological Station “Glubokoe Lake” L.A. Luferova) is tolerant (mostly) towards using (Russia). The author is grateful to his imme- a big part of my time for such ventures as this. diate colleagues from the ‘Cladocera team’ Formulation of ideas included in this book for help and discussions: O.S. Boikova, and its composition was much favored by N.M. Korovchinsky, A.A. Kotov, E.I. Bekker, creative environment at the Institute of Ecology A. Yu. Sinev, as well as to Dr Yu.B. Manteifel and Evolution of the Russian Academy of and Dr E.P. Zinkevich for advice. Sciences, and by personal attention of academi- Dr A.A. Kotov critically read the draft man- cian D.S. Pavlov, Director, and of academician uscript, suggested numerous useful additions, Yu.Yu. Dgebuadze, Laboratory Head. and used his skill for preparation of the manu- The subject is made much more complete by script and figures. the authors of chapters 16À18.

xiii CHAPTER 1

General N.N. Smirnov

1.1 SYSTEMATIC POSITION being investigated, keys to the worldwide fauna of Cladocera are indicated: “Guides to the iden- It is now thought that there are over 700 spe- tification of the macroinvertebrates of the conti- cies of the order Cladocera in the world fauna, nental waters of the World,” issues 1, many of which develop populations in enor- Macrothricidae (Smirnov, 1992); 3, Ctenopoda mous quantities and thus play a big role in the (Korovchinsky, 1992), 11, Chydorinae (Smirnov, life of the biosphere. New species are still being 1996); 17, Simocephalus (Orlova-Bienkowskaja, described. 2001), 13, The predatory Cladocera (Rivier, The Cladocera belong to the subclass 1998); 21, Daphnia (Benzie, 2005); and 22, Phyllopoda of the class Crustacea. Most Ilyocryptidae (Kotov and Stifter,ˇ 2006). There Cladocera belong to the orders Anomopoda and are also newer, general worldwide resources for Ctenopoda. Anomopoda principally comprise Ctenopods, created by Korovchinsky (2004); the families Daphniidae (e.g. the genera Daphnia, Leydigia (Chydoridae), by Kotov (2009); and Ceriodaphnia, Simocephalus,andScapholeberis), Eurycercus (Bekker, et al., 2012); as well as recent Moinidae (e.g. Moina), Ilyocryptidae regional keys. (Ilyocryptus), Macrothricidae (e.g. Macrothrix and As investigations into Cladocera are actively Streblocerus), Acantholeberidae, Ophryoxidae, developing, the aforementioned summaries are Eurycercidae (Eurycercus), Chydoridae (e.g. rapidly becoming incomplete, and more recent Chydorus and Pleuroxus), Bosminidae (Bosmina, literature should also be taken into Bosminopsis); and Ctenopoda comprise the fami- consideration. lies Sididae (e.g. Sida, Pseudosida,and Diaphanosoma)andHolopedidae(Holopedium). Others belong to the order Onychopoda 1.2 GENERAL MORPHOLOGICAL (Polyphemus,aswellasmarineandbrackish BACKGROUND water species) and the order Haplopoda with the family Leptodoridae (Leptodora). As animal functions are linked to their form, As physiological studies should be accompa- some comments on the body structure and nied by the reliable identification of the subjects organs of Cladocera are provided here. Most of

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00001-X 1 © 2014 Elsevier Inc. All rights reserved. 2 1. GENERAL the animals attributed to the order Cladocera pairs of appendages—antennules, antennae have the same principal structure, with various (biramous, with the single exception of female modifications present in different species. Holopedium), mandibles, maxillulae, maxillae Investigations into comparative and functional (may be completely reduced), mandibles, and morphology (such as, e.g. those by Fryer, 1968, five or six pairs of thoracic limbs (Figs. 1.11.4). 1974, 1991, etc.) have revealed exciting data on Cladocerans are mostly oval in shape, com- particular species, permitting a better under- pressed from the sides, but many are spherical. standing of their lifestyles. In the case of Graptoleberis, there is a curious and Cladocerans have inherited from their ances- unique combination of lateral and dorsoventral tors a weakly segmented body covered with a compression (Fig. 1.3). The appendages have chitinous, mostly bivalved, shell and bearing few numerous, but organized, setae. The posterior

C BP HT 5c EMB TMM SUS DVM DLM TMT VLM MG

DIV A2MUS

FIB DAO

PA END CG O AD RE 100 µm A1 LM OCM ODM ENP LGC K L Mand DG LDS TLS

FIGURE 1.1 General anatomy of Acantholeberis curvirostris. A1, antennule; A2MUS, antennary muscles; AD, apodeme; BP, brood pouch; C, carapace; CG, cerebral ganglion; DAO, dilator muscle of atrium oris; DG, duct of labral glands; DIV, diverticulum; DLM, dorsal longitudinal muscles; DVM, dorsoventral trunk muscles; E, compound eye; EMB, embryo; END, endoskeleton; ENP, endoskeletal plate; FIB, fibrils; HT, heart; K, keel of labrum; L, labrum; LDS, long distal setae of outer distal lobe of trunk limb 1; LGC, labral gland cells; LM, levator muscle of labrum; Mand, mandible; MG, mid-gut; O, ocellus; OCM, esophageal constrictor muscles; ODM, esophageal dilator muscles; PA, postabdominal lamella; RE, rectum; SUS, suspensory ligament; TLS, trunk limbs; TMM, 5c, transverse muscle of mandible; TMT, transverse mandibular tendon; VLM, ventral longitudinal trunk muscles. Source: Fryer (1974).

PHYSIOLOGY OF THE CLADOCERA 1.2 GENERAL MORPHOLOGICAL BACKGROUND 3

OL E CG LM Ca O HS Oe A2M ODM Mand DES LGC TMT TMM A2M SUS L 5c Endo S SA1 A2M

TL1

TL2 Ht

D Endo S C TTC Mxlle

TL3 BP

NC FCS

FG FP3 200 µm A = ACAM EX3 TL5 P = PCAM EXS5 FP4 EXS3 EX4 TL4 AVS EP5

FIGURE 1.2 General anatomy of Daphnia longispina. A, anterior carapax adductor muscle; A2M, antennal muscles; AVS, anterior vertical seta of trunk limb 5; Ca, cecum; D Endo S, dorsal endoskeletal sheet; DES, dorsal extension of ventral endoskeletal sheet; Endo S, endoskeletal sheet; EP5, epipodite of trunk limb 5; EX3, 4, exopod of trunk limbs 3, 4; EXS5, exopod seta 5; FCS, filter-cleaning spine of trunk limb 2; FG, food grove; FP3, gnathobasic filter plate of trunk limb 3; FP4, gnathobasic filter plate of trunk limb 4; Ht, heart; HS, head shield; LGC, labral gland cells; Mxlle, maxillule; NC, nerve cord; Oe, esophagus; OL, optic lobe of cerebral ganglion; P, posterior carapax adductor muscle; SA1, sensory seta of antennule; TL1, 2, 3, 4, 5, trunk limbs 1, 2, 3, 4, 5; TTC, thickened trunk cuticle. For other abbreviations, see Figure 1.1. Source: Fryer (1991).

PHYSIOLOGY OF THE CLADOCERA 4 1. GENERAL S 1 S 2 L Ra Rp I Vm a 2 Sg Vm 3 Vm o.Lm 3 II 1 b Sg 4 III c o.Lm d Sg IV 5 e V f Asg 1 Dm Vm 3 Ad Vm Vm 2 1

FIGURE 1.3 Muscles of Daphnia magna. Source: Binder (1931). end of the body (postabdomen) is bent at a right The outer surface of the chitinous shell may angle to the abdomen, or may even be reversed. be smooth, reticulated, ciliated, setose, or vari- All structures tend to undergo morphological ously honeycombed. The dorsal space under radiation, and homologous structures may occur the shell is the brood chamber into which eggs in different species in various forms, from the are laid and develop. ancestral state to their complete disappearance The inner organs are situated within the or, in contrast, enlargement and specialization. body rather loosely. The intestine may be

PHYSIOLOGY OF THE CLADOCERA 1.2 GENERAL MORPHOLOGICAL BACKGROUND 5

SSL

HS R

S A2

MVS

SSA T

50 µm

PG 50 1 µ m

VF PVS

TCB PA

PAS

FIGURE 1.4 Modification of the ventral side for movement over flat surfaces. Right, edge of valve of Scapholeberis mucronata.Left,Graptoleberis testudinaria. A1, antennule; A2, antenna; C, carapace; EX 3, exopod of trunk limb 3; HS, cuticle of head; MVS, medium ventral setae; PA, postabdomen; PAS, Postabdominal seta; PG, posterior gap; PVS, posterior ventral setae; R, thickened rim of head shield; S, Sensory setae of AII; SSA, Swimming setae of AII; SSL, setules of sealing seta of trunk limb 1; TCB, transverse chitinous bar; TL1, trunk limb 1; VF, ventral flange. Sources: right, Dumont and Pensaert (1983); left, Fryer (1968). PHYSIOLOGY OF THE CLADOCERA 6 1. GENERAL straight or convoluted. Muscles do not form compact masses and most of them can be seen individually (Fig. 1.3). The largest muscles are longitudinal bands stretching along the gut. Groups of muscles allow motion of the thoracic limbs and antennae. Small muscles rotate the eye and move the labrum and antennules. C2 The intestine is supplied with circular muscles. S The ovary (or testis) is paired and situated C1 ventrally along the gut; this is also where the fat body is situated, in contact with the ovaries (Jaeger, 1935). There are two paired remains of the coelom: the antennal gland and the maxillary gland (shell gland; Fig. 1.5). The latter is the organ of excretion, while the antennal gland has no duct and no outer orifice. On the head of most species there are head pores, leading to an organ whose function is probably ion exchange. The nervous system comprises a double chain of ganglia, with the brain located in the cephalic region. Sense organs comprise the unpaired eye, the unpaired ocellus (Figs. 1.1, 1.2, and 1.6), sensory papillae situated on the antennule and on some thoracic limbs, and numerous tactile setae. The eye or the ocellus may be absent in some species. On the basis of this general structural scheme, FIGURE 1.5 three kinds of specialization are formed: one Position of nephridia in the body of used for collecting food from substrata (Fig. 1.1), Daphnia. C1, maxillary coelomic sac; C2, antennal coelomic sac; another for filter feeding in open water (Fig. 1.2), S, ducts of maxillary coelomic sac. Source: Gicklhorn (1931a). and the last, predatory. Littoral cladocerans live among organic and mineral particles and require protection, sup- 1.3 SIZE AND WEIGHT plied by thick chitinous shells, most with sculp- CHARACTERISTICS turing; this increases the durability of their shells (Fryer, 1968). According to the same Most Cladocera species are small: from author, in littoral species the cuticle is up to about 0.3 mm to c. 6 mm. One of the largest, 12 µm thick (in Pseudochydorus). In pelagic spe- Leptodora, is c. 10 mm in length. Due to their cies, the integuments are many times thinner. small size, their surface:volume ratio is high. Further information on Cladocera may Several authors have investigated their be obtained from www.cladocera-collection.cz length:weight ratios [e.g. Ivanova and and Lampert (2011). Klekowskii, 1972 (Simocephalus); Kawabata and

PHYSIOLOGY OF THE CLADOCERA 1.3 SIZE AND WEIGHT CHARACTERISTICS 7

where q is 0.133 for Scapholeberis; 0.075 for other Daphniidae, 0.127 for Eurycercus; 0.091 for Alona and Alonella; 0.203 for Chydorus; 0.083 for Macrothrix; and 0.140 for other Chydoridae and Macrothricidae. b is 2.630 for Scapholeberis; 2.925 for other Daphniidae; 3.076 for Eurycercus; 2.646 for Alona and Alonella; 2.771

c for Chydorus; 2.331 for Macrothrix; and 2.723 for other Chydoridae and Macrothricidae. Similar equations were determined by Lynch et al. (1986):

for Daphnia ambigua,W5 5.740L2.370; 5 2.200 b for D. galeata mendotae,W 5.480L ; a d for D. parvula,W5 4.740L2.190; and 2.093 bc for D. pulex,W5 10.674L .

ce dFO Dry weight (DW) increases with length, as shown for D. hyalina (Baudouin and Ravera, fF 1972). The relationship between DW (in mg) and length (L, in mm) was determined for la og tc D. pulex by Richman (1958) to be: int proto npe 2 : DW 5 0:028L 0 022 (1.2) Isc 2 trito and (by Wen et al., 1994), for Daphnia, Isc 1 bc Simocephalus, Ceriodaphnia, and Bosmina combined, to be :

ant : DW 5 0:013L2 14 (1.3)

FIGURE 1.6 Cephalic region of nervous system. Regression equations for DW and length for Above, Eurycercus. a, antennule; b, ocellus; d, lateral organ. many planktonic and littoral species were sup- Below, Daphnia. ant, antennule; bc, bulhed cells; ce, compound eye; dFO, dorsal frontal organ; fF, frontal filament; plied by Dumont et al. (1975), for five la, lamina; lsc 1, labral secretory cells; npe, nauplius eye; Amazonian species by Maia-Barbosa and og, optic ganglion; proto, protocerebrum; te, tectum; trito, Bozelli (2005), and for three species from tritocerebrum; *, deuterocerebrum. Source: above, Leydig Mexico by Nandini and Sarrma (2005). (1860); below, modified from Weis et al. (2012). Weight at a certain length of Cladocera was also determined and presented in tabulated Urabe, 1998]. The relationship between length form by Mordukhai-Boltovskoi (1954), Kosova (L) and wet weight (W) may be described by (1961), and Sokolova (1974). In addition, Eq. 1.1 (Kurashov, 2007): Vasama and Kankaala reported length-carbon regressions in Cladocera (1990). WðmgÞ 5 qLbðmmÞ; (1.1)

PHYSIOLOGY OF THE CLADOCERA CHAPTER 2

Methods

Methods of investigating the physiology of Gajewskaja (1940, 1948) suggested a method Cladocera range from direct observation of liv- of separating large-scale cultures of Cladocera ing specimens to recording the physical and and algae because combined cultivation of chemical manifestations of particular physio- Cladocera and algae requires conflicting condi- logical processes. Detailed examination of pre- tions. Since then, methods of large-scale served or living specimens supplies useful culture have been developed further information on their anatomical background (Yalynskaya, 1961; Ivleva, 1969; Lampert, 1975; and function. Bogatova, 1992). Cladocera may be collected in the littoral In culture, littoral Cladocera should be fed and pelagic zones of large and small water with organic debris (detritus) collected at shore bodies. They should be looked for in ponds, bottoms and screened to remove foreign pools, puddles, temporary pools, roadside animals (Smirnov, 1971) or with artificial ditches, acid bogs, fountains, all kinds of artifi- detritus prepared from plants (see e.g. Rodina, cial basins, moist moss, and even in moist, 1963; Esipova, 1969, 1971; Dekker et al., 2006). moss-like growth on tree trunks. Fresh detritus should be given at least every Cultures may be started from living speci- other day (e.g. a specimen may be transferred mens. Cultures derived from a single female to a dish containing fresh detritus). are called clonal cultures and provide rela- Cultured pelagic Cladocera should be fed tively uniform material. Culture methods with algae and adequate algal cultures should have been described by various authors be maintained for this purpose. Some species either alone (e.g. Biotechnics of Daphnia culture (Daphnia magna and D. pulex) are more easily at fish farms, 1958; Dewey and Parker, 1964; cultivated than others. Ivleva, 1969; Parker and Dewey, 1969; Sterilization was used to demonstrate the Bogatova, 1980; Goulden et al., 1982; Dodson role of bacteria in the alimentation of cladocera and Frey, 1991) or in descriptions of particu- (Gajewskaja, 1938; Rodina, 1965; Esipova, 1969, lar experiments. The combined culture of 1971). The latter author used Lugol’s solution Daphnia with other cladocerans was sug- at a 1/228 dilution to reduce the quantity of gested by Bogatova (1963, 1980). bacteria in food offered to Cladocera.

Suspensions of latex beads have also been Using cyanoacrylate glue, Onbe´ (2002) used in investigations of feeding behavior attached Pseudevadne and Evadne to the tip of a (Burns 1968b, 1969; Hessen, 1985). glass capillary held in place by a stand to facil- Various kinds of intravital staining of the itate video recording. Pen˜alva-Arana et al. organ systems of Cladocera were originally (2007) reported unbiased observations using investigated by Fischel (1908). The salivary computer recording combined with their gland can be stained using neutral red and immobilization system. Bismarck brown (Cannon, 1922). In thoracic Methods of attachment were recently limb IV of Eurycercus, there is a slime gland described by Seidl et al. (2002) and used by whose secretions are stained bright blue with Pirow et al. (2004) for investigating oxygen Mallory’s stain (Fryer, 1962, 1963). Gut con- transport processes in D. magna. The latter tents have been stained with eosin, Congo red, authors immobilized fasting animals by gluing methyl red, neutral red, and uranine for the their posterior apical spine with histoacryl to a determination of pH (Lavrentjeva and Beim, bristle, which was then fixed to a coverslip 1978). with plastilin. Dye was then microinjected into Histochemical techniques have been used in the circulatory system from the dorsal side the analysis of Holopedium slime (Brown, 1970). into the space “directly downstream of the External slime may be distinguished by plac- heart.” The coverslip then formed the base of a ing a cladoceran in diluted Indian ink. thermostatted perfusion chamber in which an Preferences for environmental factors, and immobilized daphnid was able to freely move for food, may be ascertained by experiments its antennae. on living specimens. Examination of food com- Ivanova and Klekowski, (1972) achieved position is made easier by dissolving the soft immobilization of Simocephalus by placing it tissues of cladocerans with 3% sodium hypo- into the bulb of a Cartesian diver (used for chlorite (Infante, 1978). This treatment may determining oxygen consumption) and leaving also be useful for other purposes. no free space around the animal, i.e. it was too Cladocerans are very agile; therefore high- small to allow movement. speed photography has been used (e.g. Various kinds of microscopy, including Storch, 1929; Zaret and Kerfoot, 1980) to scanning electron microscopy (SEM), can be study them. Alternatively, methods to inhibit used in investigations of Cladocera. A varia- their movement have been devised. tion of cladoceran preparation for SEM was Immobilization by narcotization is discussed suggested by Laforsch and Tollrian (2000). in more detail in Chapter 12, section 12.5. Modern video microscopy and digital image Early techniques included attaching speci- processing methods take advantage of their mens to a substrate on a glass slide using transparency (Colmorgen and Paul, 1995). wax dissolved in alcohol (Scourfield, 1900b; Fluorescence analysis was originally used by Pen˜ alva-Arana et al., 2007). More recently, Pravda (1950). Porter and Orcutt (1980) used silicone grease A special set-up for optophysiological to immobilize D. magna by its head shield to recordingwasdevisedbyPauletal.(1997): observe its feeding. For the purposes of his this comprised a video recorder and PC, as study, Jacobs (1980) attached Daphnia by the well as computerized respirometry. For this caudal spine to a plasticine bed. Specimens procedure, Daphnia were placed in a glass cell thus immobilized could be observed and the of 0.45 mL volume. Pirow et al. (2001) mea- next instar was released into free water from sured tissue oxygenation using an apparatus the attached exuvium. consisting of a reversed microscope equipped

PHYSIOLOGY OF THE CLADOCERA METHODS 11 with systems for measuring hemoglobin (Hb) Spectrophotometry has been used for the oxygenation, reduced nicotinamide adenine identification of particular compounds, includ- dinucleotide (NADH) fluorescence (as an indi- ing Hb. Initially, Fox (1948) suggested that a cator of tissue oxygenation state), and the quantitative estimation of Hb content in movements of organs. Remarkable results Daphnia in arbitrary units could be obtained were obtained by Pirow et al. (2004) using against a wedge-shaped standard prepared injection of the oxygen-sensitive phosphores- from the worker’s blood. Following this, cla- cence probe Oxyphor R2 into the circulatory doceran Hb was investigated using spectral system followed by phosphorescence imaging. (Hildemann and Keighley, 1955; Hoshi et al., Surgical methods can be applied in investi- 1968) and chemical (Hoshi, 1963a, 1963b; gations of regeneration, vision, and neurose- Smirnov, 1970) methods. cretion. These are described in Angel (1967) Recent toxicological studies have assessed and in Chapter 10, section 10.2.1 and Chapter the effects of xenobiotics on particular physio- 13, section 13.4.3. logical processes. In biochemical and metabolic investigations, Photographic recording of the heart rate special procedures, such as homogenization and movement of thoracic limbs has been used (e.g. Guan and Wang, 2004b) and radiotracer (e.g. Kolupaev, 1988). Moreover, computer or chemical methods, have been used. Studies recording of behavior is now used (Pen˜alva- on homogenates using modern sensitive meth- Arana et al., 2007). Special techniques may be ods have opened up the possibility of investi- found in descriptions of the original gating metabolic pathways. investigations.

PHYSIOLOGY OF THE CLADOCERA CHAPTER 3

Chemical Composition

3.1 MOISTURE CONTENT AND content of Daphnia and Ceriodaphnia increases CALORIFIC VALUE with increased protein in their natural food (Guisande et al., 1991). The content of nitrogen The moisture content and calorific value of (N) was determined by Hessen (1990) as 8.18% some Cladocera spp. are shown in Table 3.1. DW, with little variation; the lowest values Moisture content is usually within 80À90% were determined in starved specimens. The and calorific value within 3À6 kcal/g dry content of nitrogen in five species of Cladocera weight (DW). The calorific value (kcal/g) is: was c. 8%, and of phosphorus (P) was c. 1.4% Daphnia hyalina, 6.3; Bosmina coregoni, 6.3; (Main et al., 1997). Chydorus sphaericus, 6.1; and Leptodora kindtii, It is notable that the fat content is especially 5.8 (Vijvberger and Frank, 1976). Variations in variable. Some other data on the lipid content the calorific value obviously depend mostly on (in % DW) have been reported: D. magna, the fat content. The calorific value of “lean” 17À22; D. pulex,10À40; and Bythotrephes ceder- Daphnia magna is only 60% of that of Daphnia stroemi,10À19 (Bilkovic and Lehman, 1997). containing more fat (Chalikov, 1951). Far more informative are reports on the dynamics of general chemical composition, which depends on seasonal changes in a num- 3.2 PRINCIPAL ORGANIC ber of factors, on starvation, or on other partic- CONSTITUENTS ular factors. Often, scattering of the data indicates dependence on specific factors. There have been various determinations of Indeed, if arranged by season, chemical consti- the chemical composition of some Cladocera tuents demonstrate clear composition changes, (daphnids, Moina, Bosmina, and C. sphaericus); as shown, for example, for D. pulicaria (in the general chemical composition of some cla- Fig. 3.1 of Heisig-Gunkel and Gunkel, 1982). docera is shown in Table 3.2. The chemical composition of D. pulex generally Their protein content ranges from 30% confirms this data but depends on the period upwards, their fat content is 1À20%, and their of starvation, with relative quantities of carbo- carbohydrate content is 10À30%. The protein hydrate and fat decreasing, and those of

TABLE 3.1 Moisture Contents and Calorific Values of Some Cladocera SPP

Species Moisture Calorific value Ash (% DW) Reference(s) Content (%) (kcal/g DW)

Bosmina longirostris 89 6.5 À Sherstyuk (1971)

B. longispina À 9.9 4.8 Romanova & Bondarenko (1984) Bythotrephes longimanus 84.7 8.6 5.2 Romanova & Bondarenko (1984) Ceriodaphnia affinis, adults 89.2 ÀÀStepanova (1967) C. affinis, juveniles 90.4 ÀÀStepanova (1967) C. pulchella 70À82 4À4.7 À Sherstyuk (1971) C. quadrangula À 4.9 4.6 Riccardi & Mangoni (1999)

C. reticulata 87À96 5.2À5.5 À Bogatova et al. (1971) Daphnia cucullata À 5.1À5.4 10.6À14.0 Riccardi & Mangoni (1999) D. hyalina À 4.9À5.0 12.0À12.5 Riccardi & Mangoni (1999) D. longispina 82.6 4 22 Romanova & Bondarenko (1984) D. magna 86.4À95.6 2.4À5.6 À Karzinkin (1951), Ostapenya et al. (1968), Schindler (1968), Bogatova et al. (1971), Stepanova (1968), Stepanova et al. (1971), Mityanina (1980)

D. pulex 89À95 2.8À4.9 7.6À25À8 Birge & Juday (1922), Karzinkin (1951), Malikova (1953), Ostapenya et al. (1968), Stepanova (1974) Bythotrephes longimanus À 5.2 5.6 Riccardi & Mangoni (1999) Leptodora kindtii 97.2 4.6 À Sherstyuk (1971)

Moina macrocopa 95 5 À Bogatova et al. (1971) Moina spp. 87À88 4À4.3 À Ostapenya et al. (1968), Stepanova (1974), Zhao (2006)

Polyphemus pediculus 80À86 4.4À4.9 À Sherstyuk (1971) Sida crystallina 84 5.3 À Sherstyuk (1971) Simocephalus vetulus 82À92.4 3.65À4 À Sherstyuk (1971), Stepanona (1968), Stepanova et al. (1971) Eurycercus lamellatus 80À88 4.1À4.3 À Sherstyuk (1971)

protein and ash increasing (Fig. 3.1) (Lemke was: protein from c. 140 to 400; lipids, and Lampert, 1975). Seasonal variations in the 120À180; chitin, 40À70; carotenoids, 70À230; biochemical composition of D. magna in and ash, 100À340. the Luxembourg were studied by Cauchie To understand these variations, data on et al. (1999). The variation (in mg/g DW) the dynamics of the chemical constituents in

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 15

TABLE 3.2 Composition of Some Cladocera SPP., in % DW

Species Carbon Hydrogen Nitrogen Carbohydrate Protein Lipid Author

Ceriodaphnia 49.1 7 9.8 À 54.4 5.3 Riccardi & Mangoni quadrangula (1999)

Daphnia cucullata 53.3À54.5 7.8À8 10.1 14.6 57.0À62.8 19.8À22.7 D. hyalina 50.2À52.3 7.6 11 21.3À22.9 62.2À64.0 13.1À16.5 D. pulex ÀÀ À3.3À10.9 36.4À61.7 2.8À27.9 Birge & Juday (1922) D. pulex ÀÀ À6.4 63.4 8.6 Stepanova (1968) Moina brachiata ÀÀ À8.2 63.4 17.5 Simocephalus vetulus ÀÀ À16.7 52.3 11.5

Bythotrephes 51.2 7.6 11.1 22.0 63.9 14.1 Riccardi & Mangoni longimanus (1999)

FIGURE 3.1 Changes in Daphnia pulex chemical composition as a result of starvation (left, g/Animal]

µ per animal; right, % DW). [ Horizontal axis, number of days of 200 100 starvation. Source: Lemcke and Lampert (1975).

DW Protein 150

CCarbohydratearbohydrate 100 50

[% per DW] Carbohydrate Protein

50 Lipid

Lipid 10 10 Ash Ash 01234 01234 relation to growth are also useful. McKee and of 48À62% (maximal day 8); glycogen content Knowles (1987) studied the levels of protein, steadily increased from 2.4% to 7.5%; lipid con- glycogen, lipid, RNA, and DNA during the tent varied from c. 18% to c. 15%, decreasing to growth of D. magna. They found that percentage 6.4% on day 21; RNA content varied from 8.6% DW during the first to the twenty-first day of to 6.6%, decreasing to 4% on day 21; and DNA life the protein content varied within the range content generally decreased from 0.20 to 0.14%.

PHYSIOLOGY OF THE CLADOCERA 16 3. CHEMICAL COMPOSITION

It has also been shown that during culture composition of D. pulex was determined by the protein and lipid contents of Moina macro- Malikova (1953, 1956) and that of D. magna, copa decreased in comparison with those of the Ceriodaphnia reticulata, C. sphaercus by Sadykhov initial culture, from about 25 and 0.56 mg/g wet et al. (1975). In the carotenoprotein complexes, weight (WW) to about 18 and 0.26À0.33 mg/g the following predominant amino acids were WW, respectively (Romanenko et al., 2004). found: in D. magna, alanine, glutamine, glycine, and leucine (Czeczuga, 1984); in Moina micrura, asparagine, glutamine, and glycine (Velu et al., 3.2.1 Amino Acids 2003). The presence of free intracellular amino The amino acid content of various cladocera acids in D. magna was shown by Gardner and is shown in Table 3.3. The amino acid Miller (1981).

TABLE 3.3 The Content of Different Amino Acids in Some Cladocera SPP

Amino acid Bosmina D. magna D. pulex Daphniopsis Ceriodaphnia Simocephalus Moina longirostris (mg WW/dL) (% total amino tibetana, spp. (% total vetulus (mg mongolica (%protein) (Stepanova & acids per DW) (g/100 g amino acids WW/dL) (g/100 g (Verbitsky, Naberezhniy, (Dabrowski & protein) per DW) (Stepanova & protein) 1990) 1972) Rusiecki, (Zhao et al., (Dabrowski Naberezhniy, (Zhao et al., 1983) 2006) & Rusiecki, 1972) 2006) 1983)

Phenylalanine 1.3 228À833 3.76 1.79 3.82 107À157 3.40 Tyrosine 3.1 À 4.05 3.55 3.81 60 2.80

Leucine 3.3 222À725 3.74 5.76 4.61 425À625 5.30 Isoleucine 2.1 À 2.24 3.68 2.43 À 3.40 Methionine ÀÀ 1.44 3.64 1.40 À 1.50 Valine 2.9 À 3.71 3.78 3.49 À 3.90 Alanine 2.8 157À438 3.95 6.72 4.57 283À342 4.30 Glycine 2.6 194À239 2.85 3.91 2.69 236À296 3.20

Proline 2.2 211À358 2.53 4.53 2.76 100À250 2.70 Glutamic acid 5.0 317À458 5.46 7.40 6.29 522À619 8.00 Serine 1.8 205À357 2.57 3.27 3.07 255À285 3.00 Threonine 2.6 121À279 2.93 4.00 2.99 121À164 3.20 Aspartic acid 3.2 365À474 5.22 7.46 5.94 318À371 6.40

Arginine 1.9 200À401 3.40 2.85 3.45 244À348 4.30 Histidine 1.5 À 1.25 1.64 1.46 À 1.20 Lysine 3.8 À 3.78 4.78 4.36 À 3.40 Cystine 0.7 275À423 0.71 0.49 À 100À127 0.80 Tryptophan ÀÀ À 0.43 ÀÀ 1.20

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 17

It was determined that in Daphnia, the Ribosomal DNA contains a significant frac- amino acid composition during ontogeny is tion (c. 49%) of total body phosphorus rather constant (Brucet et al., 2005). However, (Acharya et al., 2004). The growth of Cladocera it may vary rather widely depending on the involves the requirement for a greater amount culture conditions, as has been shown for of ribosomal RNA, and especially of phospho- D. magna (Stepanova and Naberezhnyi, 1970, rus (Main et al., 1997). 1972) and Moina spp. (Kokova, 1982). In well- In juvenile D. pulex, an elevated DNA con- fed Daphnia, the content of amino acids is tent was measured in the postmolt period, fol- 62.93 g WW/L, whereas in “lean” Daphnia it is lowed by an increase in RNA during the 31.21À33.52 mg WW/L. intermolt and premolt periods (Gorokhova and Kyle, 2002). It was found that the ratio of RNA:DNA in Daphnia galeata increased in 3.2.2 Mucopolysaccharide (Slime) response to an increase of P:C ratio in its food, and within 5 h (Vrede et al., 2002). Use of this The chemical composition of slime was ratio has been suggested (Markowska et al., determined for the jelly capsule of Holopedium 2011) for evaluating the condition of D. pulex, (Brown, 1970). Sulfated mucopolysaccharide assuming that higher values correspond to a was found to be present, as well as mucopoly- “good” condition. Specimens with a lower saccharide modified by carboxyl groups. It has RNA:DNA ratio have lower metabolic rates been suggested that the jelly capsule is pro- and greater longevity. duced by the mechanism that produces the exoskeleton at each molt. Slime may surround the animal (as is the 3.2.4 Carbohydrates case with common forms of Holopedium). Slime Carbohydrates comprise glycogen and chitin. is also produced by salivary glands (Fig. 3.2) and by glands of the thoracic limbs (Fig. 3.3), Glycogen as has been shown for Eurycercus (Fryer, 1962, 1963) and for Alonopsis elongata (Fryer 1968), Glycogen is a polysaccharide consisting of but not yet for other chydorids. glucose bound to protein. The content of glyco- The salivary glands may be shown by intra- gen was determined by Blazka (1966): 23% in À vital staining with neutral red or Bismarck Bosmina longirostris, 53% in C. reticulata,1 36% brown (Cannon, 1922). in Daphnia spp., and 33% in Simocephalus sp. (% of total composition, WW). Glycogen is present in developing embryos. The glycogen content in Simocephalus vetulus was determined 3.2.3 Phosphorus-Containing to be 0.7% in the gastrula, 0.91% in the nau- Substances (Nucleic Acids: DNA and plius, 0.71% in released young, 1.2% in the RNA) third instar, and 2.23% in the fifth instar Nucleic acids are high molecular weight (Hoshi, 1953). compounds (polynucleotides). The nucleic acid content comprises 4.7À5.2% of the DW of Chitin Moina spp. (Kokova, 1982). The RNA content The polysaccharide chitin is a polyacetylglu- was determined to be c. 2À6% DW in Daphnia cosamine (β-(1-4)-linked homopolymer of spp. and the phosphorus content to be N-acetyl-D-glucosamine). The acetamide group À c. 0.7 1.5% DW (Kyle et al., 2006). CH3CONH is present in the chitin molecule.

PHYSIOLOGY OF THE CLADOCERA 18 3. CHEMICAL COMPOSITION

FIGURE 3.2 Labrum of Daphnia, showing salivary glands. AG, outlet duct; Di, dilators of esophagus; EZ, substituting cells; Hy, hypo- derm of the lobe; HZ, principal cells; KE, epithelium of the head bottom; Ll, levator labri; LM, muscles of the lobe; Oe, esophagus; OeM, muscles of the esophagus; Ps, pseudosegment; SB, sensory setae. Source: Sterba (1957a).

Chitin is a major structural component of (Cauchie et al., 1995) or c. 30À70 mg/g DW arthropods. The chitinous covers of cladocer- (Cauchie et al., 1999). ans are strong and nonwettable. Cladocera, The total annual chitin production by vari- being abundant in nature, produce enormous ous cladocerans in Europe is: D. magna, quantities of chitin. The content of chitin in the 11.5 g/m2 (4.6 g/m3); D. galeata, 3.2 g/m2 body of Daphnia is approximately 15% (0.16 g/m3), D. hyalina and D. cucullata (Chalikov, 1951) or 7% DW (Andersen and combined, 0.14À0.30 g/m2 (0.09À0.2 g/m3) Hessen, 1991). In D. magna, it is 2.9À7% DW (Cauchie et al., 1995).

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 19

DR EI OR m

SL 14 µ 25 1 VLM

SL R1 2

µ GC 50µ 50 3 4 36 R EX 37 R4 GC MU5 R ME

FIGURE 3.3 Slime glands in thoracic limb 4 of Eurycercus and in thoracic limbs 1À4ofAlonopsis elongata (right). Right, Alonopsis elongata, left and middle, Eurycercus. DR, duct of reservoir; ET, entangling secretion; EX, exopodite; GC, gland cells; ME, muscle to exopodite; MUS, muscles; OR, opening of reservoir; R, reservoir; SL, suspensory ligament; VLM, ventral longitudinal muscle. Source: left and middle, Fryer (1963); right, Fryer (1968).

In contrast to chitin from copepods, chitin Hackman (1964, 499) that “[t]he biological syn- from dead Cladocera is not decomposed in thesis of chitin in insects (or other animals and bottom sediments (or at least is not fully plants) has received little attention.” decomposed). After their death, it accumulates For other crustaceans, chitin is formed from at the bottom of water bodies, sometimes glucosamine chiefly derived from glycogen, forming a high proportion of the total mass of with the acetyl group furnished by oxidation the sediment. Bottom sediment with dominant of fatty acids (Vonk, 1960; Hohnke and chitinous remains was termed chitin gyttja by Scheer, 1970). The latter authors suggested a Lundqvist (1927). general scheme of crustacean carbohydrate Chitin is not derived directly from food: it metabolism yielding, inter alia, chitin (Fig. 3.4). must be formed by the process of metabolism. In Crustacea, chitin is formed and secreted by Although chitin is specific to arthropods and the hypodermis underlying the shell (Hohnke highly accumulated by cladocerans, the meta- and Scheer, 1970). bolic pathways of chitin formation were not clearly described until recently. For insects, 3.2.5 Lipids Kuznetsov (1948, p. 336) noted that “[t]here are no actual data on metabolism yielding chi- Recent investigations have supplied abun- tin from the cycle of transformations; even dant new information on lipids. Variations in more, there are no data on the metabolism the content of lipids in Cladocera are shown in intermediate as to chitin.” It was noted by Figs. 3.1 and 3.5. Up to 17.5% DW was

PHYSIOLOGY OF THE CLADOCERA 20 3. CHEMICAL COMPOSITION

FIGURE 3.4 Carbohydrate metabolism in Crustacea. Source: Hohnke and Scheer (1970).

measured in Moina rectirostris (syn. Moina bra- In addition to their basic trophic role, chiata) (Stepanova and Vinogradova, 1970). some lipid compounds may have exceptional The fatty acid composition of Cladocera spp. is properties. Pe´rez Gutierrez and Lule (2005) indicated in Tables 3.4À3.8; that of D. pulex dried large numbers of D. pulex at room tem- grown under different conditions was deter- perature, ground them, and produced 3 kg of mined by Mims et al. (1991). fine powder. By extracting and fractionating it Oil drops are often clearly seen in the bodies they obtained four glyceroglycolipids, all of of cladocerans. Among the algae, diatoms are a which were found to be cytotoxic. prominent group that synthesize and store Lipids are discussed in more detail in lipids: after photosynthetic production of a car- Chapter 4, sections 4.1.2 and 4.4. bohydrate, they transform it into lipids and the stored lipid is seen as oil drops. The diatoms are one of the dominant groups on the bottom 3.2.6 Vitamins substrata and in phytoplankton. They are not The vitamin content of D. pulex was assessed the only lipid-producing group, but the metab- as vitamin A, 5.19 mg WW/L; vitamin B , olism of other algae is less well known. 1 2.55 mg WW/L; vitamin B , 5.69 mg WW/L In temperate latitudes of the Northern 2 (Malikova, 1956); and vitamin B , μg/g DW Hemisphere, the spring peak of diatoms produc- 12 (Stepanova and Borshch, 1970). ing lipids as their reserve substance is followed by an accumulation of abundant oil drops by Cladocera. Due to their high-energy value, lipids 3.2.7 Pigments are a prominent storage substance in Cladocera. Lipids are also used in the construction of bio- Pigments are produced during the course of logical membranes and hormones. metabolism. In Cladocera, they comprise

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 21

96 orange (carotenoid) pigments, red (hemoglobin 95 (Hb), or bacterial carotenoids in cases of infes- 94 93 tation by Spirobacillus cienkowski), green (carote- 92 noprotein in hemolymph), and dark (e.g. 91 ommochromes of eyes, or tanned protein 90 89 formed at high pH, i.e. melanins) (Green, 1966b, 1971). 6 Generally, littoral cladocera are brownish, 5 whereas planktonic species are colorless. 4 3 Orange or red coloration is also observed in 2 some species. Rarely is a species brightly col- 1 ored (Weismann, 1878). Blue spots occur in 0 Eurycercus lamellatus on the postabdomen, on 1.00 the dorsal side of the trunk, at the base of the 0.75 0.50 mandibles, and on the esophagus (Weismann, 0.25 1878; Behning, 1941; Smirnov, 1971, 1974). 0 Pseudochydorus has large brown spots on its 2.51 P. globosus 2.10 valves. Newly molted are colorless, 1.68 but during the intermolt period a brown spot 1.26 appears and increases in intensity. With exces- 0.84 sive solar irradiation, Cladocera are blackish 0.42 0 (melanistic), as are their ephippia containing 4 latent eggs. 3 Leydig (1860, p. 56) originally noted that 2 1 the blood of Cladocera may be colorless,

Ash (% ww) Joule (mg ww) (% ww)0 Fat Protein (% ww) (% ww) Water yellowish, reddish, bluish, or greenish. 25 3035 40 Green (1957) reported his observations of Daphnia with pale green blood, Simocephalus FIGURE 3.5 Seasonal changes in chemical composi- with green blood, and Megafenestra aurita tion of Daphnia pulicaria from six ponds, beginning with blue blood. These colors are caused from June. Source: Heisig-Gunkel and Gunkel (1982). by carotenoid proteins, as they produce an

TABLE 3.4 The Total Content of Lipid and Percentage Lipid Components in Some Cladocera

Species Total lipid Phospholipid Triglyceride Cholesterol Cholesterol esters (% DW) (%) (%) (%) (%)

Bosmina obtusirostris 30.6 70 13 3 12.8 Holopedium gibberum 52.4 57 8.7 18.8 17 Bythotrephes 16.4 55 11.3 5 11.3 cederstroemi

DW, dry weight. Source: Lizenko et al. (1977).

PHYSIOLOGY OF THE CLADOCERA 22 3. CHEMICAL COMPOSITION

TABLE 3.5 Content of Fatty Acids (AS % Total Fatty Acid Content)

Fatty acids Daphnia spp. Daphniopsis Bosmina Holopedium Leptodora Bythotrephes

SATURATED 12:0 0.7À2.3 À 0.8 0.1À2.1 0.4 14:0 4À10 2.92 8.9 12.0 2.2À7.4 4.4 15:0 0.6À3 0.99 1.1 0.9 0.6À2.8 0.9 16:0 21À36 11.4 24À36 22À26 17:0 20.6 0.64 17.3 15.4 19.1

18:0 0.3À9.9 2.57 ÀÀ 6.5À17.6 6.7À9 MONOUNSATURATED 16:1ω7 6.7 5.81 4.1 3.5 2.9 18:1ω6 1 18:1ω9 9.2 12.3 8.1 10.3 18:1ω7 4.1 5.0 8.1 6.0 20:1ω9 À 1.2 2.3 À

POLYUNSATURATED 18:3ω3 6.8 26.5 7.6 6.7 5.3 18:4ω3 11.8 10.4 11.0 4.1 20:5ω3 11.8 1.41 14.4 17.5 23.0 22:6ω3 0.9 2.6 2.1 2.1 18:2ω6 4.6 5.3 4.1 3.6

18:3ω6 1.6 0.6 0.8 0.6 20:4ω6 3.5 0.63 4.7 6.8 9.3

2 , not detected. Sources: Daphnia spp., Bythotrephes longimanus, Bychek and Gushchina (2001), Bychek and Gushchina (2001); Daphniopsis tibetana, Zhao et al. (2006); Leptodora kindtii, Bychek and Gushchina (2001); Bosmina coregoni and Holopedium gibberum, Bychek and Gushchina (2001). orange color when treated with desaturating Carotenoids agents. Carotenoids are lipid derivatives. Cladocerans The coloration of cladocerans (especially of receive carotenoids with algal food (Green parthenogenetic eggs) may depend on the col- 1966a), which contains significant quantities: oration of the food consumed. Information on green algae, 70À500 mg DW/L; blue-green the pigments of cladocera, as related to their algae, 140À530 mg DW/L (Lavrovskaya, 1965). different ecology, and the transformation of With reference to Simocephalus, Green ingested pigments is rather scarce (but may be (1955b) found carotenoids (orange) in a free influenced by seasonal changes and the actual state in fat globules, in the gut wall, in fat cells, composition of the food). This is an open field linked to proteins in the cytoplasm, ovary, and for further useful investigations. eggs, and as carotenoprotein (green) in blood.

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 23

TABLE 3.6 Content of Fatty Acids (IN % DW) TABLE 3.8 The Content of Fatty Acids (as % Total Fatty Acids) in Three Age Groups of Daphnia magna Fatty Daphnia D. Bosmina Simocephalus acids cucullata longispina longirostris vetulus Fatty Acids Newborn 3 Days Old Mature

14:0 7.0 2.5 2.5 3.5 C14:0 0.9 2.3 3.5

14:1 0.8 3.0 2.3 3.8 C14:1 0.5 1.5 0.9 14:2 0.1 0.8 2.8 1.4 C14:2 0.6 1.7 2.2 16:0 16.4 11.7 14.0 12.8 C16:0 13.2 21.2 24.3 16:1 5.7 14.6 10.3 12.1 C16:1 4.7 4.7 4.5 16:2 0.8 C16:2 1.5 5.5 0.3 18:0 6.8 3.7 7.4 5.1 C16:3 1.2 3.4 7.7

18:1 8.3 10.1 19.3 11.8 C16:4 1.7 1.8 3.3 18:2 15.5 4.2 4.2 6.0 C17:0 1.2 Non det. 2.8 18:3 8.2 11.3 5.7 6.8 C18:0 3.4 7.7 5.8 18:4 6.3 15.8 2.8 7.8 C18:1 53.9 9.9 9.8 20:2 1.1 0.7 1.7 2.0 C18:2 6.7 22.0 18.9 20:4 6.7 1.3 5.4 6.6 C18:3 4.6 7.7 7.7

22:1 0.6 C20:4 2.8 6.1 3.8 20:5 7.6 17.4 21.7 18.9 C20:5 0.5 1.2 1.4

22:5 5.4 0.4 Source: Bychek and Gushchina (1999). 22:6 4.1 1.5 1.0

DW, dry weight. Source: Herodek and Farkas (1967).

TABLE 3.7 The Content of Phospholipid and Neutral Lipids in Three Age Stages of Daphnia magna

Types Lipids Newborn 3 Days Old Mature

Phospholipids Phosphatidylcholine 14.40 8.75 4.06 Phosphatidylethanolamine 9.15 3.94 3.21 Sphingomyelin 1.84 0.66 0.65

Neutral Lipids Triacylglycerols 566.2 283.2 452.1 Diacylglycerols Traces Traces 74.1 Free sterols 183.6 86.2 35.5 Free fatty acids 146.9 128.0 60.8 Wax esters Traces Traces 63.4

All values in μg/100 mg WW. Source: Bychek and Gushchina (1999).

PHYSIOLOGY OF THE CLADOCERA 24 3. CHEMICAL COMPOSITION

Food FIGURE 3.6 Pathways of carotenoid transfer in Simocephalus. Globules in Carapace Quadrangles of broken lines indi- gut wall epidermis cate stages in which carotenoid is free. Source: Green (1966b). Excretion via gut lumen

Blood

Ovary

Globules in adult Cytoplasm of fat cells fat cells

Globules in Globules embryonic in egg fat cells

Cytoplasm of Cytoplasm embryonic of egg fat cells

Green (1966b) reported the principal path- grown in the light contain much more caroten- ways of carotenoid transfer, with reference to oid than those grown in the dark. Carotenoid Simocephalus (Fig. 3.6). Carotenoids obtained pigments were found to be dissolved in oil from food are passed into the blood, and from drops or bound to cytoplasmic proteins in there to fat cells, to the carapace epidermis, Daphnia (Green, 1957) and Simocephalus (Green, to the ovary, and then to the eggs. A female 1966b). may pass half of its total carotenoids to her Later, Herring (1968) found the following eggs. In Simocephalus, carotenoids are either carotenoid pigments in D. magna and other present in the cells of the gut wall and cladocerans: astaxanthin, canthaxanthin, fat body or associated with proteins of fat β-carotene, echinenone, and an unidentified cells, epidermis cells, the ovary, and eggs. ketocarotenoid. Lutein was present only in A green carotenoprotein is found in the blood. the gut wall. Herring also fed D. magna pure An intermolt cycle (Fig. 3.7) and a seasonal carotenoids with yeast and found that the cycle (Fig. 3.8) of carotenoids were demon- ingested β-carotene is transformed into echi- strated by Green (1966b). nenone, canthaxanthin, and astaxanthin. The following carotenoid pigments found in In C. reticulata, Czeczuga (1976) found astax- the gut wall, fat cells and the ovary of Daphnia anthin (quantitatively dominant), astacene, were reported by Green (1957): astaxanthin, canthaxanthin, cryptoxanthin, 4-keto-4-hydroxy- β-carotene, γ-carotene, and lutein. Daphnia β-carotene, lutein-5,6-epoxide isozeaxanthin, and

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 25 astaxanthin ester. In D. magna, Czeczuga (1984) According to his determinations, the total also found β-cryptoxanthin, isocryptoxanthin, concentration of carotenoids is c. 4 g/g DW, isozeaxanthin, lutein epoxide, and zeaxanthin. of which astaxanthin forms 27% and β-cryptoxanthin 25%. In M. micrura, astaxanthin and can- 20 thaxanthin predominate (Velu et al., 2003). In Holopedium, the carotenoid astacin was found by Sørensen (1936) and Goodwin (1960, 40 p. 127) noted that Holopedium accumulates a 15 lot of astaxanthin and that its “de novo synthesis is probably ruled out.” As it is hardly pos- fat globule 20 sible that much astaxanthin is present in the food, Goodwin suggested that it is produced Mean diameter of largest 10 by oxidation of the β-carotene and its derivatives (including zeaxanthin) ingested with 12345678 diatoms. Carotenoid protein in fat cells MoltIntermolt stage Molt Foss et al. (1986) investigated in detail the FIGURE 3.7 Intermolt cycle of carotenoid pigmenta- composition of carotenoids of D. magna and tion in Simocephalus fat cells. Lower and middle lines, in found two new ketocarotenoids. cytoplasm (different years); upper line, diameter of largest In various cladocerans, particular carote- fat globules. Source: Green (1966b). noids were found (indicated in Table 3.9)

40 20 Cytoplasm

20 10 T°C Temperature (ºC) Carotenoid protein in fat cells 0

40 Globules

20 Carotenoid in fat globules

J FMAM J J ASOND J FMAMJ J ASOND J FMAMJ J ASOND 1959 1960 1961

FIGURE 3.8 Seasonal variation in Simocephalus carotenoids. Upper, in fat cells. Lower, in fat globules. Letters indicate months. Source: Green (1966b).

PHYSIOLOGY OF THE CLADOCERA 26 3. CHEMICAL COMPOSITION

(Herring, 1968). Carotenoids are not found in Arctic and high mountain populations of Leptodora (Farkas, 1958). Daphnia are blackish (Hebert and Emery, It seems that the excess of carotenoids 1990). In addition, chydorids collected in shal- ingested with food serves no physiological low rock pools in the hot climate of Western purpose. Having received carotenoids with Australia are dark brown, as reported by B.V. their food, cladocerans just transfer them over Timms and M. Jocque´ (personal communica- to their eggs and progeny. About half of the tion). Such dark pigmentation is thought to be mother’s carotenoids are transferred to the of protective value. Scapholeberis and Dadaya eggs of each brood (Green, 1957, 1966a) but living under the surface of water are also the developing embryos do not utilize this pig- black. Hobaeck and Wolf (1991) observed ment. Moreover, the presence of free or conju- melanic populations of Daphnia at altitudes of gated carotenoids does not favor growth or the 1200À1600 m above sea level inhabiting clear- viability of eggs or adults. water lakes and ponds, whereas transparent populations came from ponds with slightly Melanins humic water. Melanin was deposited in the dorsal area of the carapace, in the head shield, Dark cuticular pigmentation, including that and the antennae. Its absorption maximum of ephippia (Gerrish and Ca´ceres, 2003), is was c. 249 nm. caused by melanins (Hebert and Emery, 1990). Melanins (a term relating to both brown and black pigments) are also present in the eyes. Hemoglobin They are thought to be products of the metabo- There is an extensive literature on Hb in lism of amino acids (tyrosine): “Chemically, Cladocera. Special investigations into Hb in the melanins are probably polymerized indole Cladocera were made by Fox (during quinones formed by the action of the enzyme 1945À1955), Hoshi (during 1949À1974), and tyrosinase on the aromatic amino acids” Green (1955, 1956b). Hb may be measured (Goodwin, 1960, p. 133). chemically or spectroscopically. The ommochromes of eyes are rapidly dis- In contrast to Malacostraca (Prosser and solved with 10% alkali solution in both fresh Brown, 1967 [1962]), Cladocera synthesize Hb, and preserved specimens. which may be present in their blood, muscles,

TABLE 3.9 The Percentage Composition of the Carotenoid Pigments in Wild Cladocera SPP

Species β-Carotene Echinenone Canthaxanthin Ketocarotenoid Astaxanthin Lutein 2nd Ketocarotenoid

Ceriodaphnia 56 5 3 7252 megalops

Daphnia pulex 4À12 7À11 5À19 5À20 24À66 4À20 Trace D. longispina 6 5 2 0 56 13 3

Eurycercus 70 160 47190 Moina micrura 0.43 À 4.60 À 39.82 21.21 À Simocephalus 6 7 8 0 38 21 0 vetulus

Note: 18.65% β-cryptoxanthin and 15.29% violaxanthin have also been found in Moina micrura (Velu et al., 2003). Sources: Herring (1968); Moina micrura, after Velu et al. (2003).

PHYSIOLOGY OF THE CLADOCERA 3.2 PRINCIPAL ORGANIC CONSTITUENTS 27 central nervous system, fat cells, ovaries, or 465 dissolved in hemolymph of parthenogenetic eggs (Fox, 1955; Green, 1955). 1.4 3 The molecular weight of Daphnia Hb is 433 400,000, with an admixture of a small amount 1.2 of 34,500 (Goodwin, 1960). In M. macrocopa, 465 molecular weight of Hb was estimated to be 1 670,000, the iron (Fe) content to be 0.317%, and 2 the number of Fe atoms per Hb molecule to be 650 0.8 38 (Hoshi et al., 1967). These authors conclude 470 490 that each Hb subunit with one Fe atom corresponds approximately to human Hb. 0.6 4 Absorbance (OD) The role of Hb is considered further in 650 Chapter 5, “Respiration.” 0.4 580 5 Myoglobin Some of the Hb is present in muscles as 0.2 544 myoglobin. Goodwin (1960) warned against 0 the use of the latter term for Crustacea, as its 400 450 500 550 600 650 700 constitution was then unknown. In some of (nm) my long-standing samples of filamentous algae and Cladocera preserved in formalin, the mus- FIGURE 3.9 Levels of different components in cles of the cladocerans are stained blackish, Simocephalus. 1, myoglobin in muscles; 2,3, lipid in tissues; 4, green granules in eggs; 5, yellow-green granules in thus indicating the presence of myoglobin. eggs. OD, optical density. Source: Karnaukhov et al., 1986. This may be attributed to tannic substances, which are known to occur in filamentous algae and produce black ink with Hb iron. However, carotenoids, and carotene protein, as shown attempts to stain similar samples with tannin microspectrophotometrically by their absorp- failed, although further attempts may be tion bands (Karnaukhov, Del Rio De Valdivia, worthwhile. and Yashin, 1986). Later, the presence of myoglobin in the Biliverdin has been discovered only in the muscles of Simocephalus was directly con- eye of Polyphemus, and not in the eye of firmed by microspectrophotometry (Fig. 3.9) Daphnia (Green, 1961a). (Karnaukhov, Del Rio De Valdivia, and Yashin, 1986). Myoglobin was completely absent from oil drops. 3.2.8 Content of Particular Elements Other Pigments or Compounds Cytochrome was shown by Fox (1955) to be The content of some elements in Cladocera present in cladoceran muscles (in Daphnia, spp. is shown in Table 3.10. The content of absorption maxima at 566 and 500 nm) and by mineral substances in the bodies of cladocera Hoshy and Akizawa (1979) (in Simocephalus,at depends on mineralization of the environment; 603, 563, 550, and 532À522 nm). in D. magna, it ranges from 12 to 37.6% DW; Coloration of Simocephalus and Chydorus and in S. vetulus, from 10 to 19.4% DW results from a combination of myoglobin, (Stepanova et al., 1971).

PHYSIOLOGY OF THE CLADOCERA 28 3. CHEMICAL COMPOSITION

TABLE 3.10 Content of Some Elements in Cladocera newborns to 44% in adults (Baudouin and Ravera, 1972). Calcium Phosphorus Iron (mg Reference(s) (% DW) (% DW) DW/L) Direct determination of the total carbon content (using the dry-combustion method of Daphnia 9.6 1.48 950 Malikova Pregl-Roth) yielded the following results (Metz, pulex (1953, 1956) 1973). By measuring the uptake and release of D. 1.75 1.56 À Baudouin & radiocarbon (γC), D. pulex fell into two groups: hyalina Ravera 1.5À10 γC/individual (ind.) and 2À22 γC/ind. newborn (1972) The radiocarbon content in Daphnia with eggs D. 5.4À6.9 1À1.56 À Baudouin & was twice that of Daphnia without eggs. About hyalina Ravera 52% of carbon was in the eggs, irrespective of mature (1972), Lin & Lui (1985) the number of eggs. The minimum carbon content per egg was 1À1.5 γC. DW, dry weight. The content of organic carbon was determined in Polyphemus pediculus by Butorina Essential and Nonessential Elements (1973): parthenogenetic females, 3À7% WW, Cladocera need some elements, while others 40À50% DW; gamogenetic females, 3.4À7.5% may be physiologically nonessential or even WW, 41À48% DW; males 4.4À5.8% WW, toxic. In addition, essential elements (e.g. cop- 42À47% DW. Moreover, it was 44.6% DW in per (Cu) or zinc (Zn)) may be toxic at concen- D. hyalina and 49.05% DW in Leptodora kindtii trations higher than are normally necessary. (Lin and Lui, 1985). Cladocera may also accumulate, without any physiological use, substances that just happen NITROGEN to be around. In natural water bodies there is always a Generally, tissues of Cladocera consist of deficiency of this element. In the bodies of biogenic elements (C, H, O, and N), macroele- Cladocera, the content of nitrogen (in % DW) ments (Ca, Na, K, P, S, Mg, Mn, Fe, Cl, and was found to be 9.7 in newborn D. hyalina Cu), and microelements (Zn, Co, I, Se, Mo, Li, (Baudouin and Ravera, 1972); c. 9 in mature and some others). D. hyalina (Baudouin and Ravera, 1972; Lin and Lui, 1985); c. 9-10 in Diaphanosoma, Holopedium, Bosmina, Daphnia, and Leptodora, CARBON with little seasonal variation (Andersen and Carbon (C) is one of the principal macroele- Hessen, 1991; Hessen and Lyche, 1991); 12.7 in ments. Although the content of carbon in most Leptodora kindtii (Lin and Lui, 1985); and c. 7.9 Cladocera is generally about 47À48% DW, in S. mucronata (Hessen and Lyche, 1991). there are variations (e.g. c. 53% in Scapholeberis mucronata) (Hessen and Lyche, 1991). Data on PHOSPHORUS the content of carbon in representatives of In aquatic environments, there are only particular genera are available. For example, traces of free phosphorus because, similar to the content of carbon in the bodies of N, it is avidly assimilated by algae. Diaphanosoma, Holopedium, Bosmina, and A major pool of phosphorus in Daphnia is Daphnia was found to be 48% DW, with little present in the exoskeleton (Hessen and Rukke, seasonal variation (Andersen and Hessen, 2000a). Phosphorus content (as P2O5) in the 1991). It increases with increased body length bodies of Cladocera was found to be 3.4À3.6% in D. hyalina, from 42.8% total C in DW in DW in D. pulex; 3.5% DW in Leptodora (Birge

PHYSIOLOGY OF THE CLADOCERA 3.3 XENOBIOTICS IN THE CLADOCERAN BODY 29 and Juday, 1922); c. 1% DW in Diaphanosoma, et al., 1999). A large part of the calcium is lost Holopedium, Bosmina; c. 1.4% DW in Daphnia with the exuvium at molting. (Andersen and Hessen, 1991; Vrede et al., 1999), with little seasonal variation (Andersen IRON and Hessen, 1991, 1999); and 1.1% DW in Iron is discussed in Chapter 5, section 5.4. D. magna (Sterner and Schwalbach, 2001). See also Table 3.11. Having summarized the available data, Brett et al. (2000) indicated 0.8À1.8% P DW for vari- LEAD ous species of Daphnia, the highest being According to Holm-Jensen (1948), lead (Pb) 1.8 for D. pulicaria and the lowest 0.81 for is accumulated “mainly passively” in the shells 1 D. pulicaria. The phosphorus content was espe- of D. magna “probably by exchange” of Ca2 1 cially high in juvenile Daphnia, which were for Pb2 . thus more dependent on sources of phosphorus in their food (Main et al., 1997). MAGNESIUM P content was found to be higher in filtra- In D. pulex, 0.5À0.9% DW magnesium (Mg) tors (.1.5%) and lower in carnivorous clado- was found, and in Leptodora, 0.95% DW (as cerans (c. 0.5% P DW) (Hessen and Lyche, MgO in ash) (Birge and Juday, 1922). 1991). The highest phosphorus content, c. 2%, was found in S. mucronata and Ceriodaphnia COPPER quadrangula. An optimum copper concentration range for D. magna was determined to be 1À35 μg Cu/L CALCIUM AND STRONTIUM (Bossuyt and Janssen, 2004), and active copper Although cladocera do not possess a strongly regulation between 0.5À35 μg Cu/L was calcified carapace, some, e.g. Daphnia, still retain observed in the body. For the metabolic role of some ability to accumulate calcium (Ca) and Cu, see also Fig. 3.10. strontium (Sr) in their shells (Porcella et al., 1967, 1969). In both littoral and pelagic species SILICA (SIO2) (Alona, Chydorus, Pleuroxus, Ceriodaphnia, A total of 2.8% DW of silicon (Si) was Simocephalus,andDaphnia), the integuments reported in a sample of D. pulex (Birge and may sometimes be reinforced by CaCO3 Juday, 1922). (Leydig, 1860, pl. II; Gicklhorn, 1925; Schmidt, 1943; Taub and Dollar, 1968; Porcella et al., SODIUM 1969). This is similar to the deposition of cal- The normal concentration of sodium (Na) in cium in the exoskeletons of ostracods or of large D. magna is 26.3 mM/kg WW (Stobbart et al., crustaceans. In D. magna, calcification was found 1977). Excessive Na is not accumulated. Na to occur by the deposition of fine grains begin- uptake and release is considered further in ning just prior to molting (Porcella et al., 1969). Chapter 8, “Osmotic regulation.” The content of calcium ranges within 2À9.9% DW in D. pulex, 3.2% DW in Leptodora (as CaO in ash) (Birge and Juday, 1922), and 3.3 XENOBIOTICS IN THE within 0.8À4.4% DW in Daphnia spp. in CLADOCERAN BODY Norwegian lakes (Wærva˚gen et al., 2002). The calcium content of D. magna ranges from 4.2% Xenobiotics affect aquatic invertebrates to 1% DW, decreasing when there is a lower either by direct toxicity of dissolved toxicants calcium concentration in the medium (Alstad or as contaminated food. The effects may be

PHYSIOLOGY OF THE CLADOCERA 30 3. CHEMICAL COMPOSITION

TABLE 3.11 Bioconcentration Factors (i.e. the Accumulation of Various Substances Over Time)

Substance Species Factor ( 3 Original Time Reference(s) Concentration)

Arsenic trioxide Daphnia magna 219 21 days Spehar et al. (1980)

Cesium-137 D. magna 84 5 days Nilov (1983)

th HgCl2 Ceriodaphnia affinis 2000 In 20 generation Gremiachikh & Tomilina (2010) Manganese D. magna 65 8 h Kwasnik et al. (1974) Neptunium D. magna 32 48 h Poston et al. (1990) Nickel D. magna 25 48 h Pane et al. (2003) Anthracene D. pulex 760 3 h Herbes & Risi (1978)

Benz(a)anthracene D. pulex 10,000 24 h Southward et al. (1978) Benzo(a)pyrene D. magna 1,000À8,000 24 h McCarthy (1983), Oikari & Kukkonen (1990)

Chlordanes D. pulex 24,000 24 h Moore et al. (1977) DDT D. magna 23,000 24 h Crosby & Tucker (1971) Estrone D. magna 228 16 h Gomes et al. (2004) Naphthalene D. pulex 100 24 h Southward et al. (1978) Pirimicarba D. magna 50 48 h, per DW Kusk, 1996 Triphenyltin chloride D. magna 290 3 h Filenko & Isakova (1979) (0.1 mg/L in water) Water-soluble oil fraction Daphnia .500 19 h Mikhailova et al. (1986)

DDT, dichlorodiphenyltrichloroethane. a2-(diethylamino)-5,6-dimethyl-4-pyrimidinyl dimethyl carbamate. immediate or delayed, be manifested as dam- noted the necessity of taking into consideration age to metabolic links in the organism, be the rate of penetration of toxicants through teratogenic, or disrupt links in the natural bio- integuments. He observed that when cladocer- cenological network. At low concentrations of ans are placed in a toxic solution, at first xenobiotics, Cladocera continue to live and they behave in their usual way (“as if nothing reproduce, but are less prolific. High concen- had happened”), then they manifest signs trations of xenobiotics either cause death or of anxiety, make desperate jerks, sink to the decrease the life span. They also disturb natu- bottom, and make occasional movements ral adaptation processes. Generally, they have there, then all movement stops, and finally the far-reaching effects at the species, population, heart stops. and biocenotic levels. Excess pollution may kill all animal life or, Numerous experiments have shown a at lower concentrations, kill animal species decrease in life span and fecundity in the pres- selectively or inhibit certain vital functions or ence of toxic substances. Beklemishev (1924) interrelations.

PHYSIOLOGY OF THE CLADOCERA 3.3 XENOBIOTICS IN THE CLADOCERAN BODY 31

NADH/UQ Reductase Cyt bc FIGURE 3.10 Proposed roles of copper in the transformation of phenathre- PHQ nequinone. Source: Xie et al. (2006). Fe-S Cyt b Inner UQ mitochondrial FMN membrane Cyt c UQH2 1+ 2+ Cu (1) Cu NADH Mitochondrial –• matrix PHQH2 O2 O2 • 2+ O– + 2H+ Cu 2 1+ 2+ (2) 1+ Cu Cu Cu H O •OH 2 2 (3) Semi-PHQ

O2 Cu+1 Cu2+ Cu+1 Cu2+ –• • O2 H O OH 2 2 (3) PHQ

In this chapter, the principal focus is on studies demonstrating how foreign chemicals become involved in their metabolism or are transformed by cladocera, and how the chemicals or excessive factors impair particular functions or metabolic links. Accordingly, measurements of the lethal dose, quantitative characteristics of fecundity, or life span are mostly not cited here. Particular substances may not accumulate homogeneously throughout the cladoceran body but may preferentially accumulate in particular organs or tissues. Thus, cadmium (Cd) taken from solution is concentrated in the exo- FIGURE 3.11 skeleton of D. magna (Carney et al., 1986). The Accumulation and removal of radiola- 109 belled strontium by Daphnia magna. Strontium removal internal distribution of Cd (cadmium-109) occurs over the course of three complete molt cycles (fol- has been determined by whole-animal autora- lowed by unlabeled medium). Source: Marshall et al. (1964). diography (Munger et al., 1999); it mainly accumulated in the diverticula of the gut, which are sites of uptake of nutrients and Ca. Cadmium the carapaces of D. magna and Ceriodaphnia accumulated by Moina (Yamamura et al., 1983) dubia (Robinson et al., 2003). was mostly bound to low molecular weight Selenium (Se) is localized primarily in the proteins (a mixture of two isoproteins). It was cytoplasm of D. pulex cells. In mitochondrial also shown to be absorbed onto the surface of matrices, excessive dense deposits were

PHYSIOLOGY OF THE CLADOCERA 32 3. CHEMICAL COMPOSITION accumulated and, with time, the mitochondria accumulated anthracene); a more slowly elimi- degenerated (Schultz et al., 1980). nated compartment (60%); and a tightly bound D. magna accumulates 95% of Sr (similar to one (8%) (Herbes and Risi, 1978). It is thought Ca) in the exoskeleton; it is eliminated at each that metabolization of anthracene by D. pulex is molting, thus falling to a minimum, and then much slower than its accumulation. Exposure of the Sr content gradually increases (Fig. 3.11) D. magna to anthracene plus ultraviolet radiation (Marshall et al., 1964). Pb is also thought to be (UVR) reduced survival and fecundity more than accumulated in the shells, probably by repla- exposure to anthracene alone (Holst and Giesy, cing calcium (Holm-Jensen, 1948). In D. magna, 1989). Exposure to UVR in the absence of anthra- nickel (Ni) is mainly accumulated in the cara- cene produced no significant effect. pace, gut, and hemolymph (Hall, 1982). There is also a dependence on temperature. Zinc-65 (65Zn) is partly accumulated in the It was shown by analyses of D. pulicaria exoskeleton and is removed at molting homogenate fractions (Heisig-Gunkel and (Winner and Gauss, 1986). Gunkel, 1982) that at a temperature of about Anthracene accumulated by D. pulex is 8C the accumulation of atrazine is correlated removed at a rate of 1.6 μg/ind./h. However, with protein content, whereas at higher tem- within Daphnia there are three “compartments”: a peratures (12À29C), it correlates with fat, and rapidly eliminated compartment (with c. 30% of protein becomes less important.

PHYSIOLOGY OF THE CLADOCERA CHAPTER 4

Nutrition

4.1 FEEDING Nevertheless, in the process of ingestion some diatoms are damaged, especially Asterionella 4.1.1 Anatomical Background (Infante, 1973; Lampert, 1978; Fryer, 1991), and Eurycercus spp. have been shown to crush Cladocera obtain their food by means of large food clumps with their mandibles their thoracic limbs, but there is a profound (Kotov, 1998). difference in the methods of food collection between littoral and planktonic Cladocera spp. Feeding in cladocera is inseparably linked to Cladocera that Collect Food from Substrata respiration. With reference to Daphnia magna,it Cladocera that collect their food from sub- has been shown that the principal part of their strata have evolved various specializations of oxygen supply is extracted from the feeding their food-collecting apparatus, as discovered current (Pirow et al., 1999a). and described for chydorids and macrothricids Although generally bilaterally symmetrical, by Fryer (1963, 1968, 1974, 1991). In general, Cladocera have asymmetrical mandibles that are bottom-living cladocera (e.g. Figs. 1.1 and 1.4, controlled by asymmetrical muscles. Fryer (1963, left), collect food from the surfaces of various p. 347) observed that “the most important man- substrata using strong, second thoracic limbs, dibular movements are those of rotation.” As push the collected mass forward toward the mandibular muscles are asymmetrical, “[t]his mouth, and remove excess water by undulat- enables the two masticatory surfaces to move ing the exopodites of their posterior limbs. relative to each other and to exploit to the full There are further specializations of this feeding their skeletal asymmetry” (Fryer, 1963, p. 351). activity in different genera and species. The To an extent, Cladocera crush their food basic (least modified) food-collecting system of clumps and algal cells with their mandibles the thoracic limbs in chydorids seems to be but Hebert (1978a) noted that physical damage that present in Eurycercus and Pleuroxus trunca- to food particles by the mandibles during tus (Fryer, 1963, 1968). ingestion is slight. For example, in the gut of The methods of food collection used by bot- Picripleuroxus striatus none of the scraped off tom-living cladocerans are diverse (Fryer, and ingested frustules of the Cocconeis diatom 1963, 1968, 1974); however, more detailed were broken (Smirnov, 1971, Fig. 233). information on the physiological aspects of

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00004-5 33 © 2014 Elsevier Inc. All rights reserved. 34 4. NUTRITION feeding is scarce, in contrast to the planktonic decomposition of the food material. It has filter-feeders. occasionally been observed that Alonella spp. The food particles collected by littoral clado- remain alive in vessels with detritus for several cera are glued together by the secretions of months when all other companion species their salivary gland (Fig. 3.2) and of glands in have perished. the thoracic limbs (the latter are known to be Van de Bund (2000) supplied what seems to present in Eurycercus and Alonopsis; Fig. 3.3) be the only direct observation that a chydorid, and then forwarded to the intestine (Fryer, Chydorus piger, reproduces especially well 1962, 1963, 1968). The salivary gland cells are when feeding on fecal pellets of Chironomus supplied with nerve endings (Zeni and riparius (“a highly significant effect” was Zaffagnini, 1988). No attempts have yet been observed), as well as on natural detritus; it is made to check whether the glands in the tho- thought that in general chironomid fecal pel- racic limbs of these genera are present in vari- lets (coprophagy) support the reproduction of ous other chydorids and macrothricids; nor is chydorids. any information available as to whether these Littoral Scapholeberis and Dadaya attach to secretions contain digestive enzymes. the surface film of water and collect pollen, Littoral Cladocera, especially those which minor particles, and, in all probability, the live on substrata or on the bottom of water unimolecular film of organic matter. Filter bodies, exist within a seemingly unlimited feeding is performed by sedentary Sida and food supply of organic debris at all stages of Simocephalus spp. decomposition, as well of algae and bacteria. Littoral Lathonura and Drepanothrix spp. Fecal pellets containing variously digested may be observed to remain motionless for organic matter also settle to the bottom and hours, lying on their backs and making no become a part of the detritus. A special place other movements than filter beating their tho- in the littoral belongs to some diatoms, mostly racic limbs. epiphytic and attached to substrata, which Chydorids and macrothricids can thrive for transfer their photosynthesis products to lipids several months (e.g. Smirnov, 1964, 1965a) and accumulate fat stocks. when fed periodically with fresh organic Undoubtedly, therefore, among the bottom- debris (or “detritus”). Dekker et al. (2006) cul- living cladocera there are some types that are tured C. sphaericus under similar conditions physiologically adapted to feeding on organic and obtained the best growth using artificial detritus at various stages of decomposition, detritus prepared from Urtica and Nitzschia from living algae to profoundly decomposed diatoms. organic matter. Such a sequence of adaptations to different decomposition stages has never Cladocera that Collect Food Suspended been specifically studied. It is likely that differ- in Open Water ent decomposition levels of organic matter In contrast to those living in the littoral supply nutrition for different species of zone, pelagic Cladocera periodically suffer Cladocera that are physiologically specialized food shortages or drastic seasonal changes in for their consumption. Unfortunately, there are food quality. In filter-feeding (mostly plank- no relevant investigations, except for observa- tonic) anomopods and in ctenopods, food par- tions that in an assembly of collected littoral ticles suspended in the outer medium are Cladocera kept in a jar with detritus its constit- filtered by filtering fans consisting of setae on uent species die off gradually and in a certain thoracic limbs (e.g. Fig. 1.2). In anomopods, sequence, which might be related to the filtering fans are formed by gnathobasic

PHYSIOLOGY OF THE CLADOCERA 4.1 FEEDING 35 setae; in ctenopods, this is done by setae on food alone, but nothing is known of any special endites in combination with a smaller area of traits of their nutritional physiology. gnathobasic setae. Predatory (carnivorous) Cladocera are also In daphnids, slime glands in thoracic limbs present in the pelagic zone. These include are not reported (Fryer, 1991) and are probably Leptodora, Bythotrephes, and Polyphemus spp., absent. the latter being common in patches of open For pelagic cladocera, there is obviously a water in the littoral zone. similar gradation of food, from fresh or recently dead algae, as well as fecal pellets, to all stages of decomposition, potentially with specialized consumers. Coprophagy in pelagic 4.1.2 Environmental Background and Cladocera was studied by Pilati et al. (2004). Food Resources These authors obtained 14C-labeled feces by feeding zooplankton with 14C-labeled algae. Algal Food A considerable clearance rate was found for In almost all waters, algae are available in such fecal matter: 0.0840.089 mL/mg/h in quantities but the composition of their popula- Holopedium gibberum and 0.026 mL/mg/h in tions depends on season, latitude, and the type daphnids. of water body. There are various sizes and Pelagic Cladocera spp. reproduce successfully types of algae, from those comprising small when feeding on algae (less efficiently when cells to those forming clumps or filaments. feeding on blue-green algae) (Daphnia), bacteria Frequently, algae are covered with slime. It has (Daphnia, Ceriodaphnia, Bosmina coregoni, C. sphaer- been shown that algal cells may be ingested icus,andPolyphemus), and yeast (Daphnia) repeatedly by D. magna before being completely (Rodina, 1950). However, Lampert (1987) demon- digested (Schindler, 1968; Kersting, 1978). strated that Daphnia grow but do not reproduce Generally, sugar is produced by photosyn- when fed on bacteria. thesis and is then transformed, inter alia, into Planktonic cladocera consume any algae glycerol and saturated and unsaturated fatty that are present in the water, as well as organic acids. Glycerin combined with fatty acids particles and bacteria. They collect their food yields fat. Kretovich (1986, p. 278) notes that by filtration using their thoracic limbs. The col- “the process of formation of fat from carbohy- lected food is directed into the mouth opening, drates may occur at a very high rate.” The where it is formed into a conveniently narrow schematic pathway from initial photosynthetic flow by rotation of the mandibles. saccharides to lipids and amino acids in algae (Arts et al., 1997) is shown in Fig. 4.1. The chemical composition of algal popula- Carnivorous Species tions depends on the specific physiology of In contrast to most cladocerans, the littoral particular groups of algae. While green algae chydorid Pseudochydorus globosus is carnivorous may be considered similar to green vascular and feeds on the dead bodies of cladocerans, plants in this respect, algae from other divi- whereas Anchistropus is specialized for feeding sions may be highly specific in their physiol- on living hydra (as recently observed by Van ogy and their photosynthetic products. For Damme and Dumont, 2007). Bythotrephes example, green algae accumulate starch and ingests the soft tissues of Daphnia spp. cellulose, with a small quantity of fat, as the (Lehman (1993). These species successfully principal products of photosynthesis; diatoms complete their life cycles when feeding on this (Bacillariophyceae) mainly produce lipids.

PHYSIOLOGY OF THE CLADOCERA 36 4. NUTRITION

7 TABLE 4.1 Percentage Fatty Acid Content of 6 EPA + DHA Freshwater Seston, Diatoms, Daphnia spp., and Fish 5 4 Fatty Seston Diatoms: Daphnia Fish: 3 Acids (Persson Cyclotella (Persson Coregonus 2 1 and Vrede, (Desvilettes and Vrede, (Sushchik, 0 2006) et al., 1997) 2006) 2008) 6 18:2 + 18:3 Total 51.8 17.7 37.1 5 SAFAs 4 3 Total 20.2 18.6 19.9 2 MUFAs 1 0 Total ω3 16.8 62.0 31.2 17.0 30 (PUFAs) 25 Total fatty acids ω 20 Total 6 6.1 4.8 9.8 5.4 15 (PUFAs) % DW10 % DW % DW Total 22.9 71.6 41.0 22.4 5 PUFAs 0 Diatoms Cryptophytes Chlorophytes Cyanobacteria Values represent % of total fatty acid content. FIGURE 4.1 Content of highly unsaturated fatty acids MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated in dominant groups of planktonic algae. DHA, docosa- fatty acids; SAFAs, saturated fatty acids. hexaenoic acid; DW, dry weight; EPA, eicosapentaenoic acid. Source: Brett and Mu¨ller-Navarra (1997). obtained a high percentage of sterile cladocerans. It was shown that feeding on bacteria, in The polyunsaturated fatty acid (PUFA) com- combination with algae, supports a long series positions of different organisms are shown in of parthenogenetic generations of D. magna;in Tables 4.14.3. contrast, cultures failed if fed either on bacteria Blue-green algae (cyanobacteria) also accu- or on sterile algae (Gajewskaja, 1940, 1948). The mulate lipids, which, however, contain toxic same conclusion was made by Taub and Dollar compounds. Cyanobacteria are characterized (1968) for Daphnia pulex, and Tezuka (1971) con- by a low eicosapentaenoic acid (EPA; 20:5ω3) cluded that Daphnia could not to survive when content (Mu¨ ller-Navarra et al., 2000) and a low fed on bacteria alone. sterol content (von Elert et al., 2002) which Daphnia thrive when fed on organic debris constrains the growth and reproduction of prepared from algae or vascular plants. Good Cladocera. growth and reproduction of D. magna and D. longispina was obtained by Esipova (1969) Bacterial Food using detritus prepared from phytoplankton Along with other food items, Cladocera con- treated with Lugol’s solution to reduce the sume bacteria; they are also internally popu- quantity of bacteria. lated by bacteria, including in their intestines. It has also been shown that D. magna feed To solve the question as to whether bacteria are efficiently on suspended bacteria of size an obligatory food component, it was necessary 0.11 μm, but that for D. galeata, D. hyalina, to develop methods for sterilizing Cladocera. and D. pulicaria feeding is less efficient To achieve this aim, Gajewskaja (1938) main- (Gophen and Geller, 1984). Heterotrophic bac- tained Daphnia and Bosmina in a 0.1% solution teria weakly support D. magna, but a culture of Rivanol (ethoxydiaminoacridine lactate) and developed well when supplemented with

PHYSIOLOGY OF THE CLADOCERA 4.1 FEEDING 37

TABLE 4.2 The Fatty Acid Composition of Some Freshwater Algae, as Percentage of Total Fatty Acids

Fatty Green Diatom Blue-Green Chrysophyceae Cryptophyceae Dinoflagellates Acids Algae Cyclotella Algae Chromulina Peridinium

14:0 0.72.3 2.0 2.117.0 11.1 1.72 7.6

16:0 9.820.4 11.6 18.529.1 5.0 11.113 29.8 16:1ω7 0.31.1 5.2 1.822.6 2.1 0.30.6 2.1 18:0 0.64.2 3.3 1.61.9 1.5 0.71.3 0.4 18:1ω9 5.036.5 1.93.2 1.3 11.1 30.0 18:1ω7 0.21.8 0.52.5 2.3 0.62.1 0.4 18:2ω6 6.722.1 2.911.4 7.1 0.91.0 0.5

18:3ω6020.4 3.7 0.3 3.4 18:3ω3 14.937.2 22.824.6 6.6 9.721.3 0.2 18:4ω3 2.53.4 2.5 26.6 21.824 4.3 20:0 0.10.2 20:1ω9 0.3 2.4 0.7 20:3ω6 5 0.4

20:4ω6 0.5 0.6 20:5ω3 35.1 0.6 0.6 15.820.5 6.9 22:0 22:4ω6 0.4 22:5ω3 3.1 22:5ω6 9.9 2.74.7

22:6ω3 6.5 2.5 4.37.2 12.2

, none or ,0.1%. Sources: Ahlgren et al. (1900); Cyclotella, from Desvillettes et al. (1997).

cholesterol or PUFAs (Martin-Creuzburg and Enteropathogenic non-agglutinating vibrios Heilke, 2011); Hydrogenophaga and Pseudomonas are consumed by D. magna but do not cause are toxic for D. magna. pathologies and are digested as food (Avtsyn Daphnia consumes methanotrophic bacteria and Petrova, 1986). using methane-derived carbon (C) (Kankaala Nevertheless, overall it has been determined et al., 2006), as well and those containing a that bacteria may make up to 87% of the total large part of carbon from this source (Taipale carbon ingestion of D. galeata (Kamjunke et al., et al., 2007). Methanotrophic bacteria and 1999). Under conditions of mixed feeding (bac- methane are highly significant sources of car- teria and algae), D. magna obtained 80% of its bon for Daphnia (Deines and Fink, 2011). carbon from bacteria and in this situation they

PHYSIOLOGY OF THE CLADOCERA 38 4. NUTRITION

TABLE 4.3 General Fatty Acid Composition of Some Algae, as a Percentage of Total Lipids

Fatty Diatoms: Green Algae: Dinoflagellates: Chrysophyceae: Cryptophytes: Blue-Green Acids Cyclotella Pediastrum Peridinium Chromulina Cryptomonas Algae: Anabaena

Saturated 17.7 21.5 36.4 17.6 15.1 40.5

Monoenes 10.7 12.0 30.6 8.1 3.0 18.6 Total 71.6 66.0 24.9 57.7 64.9 51.3 PUFA

Total ω3 62.0 53.5 24.3 36.3 59.2 38.3 Total ω6 4.8 9.5 0.6 21.4 5.7 9.8

PUFA, polyunsaturated fatty acid. Source: Desvilettes et al. (1997).

TABLE 4.4 The Chemical Composition of the Bottom Silt in Two Russian Lakes

Lake Sugars, Hemicelluloses, Cellulose Waxes and Fatty Organic Total Ash Pentosans Resins Acids Carbon Nitrogen

Glubokoe 1.0 2.2 2.3 0.2 11.9 1.1 74.2 Beloe 7.1 6.4 6.5 1.0 27.0 2.5 46.4

Values represent % absolutely dry matter. Source: Shcherbakov, 1967. assimilated c. 46% of their fatty acids from bac- profoundly decomposed, debris does not sup- teria (Taipale et al., 2012). D. magna fed solely port Daphnia in culture (Lampert, 1987). on bacterial diets die after 512 days. It is likely that different decomposition levels of organic matter supply nourishment for different species of Cladocera that are Organic Debris (Detritus) physiologically specialized for the consump- Organic debris suspended in water and set- tion of material at different decomposition tling on the bottom of water bodies consist of levels. The sequence of decomposition in sedi- decomposing algae, decomposing feces, and the ments of lakes was described by Rodina (1963, bodies of dying animals, as well as all kinds of p. 388): “The processes of decay of protoplasm allochthonous material. Detritus also harbors of animal and vegetative tissue and the pro- bacteria. Organic debris forms a major compo- cesses of oxidation and reduction are caused nent of the food of littoral and pelagic Cladocera, by different physiologic groups of bacteria: as noted originally by Naumann (1918). putrefactive, fat and carbohydrate decompo- The chemical composition of lake silt is sers, amylolytic, pectinous, cellulose decompo- shown in Table 4.4. Proteins and carbohydrates sers, denitrifying, nitrogen fixers, phosphate in the detritus (debris) are decomposed by liberators, sulfur bacteria, and yeasts.” enzymes in the feces of various animals, as has Although little is known on the subject, dif- been convincingly shown for fish by Kuzmina ferences in food choice were made use of in et al. (2010), as well as by bacteria. “Old,” i.e. combined cultures of Daphnia and Chydorus;

PHYSIOLOGY OF THE CLADOCERA 4.1 FEEDING 39

25 FIGURE 4.2 Fatty acid composition in Eurycercus Particulate matter and in its particulate food. n-, represents ω numbers. 20 Source: Desvillettes et al., 1997. 15

5 % total fatty acids % total fatty 0 16:0 18:0 16:1 18:1 BrFA 20(n-6) 22(n-6) Σ Σ 18:2(n-6) 18:3(n-6) 18:3(n-3) 18:4(n-3) 20:5(n-3) 22:6(n-3)

25 Triacyiglycerols 20 of E. lanellatus

5 % total fatty acids % total fatty 0 16:0 18:0 16:1 18:1 BrFA 20(n-6) 22(n-6) Σ Σ 18:2(n-6) 18:3(n-6) 18:3(n-3) 18:4(n-3) 20:5(n-3) 22:6(n-3) production was improved by Chydorus feeding FebruaryApril and lower in MaySeptember on the fecal remains of Daphnia (Bogatova, (48%); chlorophyll a levels increased through- 1963, 1980). out the season, from c. 100 mg/L/m2 in The seasonal periodicity of amino acid com- February to over 300 mg/L/m2 in October. The position of organic bottom material may be a phaeopigment content, a measure of algal contributory factor in the periodicity of littoral decomposition, demonstrated no definite trend, cladocera. Although not explored further, pre- fluctuating between 300 and 50150 mg/L/m2. liminary determinations by this author have With reference to the eutrophic Lake demonstrated that various amino acids are Nesjøvatn (in Norway), it was shown that present in organic bottom material, some of D. galeata obtain carbon principally from algae which are abundant and some present in low and detritus and phosphorus principally from concentrations. The amino acid composition of algae (only a minor part of total phosphorus is seston in small water bodies of the Yenissey obtained from bacteria) (Vadstein et al., 1995). Basin was also studied by Kolmakova (2010). Desvilettes et al. (1994) determined the fatty Some authors have attempted to investigate acid composition of littoral particulate matter: the food quality of littoral organic debris and the prevailing fatty acids were also dominant its changes over time. Robertson (1990) esti- in Eurycercus and Simocephalus (Fig. 4.2). mated the content of organic matter, chloro- Even when there is a considerable inflow of phyll a, and phaeopigment in River Thames terrestrial organic matter, Daphnia derive most mud in relation to the population dynamics of of their carbon from phytoplankton (Brett various cladocerans. The content of organic et al., 2009). However, this source of food may matter was higher (up to 16%) in be important for littoral species.

PHYSIOLOGY OF THE CLADOCERA 40 4. NUTRITION

It has also been shown experimentally that Fig. 4.3 shows the schematic pathway from ini- Daphnia assimilate some dissolved organic tial photosynthetic saccharides to lipids and matter (e.g. amino acids), but this alone is amino acids in algae. insufficient for their normal lifestyle (Rodina, Lipids are present in water in both a particu- 1946, 1948, 1950). late (i.e. within algae) and a dissolved state (Arts et al., 1997). In Cladocera, they are used in energy metabolism, in the construction of cell 4.1.3 Introductory Remarks about Lipids membranes, and in metabolism; further, derivatives of lipids act as sex hormones. To under- The lipid pathway in the aquatic environ- stand the next sections, it is first necessary to ment starts with lipid formation by plants statesomebasicideasanddefinitions. from initial products of photosynthesis. There are several classifications of lipids According to Harwood and Jones (1989, p. 12), (e.g. Kucherenko and Vasilyev, 1985). In one “[d]e novo synthesis [requires] the concerted of these, the following four groups are action of acetyl-CoA carboxylase and a type II distinguished: (dissociable) fatty acid synthase.” The chain length of the product may then be elongated 1. Simple lipids, which are esters of fatty acids and unsaturated bonds may be introduced. with glycerol (fats) or aliphatic alcohols

FIGURE 4.3 Schematic pathway of photosynthetic saccharides to lipids and amino acids in algae. C3-P.S., photosynthesis. Source: Arts et al. (1997).

PHYSIOLOGY OF THE CLADOCERA 4.1 FEEDING 41

(waxes). Waxes comprise true waxes [The fatty acids may be saturated, i.e. con- and esters of cholesterol, vitamin A, or taining no double bonds between the carbon vitamin D. atoms, and unsaturated, i.e. containing double 2. Complex lipids, which are esters of fatty bonds. The latter are described in the format x: acids with other alcohols, e.g. ywz (e.g. Ahlgren et al., 1990), where x is the phospholipids, glycolipids, sulfolipids, number of atoms, y is the number of double lipoproteins, or lipopolysaccharides. bonds (ω), and z is the position of the first 3. Lipid derivatives (lipoids), which include fatty double bond from the methyl end of the mole- acids (saturated and unsaturated), cule. For example, 20:0, where 20 is the num- monoglycerides, diglycerides, sterins, ber of carbon atoms and 0 (or a certain digit) is steroids (vitamin D group), alcohols with β the number of double bonds in the molecule of ionic ring (vitamin A group), phosphatides, a fatty acid. and carotenoids. Some names of lipids corresponding to a. Steroids are derivatives of cyclopentano- these designations are listed: perhydro-phenatrene. They comprise sterols and their derivatives. Ecdysteroid C14:0 myristic fatty acid (ecd) concentration in whole D. magna is C16:0 palmitic acid c. 200 pg ecd equivalent/mg dry weight C16:1 palmitoleic acid (DW) (Bodar et al., 1990b). In diatoms, C18:0 stearic acid cholesterol and C28 sterols are the major C18:1 oleic acid sterols (Soma et al., 2005). Derivatives of C18:2ω6 linoleic acid steroids are sex hormones: male C18:3ω3 α-linolenic acid hormones, testosterone and C18:3ω6 γ-linolenic acid androsterone; and female hormones, C18:4 octadecatetraenoic acid estron (progesterone) and estradiol (see C18:4ω3 stearidonic acid Section 11.3.5). C20:1 eicosenoic acid b. Phosphatides are esters of polyatomic C20:3 eicosatrienoic acid alcohols, fatty acids, and phosphoric C20:4ω6 arachidonic acid acid. They comprise lecithins, which C20:5ω3 eicosapentaenoic acid consist of radicals of glycerol, phosphoric C22:5 docosapentaenoic acid acid, choline, and higher fatty acids C22:6ω3 docosahexaenoic acid (saturated or unsaturated). 4. Various others, including vitamins E and K, and aliphatic carbohydrates. Essential Fatty Acids Generally, fat is represented as a triglycer- It is thought that almost all PUFAs are ide connected to various fatty acids (R): obtained by animals from plants and are not synthesized by animals. Thus, linoleic acid O ω α ω || (C18:2 6) and -linolenic acid (C18:3 3) are CH3 —O—C—R1 essential fatty acids, whereas EPA is not | O strictly essential. Becker and Boersma (2007, || p. 463) arrived at the conclusion that “although CH3 —O—C—R2 | O dietary fatty acids can be used for energy pur- || poses, specific fatty acids [namely, PUFAs] are CH3 —O—C—R3 etc. required to build new biomass.”

PHYSIOLOGY OF THE CLADOCERA 42 4. NUTRITION

Lipids in Cladocera Oil drops are easily observed in Cladocera and are often mentioned in the literature (e.g. Flu¨ ckiger, 1951). They are distributed throughout the body, although this has not been suffi- ciently described for representatives of various genera living in different environments. They are mostly orange in color and their size ranges from quite small to very large. The following microchemical components are found in the fat of D. magna (Jaeger, 1935): butyric acid, sodium oleate, triolein, cholesterol oleate, cholesterol stearate, stearic acid, sodium stearate, tristearin, lecithin, linseed oil, and linoleic acid. Tessier et al. (1983) found that the major lipid types in Daphnia are triacylglycerols and wax FIGURE 4.4 esters. Arts et al. (1993) confirmed this preva- Fatty acid composition in planktonic cladocerans and in their seston food. A-D denote taxa lence and indicated that the next most dominant that differ significantly in the content of the respective lipid class is phospholipids and sterols. fatty acid group. MUFA, monounsaturated fatty acids; It is likely that Goulden and Place were PUFA, polyunsaturated fatty acids; SAFA, saturated fatty among the first to discuss the quantitative distri- acids. Source: Persson and Vrede (2006). bution of lipids in Cladocera (1993). Taking into consideration that the fatty acids are either derived from food or synthesized de novo in It has been determined that Cladocera pre- the body and that acetate (derived from the dominantly contain (1223%) EPA (Persson breakdown of carbohydrates or amino acids) and Vrede, 2006) (Fig. 4.4), in contrast to cope- is necessary for their synthesis, they used [14C] pods. This difference is assumed to be a result 3 acetate or H2O precursors and determined that of their phylogenetic origin. the lipids synthesized by well-fed Daphnia (fol- Bychek et al. (2005) found that D. magna is lowing incubation of up to 4 h) make up no capable of high rates of de novo lipid radiola- more than 1.6% of the accumulated fatty acids. beling; D. magna also makes direct use of die- Food-restricted Daphnia synthesize 0.16% of tary components (such as the PUFAs linoleate their accumulated lipids; the overwhelming and α-linolenate). In addition, D. magna toler- remainder is derived from algal food. ates 24-h fasting with little change in lipoid Thus, according to Goulden and Place metabolism. (1993), daphnids selectively assimilate lipids Tessier and Goulden (1982) recorded an from food and synthesize de novo not more abundance of oil globules in Daphnia by the than 1.6% of their accumulated fatty acids. visually estimated lipid index (LI). Dodson Goulden and Place also indicated that lipids in (1989) observed that in presence of predators daphnids consist of storage (triglycerides and the fat content in the body (LI) decreases, wax esters) and structural (phospholipids and which might be related to defense from preda- sterols) lipids, and that most of the lipids are tors. Hoenicke and Goldman (1987) estimated transferred to the ovaries and then into eggs the lipid-ovary index in scores (based on and used in the development of embryos. visual scoring) in Daphnia and Holopedium and

PHYSIOLOGY OF THE CLADOCERA 4.1 FEEDING 43 found that this index varies depending on the Of the planktonic algae, the best food items composition of their natural food. for D. longispina, Bosmina, and C. sphaericus are It has also been shown that Daphnia (specifi- cryptomonads, with Cryptomonas and cally with reference to D. magna) cannot syn- Rhodomonas containing high percentages of the thesize linoleic acid (C18:2ω6) or α-linolenic PUFAs 20:5ω3 and 22:6ω3 (docosahexaenoic acid de novo (Persson and Vrede, 2006) acid) (Ahlgren et al., 1990). These PUFA were (Fig. 4.4). Thus, they depend on fatty acids also found to be common in fish. produced by plants (including essential fatty It was found that in D. obtusa fed with the acids, e.g. C20:5ω3, EPA). green alga Scenedesmus the content of lipid in Wacker and von Elert (2001) found that the body does not positively correlate with EPA (C20:5ω3) and α-linolenic acid (C18:3ω3) growth rate (Sterner et al., 1992). are not mutually substitutable resources for D. pulex fed with Aphanizomenon did not D. galeata, that their physiological functions are accumulate oil drops, although this blue-green likely to be different, and that the former is not alga is not directly toxic for them; on the other limiting for the growth of D. galeata cultivated hand, when fed with the green protococcous on seston. alga Ankistrodesmus, D. pulex did accumulate Having accumulated their fat reserves, oil drops (LI: 0 and 3, respectively) (Holm and Daphnia propagate, consume this resource, and Shapiro, 1984). Brett et al. (2006) studied the then decline (Goulden and Hornig, 1980). fatty acid composition of D. pulex fed on Normally, the fat present in the body may be cryptomonads (Cryptomonas and Rhodomonas), used by starving daphnias, as was observed Chlorophyta, and blue-green algae and found in D. magna by Flu¨ckiger (1951), and short-term that it generally matched that of their algal (24-h) starvation does not lead to profound food. However, there were some differences: in changes in lipid metabolism (Bychek et al., 2005). comparison with their food, the content of satu- Further data are presented in Section 4.3. rated fatty acids in Daphnia was substantially less and that of arachidonic acid was higher. 4.1.4 Algal Lipids According to these data, Daphnia on the whole transfers somewhat modified fatty acids Lipids are derived by Cladocera from vari- up the food chain (Fig. 4.5). ous groups of algae, and different algae vary The fatty acid composition of some freshwa- in their value as lipid sources for Cladocera ter algae is shown in Table 4.1. The general- (Fig. 4.3). Although diatoms supply the bulk of ized composition of fatty acids in different lipids for their consumers, their lipids are not algae is shown in Tables 4.1 and 4.3 and in necessarily the best types for Cladocera. Fig. 4.1. A high content of lipids in freshwater Diatoms are not alone in having the peculiar diatoms, i.e. 13.6% DW, has been determined trait of not producing and accumulating starch. by Birge and Juday (1922, p. 164): “The diatom Fogg (1953, p. 105) noted that “starch evidently sample contained a larger percentage of ether does not occur in the Xanthophyceae, extract than any other sample of plant Chrysophyceae or Bacillariophyceae, and the material.” same three classes have a general tendency to Blue-green algae (or cyanobacteria) are also store fats as reserve materials rather than poly- a source of lipids for Cladocera. Based on saccharides.” Sedova (1977) added that diatoms extensive observations and analyses, Sushchik and dinoflagellates are especially rich in lipids. et al. (2002) concluded that the development Particular groups of algae are characterized by of planktonic D. longispina and D. cucullata specific spectra of fatty acids (Sushchik, 2008). mostly depends on 18:3ω3 produced by

PHYSIOLOGY OF THE CLADOCERA 44 4. NUTRITION

FIGURE 4.5 Fatty acid composition of Daphnia after consuming various kinds of phytoplankton and of phytoplankton groups. Chloro, Chlorophyceae; Crypto, Cryptophyceae; Cyano, blue-green algae. ARA, arachidonic acid (20:4ω6); DHA, docosahexaenoic acid (22:6ω3); EPA, eicosapentaenoic acid (20:5ω3). Source: Brett et al. (2006). blue-green algae. However, at the same time, some protein, pigments, and vitamins); they blue-green algae contain compounds that are live in siliceous (glass) frustules; and they do toxic for Cladocera (see Section 4.2.4). not synthesize starch and cellulose from the Not all PUFAs have a positive role: primary carbohydrate products of photosyn- γ-linolenic acid (a PUFA obtained commer- thesis. In contrast, they can exploit the reaction cially for experiments) is acutely toxic (!) for of fat formation that is generally characteristic D. magna at a concentration of 9 μg/mL of plants: their metabolism is largely confined (Reinikainen et al., 2001). to this process. Writing specifically on diatoms, Rabinovich The Physiology of Diatom Photosynthesis (1945, 1951) commented that “the oil deposits Of all the algal groups, diatoms of the diatoms .... must be considered as (Bacillariophyceae) are the most prominent products of comparatively slow secondary producers of lipids. However, they also have a transformations” (1945, p. 43) of primary very peculiar metabolism: they transform the photosynthesis products. The metabolism of bulk of the initial monosaccharide produced in diatoms is better understood than that of other photosynthesis into lipids (with the addition of types of algae. The lipid content of diatoms

PHYSIOLOGY OF THE CLADOCERA 4.2 FEEDING CHARACTERISTICS 45

0.3 Melosira and discovered a prevalence of palmi- r2 = 0.93 tic acid (16:0; 12.431.8% of total fatty acids) 0.2 and palmitoleic acid (16:1; 23.331.7%). The unsaturated acid content is 53.6% and 36%, 0.1 respectively, and the saturated acid content is

Growth rate/day Growth 21.2% and 35.1%, respectively. 0.0 Accumulation of the essential, unsaturated 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.0 1.6 fatty acids 20:5ω3 and 22:6ω3 in benthic (per- μg EPA/I iphytonic) diatoms from March to January was FIGURE 4.6 Relationship between available eicosa- assessed by Gladyshev et al. (2005). They pentaenoic acid and Daphnia growth (Lake Scho¨see, found that the maximum of both types, c. Germany). EPA, eicosapentaenoic acid. Source: Brett and 40 μg/m3, occurs in August. Sushchik (2006) Mu¨ller-Navarra (1997). noted that some diatoms (Cyclotella) are not rich in essential PUFAs and that some blue- reaches about 35% of dry matter (Barashkov, green algae (Anabaena) have a high content of 1972), mostly consisting of unsaturated fat: these fatty acids, contrary to the generally according to Farkas (1970), these lipids are rich accepted viewpoint, and thus are an important in 14:0 and relatively poor in 18:2 and 18:3 source. Sushchik (2008) also concluded that fatty acids. Data on the lipid composition of diatoms are characterized by an increased con- diatoms are shown in Tables 3.4 and 4.1: tent of 14:0, 16:1ω7, and specific C16 PUFAs, Daphnia growth (in the Scho¨see, Germany) was and by a low content of C18 PUFAs. shown to be proportional to the available EPA Some other groups of algae also synthesize (Fig. 4.6) (Brett and Mu¨ ller-Navarra, 1997). lipids, but information on their metabolism and In the freshwater diatom Navicula pelliculosa, accumulation of stock products is insufficient. the total lipids content includes the following There have been no reports on the controlling major components: sulfoquinosyl diglyceride, mechanism that channels photosynthesis to digalactosyl diglyceride, monogalactosyl lipid formation in diatoms. diglyceride, phosphatidyl glycerol, lecithin, In some lakes (e.g. Myvatn and Kronotskoe), and phosphatidyl inositol (similar to green diatoms are highly dominant: as evidence, the algae). The major fatty acid constituents are ground surrounding the lakes consists almost palmitoleic acid, palmitic acid, EPA, and eico- entirely of diatom frustules (diatomite). Thus, satetraenoic acid (Kates and Volcani, 1966). the trophic chain in such lakes is based princi- The concentration of linolenic acid (octadeca- pally on thee lipids produced by diatoms. trienoic acid), on the other hand, is low. Opute (1974) confirmed that (1) the lipids of freshwater diatoms (Nitzschia and Navicula) consist mainly of triglycerides, monogalactosyl 4.2 FEEDING CHARACTERISTICS diglyceride, digalactosyl diglyceride, sulfoqui- novosyl diglyceride, phosphatidyl glycerol, 4.2.1 Quantitative lecithin (phosphatidylcholine), and phosphatidylethanolamine; and (2) the major fatty acids Rate of Mandible Rolling are palmitoleic, palmitic, EPA, and eicosate- Cladocera eat almost continuously. traenoic acids, with 16:0 and 16:1 prevailing. The rate of movement of the mandibles is c. Volkova (1980) also studied fatty acids in 250 beats/min in Leydigia leydigii (Fryer, 1968), the freshwater diatoms Stephanodiscus and 177 beats/min in Streblocerus serricaudata

PHYSIOLOGY OF THE CLADOCERA 46 4. NUTRITION

(Fryer, 1974), and 110190 beats/min in carinata, the beat frequency of the thoracic limbs Daphnia rosea (in direct proportion to yeast is 327 beats/min for a body length of 0.6 mm; concentration) (Burns, 1968a). In P. globosus, this increases slightly to 360 beats/min for a “rolling and swinging take place too rapidly to body length of 2.15 mm, and then decreases to permit visual determination of the rate” 310 beats/min for a body length of 2.6 mm. The (Fryer, 1968, p. 335). beat frequency decreases by c. 41% in response In Daphnia, Starkweather (1978) found well to natural water toxicity caused by algae such defined daily patterns in the rate of mandible as Cyclotella or Anabaena (Forsyth et al., 1992). At rolling, with distinct nocturnal minima. In the too high a food concentration, D. magna daytime, the rate of mandibular beating in decreases the rate of strokes of its thoracic limbs D. pulex was c. 140 beats/min, whereas at night and discards the food collected in the food (01:0007:00) it was c. 80 beats/min. This rate groove if the intestine is full. decreased in larger specimens, from 180 beats/ According to Lampert and Bredeberger min for a body length of 2.0 mm to 80 beats/ (1996), at low concentrations of food particles, min for a body length of 3.4 mm. According to Daphnia adapts to low food levels by slightly Murtaugh (1985), the rate of mandible move- decreasing the appendage beat rate (from c. ments may be taken as a measure of feeding 300550 to 300450 beats/min in D. pulicaria); intensity. In D. pulicaria, it ranges from a mean simultaneously, phenotypical enlargement of of 1.3 Hz (beats/sec) at a lower food concentra- the area of its filtering screens occurs. tion to 2.1 Hz at a higher food concentration. Broken diatom frustules have been found in Water Clearance Rate the intestine of some Cladocera. Therefore, LITTORAL CLADOCERA mandibles may contribute to the breaking up of food items. The water clearance rate in Eurycercus was determined to be 20.5 mL in 24 h (Marcolini, Rate of Thoracic Limb Strokes 1953) or up to 30.6 mL in 24 h for each speci- In Cladocera, water flows created by the tho- men (Krychkova, 1969). racic limbs are important for both feeding and respiration. In Anomopoda and Ctenopoda the PELAGIC CLADOCERA thoracic limbs move in a metachronal rhythm, Determination of filtering rates differs accord- i.e. with a certain phase lag for the movement ing to different authors. For Cladocera, filtering of every subsequent pair (Cannon, 1933). rate generally ranges from 0.9 to 135 mL in 24 h, The stroke rate of the exopodites of thoracic per specimen, depending on food abundance limbs, which creates a feeding and respiratory and other factors (Monakov, 1998). The filtration current, is 290300 cycles/min in chydorids rate of D. magna ranges from 0.3 (and lower) to (Smirnov, 1971), 170 cycles/min in C. sphaericus, 1.8 mL/h, per specimen (Gulati, 1978). over 300 cycles/min in D. longispina,60244 Darchambeau and Thys (2005) observed clear- cycles/min in Ceriodaphnia (Harnisch, 1949), and ance rates of 2.513.5 mL in 24 h, per specimen 150470 cycles/min D. magna (McMahon, 1968). for D. galeata in Esch-surSuˆre Reservoir The rate of strokes varies both individually (Luxembourg). In the latter study, the clearance and under different environmental conditions rate always correlated negatively with phospho- (e.g. in Daphnia)(Pen˜alva-Arana et al., 2007). In rus concentration in the food. D. pulex, the rate of strokes of the thoracic limbs As summarized by Sushchenya (1975), the increases at decreased oxygen concentrations filtering rates of different species [in mL per (Wolvecamp and Waterman, 1960). In Daphnia individual (ind.) per 24 h/mL per mg DW per

PHYSIOLOGY OF THE CLADOCERA 4.2 FEEDING CHARACTERISTICS 47

24 h at 20C] are 0.25115/187780 for (Sushchenya, 1958a, 1958b). However, at a cer- Daphnia, 1.215.6/5003800 for Diaphanosoma, tain upper limit of food particle concentration 140/1255000 for Bosmina, 1/560 for (over 8 g/m3 for D. longispina), regulation of Ceriodaphnia, 8.6135/538860 for 26/2760 for the filtration rate is no longer possible Moina, and 3133/4703021 for Sida, (Monakov and Sorokin, 1960). In D. magna, Simocephalus. Filtering rates (in μL/μg/DW/h) food consumption is not increased further are 28.3 in D. hyalina and 49.5 in D. cucullata when the food concentration reaches 2 3 106 (Steiner and Kasprzak, 2000). cell/mL of Chlorella or 1 3 106 cell/mL of yeast The filtering rate (in mL/24 h, per specimen) (Rigler, 1961b; McMahon and Rigler, 1963, was determined to be 0.95.15 for D. pulex 1965); the feeding rate increases with increas- 0.71.8 mm long, increasing with size in a cur- ing Chlorella concentration up to a maximum vilinear fashion (or 300180 mL/mg DW/day) and then does not increase further at higher (Richman, 1958); for a length of 2 mm, it was concentrations (oxygen uptake behaves in a about 5 mL in the daytime, increasing at night similar way) (Kersting, 1978). This concentra- up to about 20 mL (Haney, 1985). tion was termed the incipient limiting level In ctenopods, as summarized by (McMahon and Rigler, 1965). Korovchinsky (2004), it is 1248 mL/ind./24 h In hungry D. magna, filtering rates (in mL/ in Diaphanosoma brachyurum s.l., 1058 mL/ ind./h) and feeding rates (in μm3/ind./unit ind./24 h in Holopedium,2657 mL/ind./24 h time) are higher than in well-fed specimens in Sida, and 32252 mL/ind./24 h in Penilia. (Ringelberg and Royackers, 1985). D. magna While feeding on Chlamydomonas, filtering quickly increase their respiratory rate with rates are 364 μL/ind./h in Bosmina longirostris, increasing concentration of food algae 399 μL/ind./h in D. longispina,408μL/ind./h (Lampert, 1986). in Ceriodaphnia quadrangula, and 403 μL/ind./h Obtaining food particles from suspension in C. sphaericus (Lair, 1991). was shown, at least partly, to occur not only A very wide scattering of the experimen- through mechanical screening. Gerritsen and tally obtained values is observed. This may Porter (1982) believe that collecting food parti- depend on environmental factors, particular cles also depends on viscosity and the electric experimental conditions, or the specific clone charge of both the filtering limbs and the food tested. Nevertheless, Knoechel and Holtby particles. The filtering rate also depends on (1986) estimated that filtering rate are mainly oxygen consumption, and this relationship dependent on body length and comply with seems to be species dependent. In contrast to the following equation (Eq. 4.1) D. galeata, oxygen consumption in D. magna rapidly declines at concentrations below c. : F 5 11:695L2 480; (4.1) 3 mg/L (Heisey and Porter, 1977). In their analysis of food filtration by daph- where F is individual filtering rate in mL/day, nids, Gerritsen and Porter (1982) measured the and L is body length in mm. Reynolds number (Re): for D. magna, the Re for The rate complies with this equation in mor- filtering combs of limbs III and IV was 2 2 phologically different forms such as Bosmina, 0.4 2 2.0, for a single seta it was 10 210 4, 2 2 Ceriodaphnia, Chydorus, and Daphnia spp. In and for a setule it was 10 310 4. The region Daphnia species, the exponent is 2.48. of reduced flow around a setule extends far In Daphnia, Diaphanosoma, and Simocephalus, beyond the next setule. Thus, there is little the filtering rate is inversely proportional to flow between setules and, according to these the concentration of algae in the water authors, the appendage functions as a solid

PHYSIOLOGY OF THE CLADOCERA 48 4. NUTRITION wall. As Daphnia collect smaller particles (i.e. depends on the length (L) and is described by smaller than the mesh size of the limbs) at a Eq. 4.2 (Pott, 1982): greater rate than larger particles, it was con- ðμ = 3 Þ5 : 3 2:105½ ðμ 3 Þ cluded that the mechanism of food collection I gC h animal 0 214 L C g animal : does not involve direct sieving. It was 52:9573L2 962ð4:2Þ assumed to be food capture by electrostatic attraction. In support of this, neutralization of The daily ration of pelagic species (in % the negative surface charge of small particles WW) was determined to be 17140 in Daphnia, further increases the retention efficiency. 16101 in Ceriodaphnia, 129285 in However, subsequent authors, first of all Simocephalus,3074 in Moina brachiata Fryer (1991), confirmed the fundamental sig- (Ostapenya et al., 1968; summarized in nificance of direct filtration. It was shown in Smirnov, 1969, 1975), up to 750 in juvenile Daphnia that the strokes of the thoracic limbs Moina macrocopa fed on Chlorella (Vorozhun, are repetitive, with short delays between 2001), 5.721 in Sida,2949 in D. cucullata, and pumping actions; in the presence of food the 3.524 in D. longispina (Kryuchkova, 1989). delays between pumping sessions become lon- Sushchenya (1975) reported the range of daily ger (Penãlva-Arana et al., 2007). rations to be 67129% WW, but also described Abrusań (2004) increased the viscosity of the data of various authors as ranging from the medium using a sucrose polymer pro- 12.5 to 246% WW. He further commented that duced by Leuconostoc or with Ficoll. Increasing his own previous data (Sushchenya, 1958a, the viscosity of the medium at a constant 1958b) was strongly underestimated. temperature decreased the growth of three The daily ration (i.e. ingested food; in % Daphnia species (except for D. galeata) WW) of those feeding on detritus has been (Abrusań, 2004). It is likely that the decrease determined to be 69 in D. magna,70inD. long- was due to interference with filtration. The lat- ispina, 179 in Simocephalus vetulus,23in ter is thought to occur at an Re of approxi- C. quadrangula, and 51 in Moina rectirostris 2 2 mately 10 110 4. (syn. M. brachiata) (Esipova, 1971). Generally, the daily ration for pelagic Cladocera is estimated to range from 0.8% to 247% WW, Quantities of Food Consumed depending on the food, and probably also on LITTORAL CLADOCERA the methods used (Monakov, 1998). The average daily ration for Leptodora (a predator) is There is little information on littoral Cladocera. In chydorids, the daily ration (in μg estimated to be 30% of its WW (at 17 C) DW/specimen/24 h) was determined to be (Hillbricht-Ilkowska and Karabin, 1979). 80 in C. sphaericus (Aksenova et al., 1969), 250 Food requirements (or rations) may also be in Acroperus (Smirnov, 1971), and 2500 in estimated using the oxycalorific coefficient. Eurycercus lamellatus (Smirnov, 1962). E. lamel- Investigations into the diurnal rhythm of latus ingest periphyton within the range of feeding rates have indicated that the feeding 0.5 2 462 μg DW/specimen/ 24 h (Balayla and rate in Daphnia and Holopedium spp. is higher Moss, 2004). and less variable at night than during the day (Haney, 1985). Ingestion rates of algae for D. longispina are PELAGIC CLADOCERA different for different groups of algae The absolute quantity of food ingested (I) in (Schindler, 1971): highest values (μg/h/10) the case of Daphnia pulicaria fed on Scenedesmus, were determined for Gloeocystis (green alga),

PHYSIOLOGY OF THE CLADOCERA 4.2 FEEDING CHARACTERISTICS 49

11.0; Coelastrum (green), 16.8; Asterionella (dia- are efficiently assimilated by Daphnia, whereas tom), 17.7; and Oscillatoria (blue-green), 22.7. P-limited cells passed through the gut largely Movements of the thoracic limbs are reduced intact. by nicotine, epinephrine, cyanide, strychnine, In Cladocera, food rations (i.e. ingested metrazol, and carbon dioxide (i.e. the corre- food) may reach high values. In juvenile sponding striated muscles are affected) in M. macrocopa fed on Chlorella, the ration D. magna (Sollman and Webb, 1941). reached 750% body weight (Vorozhun, 2001) At increasing concentrations of available food, in adult E. lamellatus fed on detritus, 2500% the rate of oxygen consumption by D. magna body weight (Smirnov, 1962). increases: this phenomenon is known as specific If data on oxygen consumption are avail- dynamic action of food (Barber et al., 1994). able, then the extent of redundant food con- It should be noted that the values provided sumption may be estimated using the above vary greatly depending on the dynamics oxycalorific coefficient (described, e.g. in of environmental factors. See also below on the Dediu, 1989): its value is 4.86 cal/mL O2 or redundancy of food intake (Section 4.5.3). 3.38 cal/mg O2, or 14.2 J/mg O2 (1 mL 5 O2 1.6 mg O2). Multiplying the values of oxygen consumption by the oxycalorific coefficient 4.2.2 Excessive and Insufficient Diets yields the minimum physiological energy demand (assuming that the food is physiologi- Luxury Consumption (Superfluous Feeding) cally balanced). The ratio of the ration value High concentrations of available food obtained by direct determination to that deter- (above a certain level) may lead to excessive mined respirometrically is termed the excess consumption of food particles (overcollection) index (Gajewskaja, 1948). (Porter et al., 1982). The term luxury uptake was For E. lamellatus, the daily relative ration initially used to describe the acquisition of a determined directly was 2500, and 220 when nutrient in excess of current demands (see determined respirometrically (Smirnov, 1962). Sterner and Schwalbach, 2001). It is now Thus, the excess index is c. 11 for adults; it is understood to mean the consumption of food higher for juveniles. in quantities exceeding physiological require- As a result of staying within the intestine ments. In this situation, the quantities of food for only a short period, especially under condi- material passing through the intestine increase tions of “excessive” feeding, algal cells are but less of the food is digested, i.e. some food digested incompletely or may even remain is discarded unused. alive. The latter was demonstrated for algae Redundancy of food consumed under con- ingested by Daphnia (Porter, 1975), especially ditions of excess available food (Oocystis) has for algae covered with gelatinous sheaths. been noted, e.g. in D. magna (Myrand and de la Nou¨ e, 1983); it is thought that overcollection may lead to reduced assimilation rates Insufficient Food (Schindler, 1968; Porter et al., 1982). Daphnias that are undernourished through- Sterner and Schwalbach (2001) applied the out their life live about 40% longer than well- term luxury consumption to the storage of phos- fed daphnias (Skadovskiy, 1955). If starving phorus during favorable periods to be used daphnias are given abundant food, they catch during times of less food availability. A signifi- up with well-fed daphnias and finally produce cant fact discovered by van Donk and Hessen the same quantity of eggs. However, if well- (1993) is that P-saturated cells of green algae fed daphnias are given limited food, then their

PHYSIOLOGY OF THE CLADOCERA 50 4. NUTRITION fecundity decreases by 2.5 times, their longev- found not to be able to consume suspended ity decreases, and they manifest signs of senes- bacteria by filtration. cence. Jacobs (1961) supplied cultures of In a suspension of latex beads, Holopedium Daphnia with a low (1/100 dilution) concentra- selected the largest particles and Diaphanosoma tion of Chlorella vs. standard cultures, and was almost nonselective (Hessen, 1985). observed that under such conditions growth was slower, a much longer time was needed to Postabdominal Rejection reach the first instar, maturity was not reached Cleaning of the thoracic limbs by the post- at the fifth instar, and no eggs were deposited. abdomen was observed in chydorids and Accumulated saturated fatty acids (20:0, Daphnia rosea (Burns, 1968a, 1968b). Rejection EPA) are used by D. magna during periods of of too large or too abundant algae by D. magna inadequate food (Becker and Boersma, 2005). was observed by Porter and Orcutt (1980). D. magna receiving a low quantity of food and Suspended clay increases the frequency of low-P algae produce neonates with an postabdominal rejection and decreases the rate increased lipid content (Boersma and Kreutzer, of thoracic limb beating (Kirk, 1991). In 2002). Daphnia, Burns (1968a, 1968b) also noted that It has been shown experimentally that the action of the thoracic apparatus was inter- under low-food conditions, Daphnia species rupted during postabdominal rejection. develop larger filtering screens on their thoracic limbs (Lampert, 1994). Labral Rejection Labral rejection is the rejection of food intake 4.2.3 Qualitative Characteristics: by deflection of the labrum, thus covering the mouth. This has been shown in D. rosea Selectivity (Burns, 1968a, 1968b). The presence of selectivity in filter-feeding daphnids was shown experimentally by Regurgitation Gajewskaja, (1949). Selectivity was quantita- Regurgitation has been observed by Rankin tively investigated and used to estimate the (1929) only in Simocephalus fed with palmella ecological advantages of particular species algae stained with methyl violet. (DeMott, 1982).

Morphological Background 4.2.4 Feeding is Influenced by Environmental Factors The distance between filtering setae on the thoracic limbs may be one cause of selectivity. In D. pulex and D. galeata mendotae, about This distance was determined to be from 85% of the daily filtering occurs during the 0.2 μminDiaphanosoma to 4.7 μminSida crys- dusk-dawn period; filtering rates are lowest in tallina (Geller and Mueller, 1981). In accor- deep water during the daytime, increased with dance, Diaphanosoma, C. quadrangula, reduced depth (ascent), decreased at midnight, D. cucullata, D. magna, and juvenile D. hyalina and increased again before their descent in the were found to be efficient consumers of bacte- morning (Hancy and Hall, 1975). Other ria, and D. hyalina (adult), D. galeata, D. pulicar- authors (Starkweather, 1975; Steiner and ia, B. coregoni to be low efficiency consumers of Kasprzak, 2000) noted that in daphnids indi- bacteria. S. crystallina and H. gibberum were vidual filtering rates are higher at night.

PHYSIOLOGY OF THE CLADOCERA 4.3 DIGESTION 51

With reference to C. quadrangula, it has been (if the concentration of copper ions was not shown (Gladyshev et al., 1997) that feeding above 0.1 mg/L). discontinues in the dark and that rhythmical changes of feeding intensity occur with a periodicity of 23h. C. quadrangula grown under 4.2.5 Impact of Xenobiotics on Feeding conditions of 12 h light:12 h darkness and The filtration rate of D. magna is decreased transferred to constant light manifested two by 52% (of the control) in the presence of maxima in the circadian rhythm of grazing; 0.15 mg Cd/L (Domal-Kwiatkowska et al., this does not occur when they are maintained 1994); that of D. pulex (feeding on a green alga under constant light (Kolmakov et al., 2002). Scenedesmus) is decreased by 40% following preexposure to glyphosate and by 30% following preexposure to p,p0-dichlorodiphenyl- Influence of Suspended Minerals dichloroethylene (Bengtsson et al., 2004). The Mineral suspensions, both natural and decreased filtration rate in D. magna consum- industrial, interfere with the filtration process ing fluorescing microbeads was used to esti- (Dubovskaya et al., 2003; Gladyshev et al., mate the acute toxicity of metals (Ginatullina 2003) by decreasing the feeding efficiency and Kamaya, 2012). (Rylov, 1940), which leads to decreased fecun- The rate of filtration and of ingestion in dity and other biological parameters of Daphnia decreased in the presence of the insecti- Daphnia, Moina, and Ceriodaphnia (Gorbunova, cide methylparathion (Ferna´ndez-Casalderrey 1988, 1993). et al., 1993) and the acaricide tetradifon Turbidity becomes lethal at 6400 mg/L (at (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) , μ particle sizes 5 m) (Gorbunova, 1988, 1993). (Villarroel et al., 1998). In the case of tetradifon, Al2O3 nanoparticles cause partial mortality of the reduction was 50% in comparison with the . Ceriodaphnia dubia at concentrations 200 mg/L, control, which occurred at 0.02 mg/L for filtra- which is aggravated by the presence of other tox- tion and 0.24 mg/L for ingestion. icants (Wang et al., 2011). In D. magna exposed to the insecticide Suspended clay decreases the rate of beating cypermethrin (at 0.1 μg/L) or the fungicide of thoracic limbs, decreases the algal ingestion azoxystrobin (at 0.5 mg/L), the limb filtering rate by 60 70% at low algal concentrations, rate, the mandibles rolling rate, and the heart and increases the frequency of postabdominal rate decreased (Friberg-Jensen and Nachman, rejection in Daphnia (Kirk, 1991). According to 2010); in contrast, all of these parameters Kirk, the rejected boluses contained both clay increased in the presence of 1 μg/L and algae. At 50 mg/L suspended clay, the rate cypermethrin. of beating of the thoracic appendages decreased from 246 to 93 beats/min, the rate of mandible movements from 83 to 34 beats/min. 4.3 DIGESTION In addition, a negative impact on D. hyalina was observed by the presence of suspended 4.3.1 Anatomical Background particles at a concentration of 25 mg/L (Rellstab and Spaak, 2007). The cladoceran intestine consists of a stomo- Suspended ground material decreases the daeum (foregut or esophagus) passing into the sensitivity of the investigated Cladocera spe- mesenteron (midgut) and proctodaeum (hind- cies to phenol (Gorbunova, 1993); in contrast, gut) (Hardy and McDougall, 1984). The inner the toxicity of copper was reduced by turbidity epithelium of the foregut and the hindgut is of

PHYSIOLOGY OF THE CLADOCERA 52 4. NUTRITION ectodermal origin (embryologically), whereas On the dorsal side of the midgut, a pair of that of the midgut is of endodermal origin blind diverticula (hepatic ceca) is present in (Chatton, 1920). The stomodeum and hindgut Daphniidae, Ophryoxus (Macrothricidae), and (proctodeum) have a cuticular lining. The intes- in some Eurycercus species. As reviewed by tine may be convoluted or have no convolutions. Korovchinsky (2004), in ctenopods a pair of A blind cecum may be present on the ven- short dorsal ceca is present in Holopedium and tral side of the midgut, before the beginning of Latona parviremis; Sida and Limnosida have one the hindgut, as in chydorids and Acantholeberis dorsal cecum; Diaphanosoma has no dorsal (Fig. 4.7) (Fryer, 1970). Food is transferred ceca. Chydorids also have no dorsal ceca. from the midgut to the blind cecum and accumulates there before being finally evacuated Glands (Smirnov, 1969, 1971). The ventral blind cecum The labrum comprises paired salivary glands. and its function were first described by Claus In Daphnia, the salivary gland on each side con- (1876). In many species of chydorids, the blind sists of a defined number of cells: 2 main cells, 2 cecum on the ventral side of the intestine is substitutional cells, and 32 basal cells (Fig. 3.2) rather long, as in Alona intermedia (Tollinger, (Sterba, 1957a). The labral gland of Simocephalus 1909) and species of the genera Pleuroxus, was described by Cannon (1922). Chydorus, Graptoleberis, Acroperus, and Slime glands are present in the thoracic Camptocercus (Smirnov, 1971). The equivalent limbs: in limb IV of Eurycercus, in which the cecum is short and wide in Eurycercus and slime gland secretions are stained bright blue Leydigia. with Mallory’s stain (Fryer (1962, 1963), and in

FIGURE 4.7 The ventral blind cecum of Acantholeberis curvirostris, showing its filling and evacuation. Source: Fryer (1970).

PHYSIOLOGY OF THE CLADOCERA 4.3 DIGESTION 53

Alonopsis elongata (Fig. 2.1) (Fryer, 1968). The 4.3.2 Peritrophic Membrane secretions of this gland aid the accumulation of food particles. No other species of chydorids The food collected in the intestine is sur- or other cladocerans have yet been investi- rounded by a peritrophic membrane; this gated for the presence of slime glands in tho- membrane has been shown in Daphnia racic limbs. (Chatton, 1920; Fox, 1952; Quaglia et al., 1976) The inner surface of the gut is folded (Fryer, and Acantholeberis (Fryer, 1970). The tubular 1969) and densely covered with numerous peritrophic membrane surrounds the food and microvilli (Fig. 4.8) (Quaglia et al., 1976; extends through the midgut and hindgut. Avtsyn and Petrova, 1986). The microvilli are Chatton (1920) investigated the peritrophic long, occasionally branching, and situated membrane in Daphnia in detail and demon- close to each other, as shown in Daphnia strated that two peritrophic membranes are (Fig. 4.9) (Quaglia et al., 1976). The fine struc- present. The posterior peritrophic membrane is ture of the Daphnia gut is described by Schultz formed, according to Chatton (1920), by and Kennedy (1976a). They conclude that delamination of the proctodeal cuticle and is enzyme secretion is holocrinous. Microvilli attached to the posterior circular furrow also line the inside of the digestive diverticula. between the midgut and the hindgut. It sur- Within the gut lumen of Daphnia, the pH is rounds the posterior part of the anterior peri- 5.66.2 (Lavrentjeva and Beim, 1978). trophic membrane (Fig. 4.10).

FIGURE 4.8 Cross section of the gut of Daphnia showing plications and villi. A, anterior part; one arrow, secreting epitheliocytes (holocrine type of secretion); two arrows, peritrophic membrane; three arrows, chitinous membrane. B, middle part; secreting epitheliocyte; one arrow, macrolemmocrine secretion; two arrows, secretion granules. C, middle part; epitheliocytes with villi. D, protruding secreting epitheliocyte (macroapocrine type of secretion). Source: Avtsyn and Petrova (1986).

PHYSIOLOGY OF THE CLADOCERA 54 4. NUTRITION

FIGURE 4.9 Villi in the gut of Daphnia in longitudinal and transverse section. Source: Quaglia et al. (1976).

The inner ectodermal lining of the esophagus and the entodermal lining of the midgut are also separated by a circular furrow. Both linings are in contact but are not fused. The circular furrow seems to be the site of formation of the anterior peritrophic membrane (Fig. 4.11). Chatton, however, believes that the anterior peritrophic membrane is a lining of the esophagus that is disconnected at the mouth but connected in the area of the furrow between the esophagus and the midgut, turned inside out, and extended by secretions of the midgut. This membrane fully surrounds the flow of food and feces to the anus. It isolates the midgut mucosa from direct contact with ingested food and acts as a kind of dialyzer. The food within the peritrophic membrane FIGURE 4.10 The posterior peritrophic membrane of of Daphnia is moved to and fro by antiperistal- Daphnia magna surrounding the anterior peritrophic sis of the intestinal wall (Chatton, 1920, Fox, membrane. A.r., Amoebidium on the inner side of the rec- 1952). tal peritrophic membrane; C.r., cuticle; M.P.a., part away from anus; M.P.r., rectal peritrophic membrane; S.ep., It is not quite clear whether peritrophic emargination in the gut tissue. Source: Chatton (1920). membranes are discarded with each act of defecation or at each molting. This could easily be observed in representatives of various genera,

PHYSIOLOGY OF THE CLADOCERA 4.3 DIGESTION 55 4.3.3 The Fat Body The fat body is a special organ involved in fat metabolism, as well as having other functions. It has a variable structure. In addition to their role in fat metabolism, fat cells play a role in glycogen metabolism and are the sites of hemoglobin (Hb) formation (Goldmann et al., 1999). The fat body, formed from special cells containing oil drops, was first observed in the Cladocera by Leydig (1860, p. 5152). It may be slightly yellowish or reddish. Leydig also noted that the appearance of these cells depends on the season and the developmental stage. In addition, Hardy (1892) believed that blood cells, which absorb fat, may attach to the FIGURE 4.11 The anterior peritrophic membrane (p. a.) of Daphnia magna in the place of transition of the inner substrata and form fat tissue. esophagus to the midgut, in cross section. C.oe., separat- Special investigations into the fat body of ing ectodermal cuticle of esophagus; M.P.a., peritrophic Cladocera were initiated by Jaeger (1935) and membrane; P, Pansporella in space between the peritrophic continued by Sterba (1956a). Jaeger noted that membrane and the gut wall; S.es., entero-stomodeal emar- oil drops in Cladocera are present not only gination. Source: Chatton (1920). inside the body but they are also included in special cells. According to Jaeger (1935), the fat body is connected to the ovaries and is situ- but has not yet been done. Chatton (1920) ated ventrally along the gut (Fig. 4.12). The noted that at strong contractions of the rectal sequence of development of the fat body and opening, the posterior part of the anterior peri- the ovaries is shown in Fig. 4.13. trophic membrane may be torn off. The structure of the fat body is rather irregular According to Hansen and Peters (1997/ and complex. Some of its cells are scattered and 1998), the peritrophic membranes are formed attached to various organs and membranes. by secretion of the midgut epithelium. They According to Jaeger (1935), the cells of the fat consist of a network of chitin-containing body are situated near to muscles at sites where microfibrils embedded in a matrix of proteins, they contact the membranes that direct blood glycoproteins, and proteoglycans. The thick- flow, on intestinal muscles, and in the abdomen. ness of the peritrophic envelope is 280 nm. The In the abdomen, they comprise the plate between lumen of a peritrophic membrane is termed adductors of the abdomen and two plates on the endoperitrophic space and the space outside both sides of the gut, joining at the abdominal the peritrophic membrane is called the ectoperi- membrane. In thoracic limbs, the fat body trophic space. Hansen and Peters (1997/1998) extends to the base of epipodites and into endo- experimentally determined that the peritrophic podites, but not into exopodites. Plates of the fat membrane of D. magna is permeable to dex- body are present in valves. trans with a molecular weight of up to Fat cells are present on both sides of the 2000 kDa (a Stokes radius of 31 nm) and to membranes that separate blood flows, except latex beads with a diameter 139 nm. for the dorsal membrane (Jaeger, 1935). They

PHYSIOLOGY OF THE CLADOCERA 56 4. NUTRITION are clearly separated from the membranes. The are present in the ovary, the fat content of the head does not contain fat cells. fat body increases. After the start of yolk for- In Cladocera, fat is concentrated in the cells mation in the ovary, the fat body decreases in of the fat body. As long as germinal groups size (Jaeger, 1935; Sterba, 1956a). The cells of the fat body are basophilic; their only inclusions are fat drops; and there is no intercellular substance. Due to the accumulation and expenditure of fat, the cells of the fat body are in either a fat-free or a fat phase. With fat expenditure, the fat cells are completely reduced. Jaeger (1935) observed that during this process part of the cell may be reduced. At the same time, substituting cells are formed. Restoration of the fat body occurs through the action of blood cells (Jaeger, 1935): blood cells loaded with fat may settle onto tissues and turn into cells of the fat body. Jaeger (1935) did not observe any division stages in the substituting cells or the fat cells, but assumed that division stages are initially present in both. Sterba (1956a) presented data on “the life cycles” of fat cells in response to feeding on material rich in fat or rich in carbohydrate (Fig. 4.14). In Daphnia, fat globules and other particles are carried from the gut lumen through the gut wall by blood corpuscles (Hardy, 1892) and via cells of the anterior region of the midgut (Hardy and McDougall, 1894). According FIGURE 4.12 Position of fat body in Daphnia. Fat to Jaeger (1935), osmic acid stains both fat body is shaded. En., endopodite; Ep., epipodite; Ex., exo- in Daphnia blood cells and minute fat podite; Seitl.Fl., lateral lobe. Source: Jager (1935). particles (hemokonia) in hemolymph, thus

Maxim.

Stark

Gering

Ausgangs zustand 0 1020304050

FIGURE 4.13 Sequence of fat body (continuous line) and ovaries (interrupted line) development. Abscissa, days. Source: Sterba (1956a).

PHYSIOLOGY OF THE CLADOCERA 4.3 DIGESTION 57 making the hemolymph turbid. This indicates Despite much information on lipids in a distribution of fat from the alimentary canal Cladocera now being available, no further throughout the body both in blood cells and investigations have been made into the fat as minute drops. distribution.

FIGURE 4.14 Fat cell cycle in Daphnia. Left, with food rich in carbohydrate; right, with food rich in fat. Black drops indicate fat. Lower arrow to the right indicates the response to food shortage. Source: Sterba (1956a).

PHYSIOLOGY OF THE CLADOCERA 58 4. NUTRITION

4.3.4 Histo-Hemolymphatic Intestinal Atropine blocks the actions of acetylcholine Barrier and physostigmine (Obreshkove, 1941a). Inhibition of peristalsis by atropine has also Beim and Lavrentieva (1978; 1981) investi- been shown in S. crystallina (McCallum, 1905), gated the pH in the intestinal canal of Daphnia and by epinephrine, quinine, quinidine, metra- by staining it with eosin, Congo red, methyl zol, and cocaine in D. magna (Sollman and red, neutral red, and uranine. The stained Webb, 1941). 1 material passed through the gut in 20 min. In Daphnia, calcium ions (Ca2 ) cause con- During this time, these fluorochromes did not tracture of the intestine, whereas potassium 1 stain hemolymph, oil drops, or the brood ions (K ) cause relaxation of intestinal muscles pouch. Therefore, the authors suggested the (Ermakov, 1936). presence of a histo-hemolymphatic intestinal barrier to these stains. They also stated the possibility that organic toxicants may stay within 4.3.6 Defecation the intestine and not penetrate the gut wall. See The formation of feces is confined to the also the data of Fonviller and Itkin (1938) on posterior region of the midgut and to the proc- the permeability of integuments in Section 8.3. todaeum (Hardy and McDougall, 1894). These authors noted that peristalsis in the procto- 4.3.5 Peristalsis daeum is independent of the central nervous system as it takes place in the isolated Peristalsis and antiperistalsis of the gut can proctodaeum. be easily observed and were reported for Defecation occurs very frequently in Daphnia as early as 1894 by Hardy and Cladocera. About 1020 min after the intestine McDougall. Hardy and McDougall note espe- is filled, it is evacuated. Defecation was ini- cially (1894, p. 45) that peristaltic movements tially described in Daphnia by Hardy and of the proctodaeum “undoubtedly lead to the McDougall (1894), who noted the presence of a entrance and exit of water.” sphincter muscle at the junction of the midgut Rankin (1929) observed strong reverse peri- and the hindgut. stalsis in D. magna and Simocephalus serrulatus In chydorids and Acantholeberis, food is fed on stained food. passed through midgut, transferred to the The caeca situated on the dorsal side of the blind cecum, and evacuated when the cecum anterior gut also make rhythmic contractions, is filled and dilated (Smirnov, 1969, 1971; 610 times/min in D. magna and much less Fryer, 1970). The discharge of feces is followed frequently in S. serrulatus (Rankin, 1929). by anal water intake. With regard to littoral Peristalsis is stimulated by pilocarpine hydro- species, the intestine is evacuated every 7 min chlorate and some other purgatives in Sida in E. lamellatus (Smirnov, 1962) and every (McCallum, 1905) and by pilocarpine, mecholyl, 1119 min in Acantholeberis (Fryer, 1970). physostigmine, and guanidine in D. magna Fryer (1969, p. 371) reported that L. leydigii (Sollman and Webb, 1941). Strong contractions of “was watched for 2 min 5 s as it fed, during the intestine are caused by acetylcholine or phy- which time it discharged 15 fecal ribbons. sostigmine (eserine) in D. magna (Obreshkove, Rough calculations indicate that at such a rate 1941a) and by acetylcholine, carbaminoylcholine, of ejection the entire alimentary canal could be and prostigmine in Simocephalus (Mooney and evacuated in less than 6 min, and possibly con- Obreshkove, 1948). siderably less.”

PHYSIOLOGY OF THE CLADOCERA 4.4 DIGESTION OF PARTICULAR SUBSTANCES 59

For daphnids (mainly planktonic species), 4.4 DIGESTION OF PARTICULAR the intestine is evacuated every 3135 min in SUBSTANCES D. magna, D. longispina, and S. vetulus, and 2024 min in C. quadrangula and M. brachiata Cladocera feed on various algae, bacteria, (Esipova, 1971). In Bosmina, it takes 10 min for and organic detritus at various stages of the intestine to fill with food; the average daily decomposition. These food sources contain ration is about 100% of its weight; and a por- protein, carbohydrates, lipids, and indigestible tion of food is digested in 3035 min materials. Protein, lipids, and soluble carbohy- (Semenova, 1974). In M. macrocopa, the intes- drates are definitely digested. A diagram of tine is filled in about 20 min and fully evacu- the intake and transformation of food sub- ated in less than 50 min (Morales Ventura stances in an adult daphnid is shown in et al., 2011). In periods of food scarcity, the Fig. 4.15 (based on Hallam et al., 1990). intestine remains full for longer periods (Fryer, Digestion and assimilation of food occurs in 1968). Such a short retention of food in the the mesenteron. This tissue is not differenti- intestine is somewhat compensated for in chy- ated, but Hardy and McDougall (1894) drew dorids and in many macrothricids by elonga- attention to the unique and remarkable fact tion of the intestine, through making that digestion takes place in the middle region convolutions. and absorption in the anterior region. Feces form cylindrical columns that do not Antiperistalsis and peristalsis are involved in immediately dissolve in water; they settle to this process. Feces formation occurs in the pos- the bottom. terior region. Further comparative studies of living clado- The chemical composition of D. magna is cera are desirable. much influenced by the chemical composition Intestinal evacuation in D. magna is acceler- of its food (Stepanova et al., 1971). Thus, the μ ated by incubation in 100 250 MNa2CO3, highest content of protein, fat, and carbohy- 100 μM NaNO3, or 1 μM KCl (Gophen and drate was found in Daphnia fed on yeast. Gold, 1981). Gut clearance in D. magna is more Cladocera also may accumulate various sub- efficient when green algae are available than in stances, including xenobiotics. clear water. The enzymes that take part in cladoceran Despite food being retained in the intestine digestion have been investigated since 1929 only for short periods, cladocerans grow nor- (Rankin, 1929). An amylolytic enzyme has mally and produce young. Thus, digestion for been identified in D. magna and S. serrulatus a short period provides sufficient material to (Rankin, 1929), and proteinase and polypepti- sustain normal life. dases were reported in D. pulex (Hasler, 1937). In the anterior part of the intestine, holocrine In Polyphemus (a mixed herbivore and carni- secretion takes place; in the middle part, macro- vore), proteinases, amylases, and lipases were lemmocrine and macroapocrine secretion occur identified (Dehn, 1930; Hasler, 1937). (Avtsyn and Petrova, 1986). We must remem- Much later (Schoenberg et al., 1984), endoge- ber that the holocrine secretion is transforma- nous cellulase was detected in the gut of tion of the whole cell for secretion and apocrine Acantholeberis, Daphnia,andPseudosida;asno secretion involves breaking off the ends of microbial gut flora was found, this enzyme gland cells for secretion, while the functioning enables cellulose to be digested. Cellulose diges- part of the cell with the nucleus is retained and tion has also been reported in Daphnia by continues the same kind of secretion. Lampert (1987) and De Coen and Janssen (1997).

PHYSIOLOGY OF THE CLADOCERA 60 4. NUTRITION

FIGURE 4.15 Metabolic flow diagram for an individual adult daphnid. Source: Hallam et al. (1990).

4.4.1 Protein Metabolism Proteases (trypsin, chymotrypsin, elastase, and Protein is obtained by Cladocera from algae cysteine protease) were also found in whole body and seston. Indeed, it was shown with refer- homogenates of M. macrocopa (Agrawal et al., ence to D. obtusa that the amounts of released 2005a). Their activity was significantly inhibited nitrogen and phosphorus are generally propor- by an extract of the blue-green alga Microcystis tional to the amounts ingested with food algae aeruginosa.InD. carinata, proteinase activity was (Sterner and Smith, 1993). on average, 0.42 U/mg protein/min, that of chy- The principal final product of protein motrypsin was 0.49 U/mg protein/min, and that metabolism in Cladocera is ammonia (Parry, of trypsin was 0.21 U/mg protein/min 1960); of the secondary products, the most (Kumar et al., 2005b). Peptide metabolites of abundant is urea. Microcystis that inhibit the trypsin-like activity in Proteolytic enzymes (trypsin, β-galactogenase, Daphnia were identified by Czarnecki et al. and esterase) were found and measured in (2006). homogenates of D. magna by De Coen et al. For D. magna, algae are a better source (1998). The activities of these enzymes decreased of nitrogen than bacteria (Schmidt, 1968). during 90-min incubations with various in- According to this author, an adult D. magna organic and organic toxicants. Von Elert et al. consumes 0.370.47 μg N from green algae per (2004) also identified proteases in homogenates 24 h per μg body N and excretes 0.170.19 μg/ of D. magna: two major ones (trypsin and chy- 24 h of N per μg body N. Release rates in motrypsin) and nine others. The two major pro- Daphnia were measured: nitrogen was 33.42 teases account for up to 83% of the proteolytic and phosphorus was 11.24 μg/mg C/h. No activity of the gut contents. Trypsin is strongly inter-species differences were found (Pe´rez- inhibited by N-p-tosyl-lysine chloroketone and Martı´nez and Gulati, 1998/1999). 4-amidinophenylmethanesulfonyl fluoride; chy- It has been suggested that Hb functions as motrypsin is strongly inhibited by chymostatin. both a respiratory protein and a protein store Both activities have alkaline optima. (Rudiger and Zeis, 2011).

PHYSIOLOGY OF THE CLADOCERA 4.4 DIGESTION OF PARTICULAR SUBSTANCES 61

4.4.2 Phosphorus Metabolism Macroergic phosphorus compounds (adenine nucleotides: ATP, ADP, and AMP) were found In Cladocera, phosphorus forms part of (Romanenko et al., 2004) to decrease consider- nucleic acids and phospholipids. Other poten- ably (by two to three times) during the growth tial pools of phosphorus in Cladocera are of an enrichment culture of M. macrocopa. minor metabolites (adenosine triphosphate and With P-deficient food (C:P of c. 700), adenosine diphosphate), reduced nicotinamide D. magna had a higher alkaline phosphatase adenine dinucleotide, reduced nicotinamide activity compared with the amount present fol- adenine dinucleotide phosphate, and calcium- lowing phosphorus-rich food (C:P of c. 100); associated phosphorus (hydroxyapatite) in poor phosphorus nutrition also lowers the integuments (Vrede et al., 1999). In D. galeata, activity of alkaline phosphatase in released 35 69% of the total phosphorus content is materials (containing dissolved enzymes). associated with nucleic acids (Andersen and In D. pulex, the daily renewal rate of phos- Hessen, 1999; Vrede et al., 1999). phorus in the body ranges from 35% to 60% Cladocera are commonly deficient in phos- (i.e. 3560% of phosphorus is turned over phorus. Inorganic phosphate is taken from each day) (Lehman, 1980). Most of the liber- solution by D. schødleri, mostly through the ated phosphorus is from freshly assimilated epipodites (Parker and Olson, 1966). With a material, although a fraction is from phospho- longer exposure, phosphorus accumulates in rus incorporated into tissues. Phosphorus con- muscles, a cerebral ganglion, and the ovaries. μ tent in D. pulex was found to be higher when it The addition of 3 M AgNO3 to the surround- is fed with green algae rich in phosphorus ing water reduced the uptake of phosphorus (Lehman and Naumoski, 1985). as a result of silver impregnating the surface of The phosphorus release rate in D. galeata epipodites. varies from 0.161.18 μg P/mg C/h in juve- Having determined that D. magna can niles and 0.060.96 μg P/mg C/h in adults extract phosphorus from food both rich and (Vadstein et al., 1995). scarce in phosphorus, Sterner and Schwalbach (2001, p. 415) asked the following further questions: “In what chemical form is phosphorus 4.4.3 Lipid Metabolism stored? Where is it stored? How does it relate to the entire phosphorus budget? Are some There is an extensive record of investiga- species better able to exploit temporal variations into the role of lipids in Cladocera digestion due to enhanced storage capabilities? tion. Some publications report the chemical And, perhaps most fundamentally, how composition of lipids in Cladocera, whereas should we relate growth studies on simplified, other studies investigate the sources of lipids constant foods to in situ conditions where in Cladocera and try to answer the question of these parameters vary in time and space?” whether Cladocera transform the ingested According to Vrede et al. (1998, 1999), about lipids. 67% of phosphorus is located in the body of The lipid composition of Cladocera is Daphnia, 24% in eggs, and 14% in the carapace. shown in Tables 3.43.8. Total fatty acid con- The latter percentage was confirmed by Vrede centrations in herbivorous cladocerans is et al. (1999) for the carapaces of D. magna and 117147 mg/g C, and 104 mg/g C in preda- D. galeata. Phosphorus present in the carapace tory Bythotrephes (Persson and Vrede, 2006). of Daphnia is discarded during molting and is Cladocera obtain lipids from their food. thought to be inorganic (Vrede et al., 1998). Goulden and Place (1993) determined that de

PHYSIOLOGY OF THE CLADOCERA 62 4. NUTRITION novo synthesis of fatty acids by daphnids is less As shown in Daphnia, PUFAs are compo- than 2%. According to D’Abramo (1979), nents of cell membranes and precursors of Cladocera, in particular M. macrocopa,are eicosanoids (hormone-like mediators) (Schlotz entirely dependent upon the sources of fatty and Martin-Creuzburg, 2011). acids in their diet. Farkas et al. (1981) found that Lipid composition has been determined in D. magna cannot form docosapolyenoic acid. total lipid extracts from whole dried Cladocera Hardy (1892) observed that in Cladocera fat (Arts et al., 1992; 1993; Sushchik et al., 2002). globules and other particles are carried from According to von Elert et al. (2002), D. galeata the gut lumen through the gut wall by blood can convert docosahexaenoic acid and C18 cells. However, these observations do not seem PUFAs into EPA (20:5ω3). α-Linolenic acid is to be followed up. Hardy and McDougall the limiting PUFA, as EPA cannot be converted (1894) also noted that fat globules are ingested into α-linolenic acid. Thus, these algal sources by columnar cells of the anterior region of the (i.e. either α-linoleic or EPA) are usable and midgut, including the liver diverticula. nonsubstitutable for Daphnia,andα-linolenic Farkas et al. (1984) suggested that D. magna acid may be a source of EPA. cannot overwinter in an active state owing to a Following feeding with EPA-free or EPA- failure to adapt the phospholipid composition enriched Scenedesmus (green alga), 18:3ω3, of its membrane and its physical state to the 18:4ω3, 20:3ω3, and 20:4ω3 tissue concentrations temperature (see Section 15.1). were higher in D. galeata than in D. hyalina, Pelagic Cladocera show seasonal changes in indicating that assimilation and biosynthesis of the lipid content that are clearly dependent on PUFAs is higher in D galeata (von Elert, 2004). the available algal food. In Humboldt Lake As Schlechtriem et al. (2006) summarized: (Canada), triacylglycerol was found to be the Daphnia feeding on highly unsaturated fatty principal component in D. pulex, especially acids [HUFAs; e.g. EPA (20:5n3) or arachidonic during SeptemberMay, after the decline of acid (20:4n6)] become enriched with these blue-green algae (Arts et al., 1992). An abun- fatty acids; docosahexaenoic acid (22:6n3) is not dance of bacteria following the collapse of a accumulated but is converted to EPA; and blue-green algal bloom resulted in increased α-linolenic acid (18:3n3) and linoleic acid lipid content in C. sphaericus and Diaphanosoma (18:2n6) are used as precursors of EPA and ara- leuchtenbergiana. The authors concluded that chidonic acid (ARA). there is an outstanding role for cryptophytes (Cryptomonas and Rhodomonas) as lipid sources, while Daphnia were at near starvation during Littoral Cladocera the blue-green algal bloom. The sources of lipids in littoral Eurycercus Significant seasonal changes in the relative were determined by Desvilettes et al. (1994, content of fatty acids were measured in 1997). These authors found a striking similarity D. galeata, Leptodora, and Bythotrephes in the between the lipid composition of Eurycercus Middle Volga (Fig. 4.16) (Bychek and and its seston food (Fig. 4.16). The food of Guschina, 2001). For example, the content of Eurycercus consisted of Cryptomonas, diatoms, linolenic acid in Daphnia was especially high in dinoflagellates, ciliated strombiids, and bacte- July. Further, the content of C16:0 (palmitic ria. Cladoceran triacylglycerols comprise odd- acid) was especially high from June to August, branched fatty acids, a high percentage of ω6 similar to that in seston, in both herbivorous PUFAs, and significant levels of 18:4ω3, 20:5ω3, Daphnia and the predators Leptodora and and 22:6ω3. The latter three were thought to Bythotrephes. be derived from Cryptomonas, whereas the

PHYSIOLOGY OF THE CLADOCERA 4.4 DIGESTION OF PARTICULAR SUBSTANCES 63

30 FIGURE 4.16 Seasonal changes in the content of lipid classes in Daphnia pulex. AMPL, acetone-mobile polar lipids; PL, 25 phospholipids; ST, sterols;. TAG, triacylgly- ) cerols. Abscissa, months. Source: Arts et al. –1 20 (1992).

g. animal g. 15 TAG µ

10 ST+AMPL PL

Lipid class ( 5

OTHER 0 MJ JASONDJFM odd-branched fatty acids came from consumed found to be directly proportional to the fat in bacteria. their food algae (green algae, Ankistrodesmus No similar investigations have been done in and Selenastrum; Fig. 4.17)(Cowgilletal.,1984); any other littoral Cladocera. this effect lasted for five generations. Only C14:0, C14:1, and C18:0 were more concentrated Pelagic Cladocera in Daphnia than in their algal food (Cowgill There is good evidence that PUFAs are et al., 1984). In Daphnia fed with yeast, the important components of Cladocera food. monounsaturated fatty acid (MUFA) concentra- Similarities between phytoplankton fatty acids tion was the highest, compared with those fed and the neutral lipids of Daphnia were noted with other foods; the MUFAs were mainly by Bourdier and Bauchart (1986/1987), namely C16:1ω7 and C18:1ω9. If the food was Chlorella an abundance of 14:0, 16:0, 16.1, and 18:0 (from seawater), the PUFA concentration was PUFAs. The growth of Daphnia is favored by comparatively high (Huang et al., 2001). For ω3 HUFAs from lipid reserves or the diet Daphnia rosea, HUFAs comprise a highly favor- (DeMott et al., 1997). able food component (Park et al., 2003). A very high correlation was found between In D. cucullata, the level of PUFAs 22:5ω6 D. galeata growth and the seston content of and 22:6ω3 was determined to be 1.76% by EPA (20:5ω3), a HUFA (Mu¨ ller-Navarra, weight of total fat (lipids), 23.9% of monosatu- 1995a). Sixty-nine percent of the variation in rated fatty acids (18:0, 9.3%), and 28.4% of sat- D. magna growth was explained by the algal urated fatty acids (16:0, 12.6%) (Farkas, 1970). ω3 PUFA content (Park et al., 2002). In D. pulex, triacylglycerols are the dominant The lipid content of some planktonic clado- lipid class, followed by phospholipids and cerans, and its fractional components, was sterols (Arts et al., 1993). determined by Lizenko et al. (1977) (Table 3.7), Weers et al. (1997) found that C16:4ω3 and who showed that fatty acid percentages C22:5 were in lower abundance in D. galeata change with age (Table 3.8). than in their algal food. For various different The total lipid content of D. magna depends amounts in their food, Daphnia tends to con- on the composition of its food (Cowgill et al., tain high levels of EPA (C20:5ω3). Daphnia 1984). The quantity of fat in D. magna was demonstrates a low rate of [14C]linoleic acid

PHYSIOLOGY OF THE CLADOCERA 64 4. NUTRITION

FIGURE 4.17 Fat content of Daphnia vs. fat content of the consumed algae. Source: Cowgill et al. (1984).

(C18:2ω6) and [14C]linolenic acid (C18:3) con- 13 mg/L (Makhutova et al., 2009); EPA is an version into C20 PUFAs. Thus, the latter are ω3-type PUFA. trophically important. There is a differential response of D. magna Brett and Mu¨ ller-Navarra (1997) and to different fatty acids consumed in food, as Demott and Muller-Mavarra (1997) found that shown by Becker and Boersma (2005, 2007). the efficiency of cladoceran growth is propor- PUFAs are predominantly stored, whereas sat- tional, first of all, to the availability of fatty urated fatty acids (20:0; EPA) are metabolized. acids, and, in second place, to HUFA, and In eggs, EPA is present in higher concentra- much less so to phosphorus, nitrogen, or car- tions than arachidonic acid. The accumulated bon (Fig. 4.5); EPA is the most significant food saturated fatty acids are used during periods component. Zooplankton growth and egg pro- of inadequate food supply, and are also passed duction strongly correlates with the 20:5ω3to into the eggs. Egg production is a major drain carbon ratio (Mu¨ ller-Navarra et al., 2000). on Daphnia fatty acids. Changes in fatty acid Daphnia growth is favored by HUFAs from concentrations are smaller compared with lipid reserves or the diet (DeMott et al., 1997). phosphorus changes. PUFAs present in phytoplankton, EPA espe- In newborn D. magna, there is a high cially, are important food ingredients for content of free sterols and phospholipids somatic growth and egg formation in Daphnia (phosphatidylcholine, phosphatidylethanol- (Ravet et al., 2003). Daphnia growth is also amine, and sphingomyelin) (Bychek and favored by EPA at a threshold concentration of Guschina, 1999). These authors also found that

PHYSIOLOGY OF THE CLADOCERA 4.4 DIGESTION OF PARTICULAR SUBSTANCES 65 the content of triacylglycerols decreases with 4.4.4 Carbohydrate Metabolism the growth of D. magna fed on Chlorella. Fatty acid desaturation is lower in newborns, and The general scheme of carbohydrate metab- wax esters are only detectable in adults. Fatty olism of Cladocera conforms to that shown in acid content has been determined by Lizenko Fig. 3.3 (Hohnke and Scheer, 1970). The diets et al. (1977) (Table 3.4) and Bychek and of Cladocera mostly contain an excess of Guschina (1999). carbon. In Daphnia spp., the EPA content varies least In D. magna and S. serrulatus, the presence among the ω3 PUFAs; less so than in the food of an amylolytic enzyme was demonstrated by available to them (Mu¨ ller-Navarra, 2006). Rankin (1929). The ingested starch, stained Mu¨ ller-Navarra fed Daphnia with a Nitzschia blue with iodine, changed color to pinkish as it culture and published the fatty acid profiles of passed down the gut, thus indicating a change this alga and of Daphnia:inDaphnia, they from starch to dextrin. reported a higher content of the fatty acids Littoral Cladocera that are present in high concentrations in the diatom. The only available data on carbon assimila- On a PUFA-rich diet, D. magna accumulate tion by littoral Cladocera are likely to be those more ω3 PUFA at 15C than at higher tempera- obtained by Infante (1973). E. lamellatus fed μ μ tures (Sperfeld and Wacker, 2011). with Scenedesmus incorporated 12 g C/100 g μ μ Copper may induce lipid accumulation, C animal/24 h; much less (2.5 g C/100 gC especially at high concentrations such as animal/24 h) was incorporated when it was 12 μg/L Cu in later generations (e.g. the four- fed with Staurastrum. teenth) of D. magna (Bossuyt and Janssen, 2004). Pelagic Cladocera Carbon assimilation in filter-feeders Abnormal Fat Metabolism (Daphnia and Sida) is 1.74 μg C/100 μg C ani- Fat droplets are commonly present in the mal/24 h, depending on the type of algal food body of cladocerans and are used as a stock (Infante, 1973). Stable isotope analysis showed material. However, Daphnia that consume food that D. hyalina derives almost all of its body deficient in a vitally necessary material may carbon from algal sources (Grey and Jones, accumulate pink fat droplets and die soon 2007). thereafter. Flu¨ ckiger (1951) described a case in Urabe (1991) determined the distribution of which in a culture of D. magna fed with green carbon use in subsequent instars of B. longiros- algae, the daphnias became filled with pink fat tris and showed that the proportion of assimi- droplets grouped abundantly around the intes- lated carbon used for respiration increases in tine (from the first third section to the anus), in subsequent instars and that used for growth thoracic limbs, and around nephridia. In such decreases (Fig. 4.18). animals, reproduction stopped, the ovaries Cellulose is also digested and assimilated could not be discerned, molting happened with an efficiency of , 11.5%, as found for very rarely, and they died within 2 days. The Acantholeberis curvirostris, D. magna, and addition of yeast or egg yolk normalized the Pseudosida bidentata (Schoenberg et al., 1984). situation and the animals continued with par- Carbon assimilation in D. magna was estimated thenogenetic reproduction. However, it was to be approximately 1.8 μg C/ind./h (Myrand not possible to conclude which substance defi- and de la Nou¨ e, 1983). Carbon release per sin- ciency caused the metabolic disturbance. gle Daphnia containing 1 γC was calculated to

PHYSIOLOGY OF THE CLADOCERA 66 4. NUTRITION

0.05 mg Cl–1 excretion of dissolved organic carbon was 1.00 higher in Daphnia fed on the high C:P algae 0.75 (13.4% of body C/day), in comparison with 0.50 Daphnia fed on low C:P algae (5.7% of body 0.25 C/day). 0.00 Lampert (1978) determined that 1017% of 0.10 mg Cl–1 the carbon contained in algae ingested by 1.00 D. pulex is transformed and liberated as dis- 0.75 solved organic carbon. Most of the carbon is 0.50 liberated into the environment from algal cells: 0.25 4% from algal cells swallowed whole; and the 0.00 rest resulting from Daphnia secretions and 0.25 mg Cl–1 leaching from their feces. 1.00 Thus, D. magna must dispose of excess 0.75 ingested carbon by means of respiration and a 0.50 higher excretion rate of dissolved organic car- 0.25 bon in order to maintain their carbon balance 0.00 and support their homeostatic carbon content. Juvenile D. magna liberate 5572% of their car- 2.50 mg Cl–1 M 1.00 bon as dissolved organic carbon and 937% of 0.75 R the total carbon loss as carbon dioxide (CO2). 0.50 Carbon lost by D. magna as dissolved B organic carbon consists mainly of the high 0.25 G 0.00 molecular weight organic fraction (He and 1234567 Instar number Wang, 2006a). For adults, Total loss of organic carbon and high-molecular organic com- FIGURE 4.18 Carbon allocation in Bosmina longiros- pounds were 4464% and 2047%, respec- tris. Proportion of carbon allocated to body growth (G), reproduction (B), respiration (R), and molting (M) at four tively. The release of some excess carbon as food concentrations. Source: Urabe (1991). CO2 by D. magna was also noted by Jensen and Hessen (2007). be c. 0.26 γC/day (Metz, 1973). Blue-green algae are characterized by a low sterol content, 4.4.5 Metabolism of Various Elements which leads to a low carbon transfer efficiency In Cladocera, the metabolic significance of into Daphnia; this constrains cholesterol syn- some elements has been investigated. It has thesis and thus growth and reproduction (von been shown for D. magna that Na, Ca, Fe, Se, Elert et al., 2002). The obvious excess of carbon Zn, and Cu are the essential elements, while in the food of D. magna and its fate during cadmium and U are not (Barata et al., 1998). digestion were investigated by Darchambeau et al. (2003). Daphnia were fed cultures of the green alga Selenastrum that differed with Calcium respect to their C:P ratios (400 and 80). The Cladocera derive calcium from food and respiration rate of Daphnia fed with high C:P water. The lowest level of calcium in water algae was significantly higher than that of in which D. magna has been recorded is Daphnia fed with low C:P algae. The rate of 0.10.5 mg/L (Hessen and Rukke, 2000a,

PHYSIOLOGY OF THE CLADOCERA 4.5 ASSIMILATION 67

2000b), and 02 mg/L for D. galeata (Rukke, maximum after 12 h, and depuration is slow 2002). There are also interpopulational differ- (Schultz et al., 1980). ences in tolerance to a low ambient calcium concentration, as has been determined for D. galeata (Rukke, 2002). The calcium content of D. magna decreased 4.5 ASSIMILATION from 4.2% to 1% DW over a range of 0.250.013 mM Ca in the culture medium; sat- By averaging data from various authors, urated calcification was reached at a calcium Sushchenya (1975) calculated the average concentration of . 0.13 mM; about 40% of cal- assimilation of food consumed by freshwater cium is lost with an old exuvium (Alstad et al., planktonic cladocera to be 58.4% of that 1999). ingested. In addition, the percentage assimilation of consumed food (green algae, bacteria, Strontium pink yeast, and bacteria) was determined Strontium is concentrated by cladocera to a radiometrically to be 4056.7% in Daphnia and much lower degree than is calcium (Marshall Simocephalus (Fedorov and Sorokin, 1967). In et al., 1964). Similar to calcium, 95% of stron- Daphnia, assimilation is highest for yeast and tium is accumulated in the exoskeleton and is assimilation is highest in Simocephalus fed bac- eliminated at each molting, thus dropping to teria. The assimilation efficiency of D. pulex is minimum; thereafter, the content of Sr gradu- 35% for Ankistrodesmus falcatus (a green alga) ally increases, as shown in D. magna by and 11% for Aphanizomenon flos-aquae (a cyano- Marshall et al. (1964) (Fig. 3.10). bacterium) (Holm et al., 1983). Bythotrephes ingests the soft tissues of consumed Daphnia Selenium and the assimilation efficiency is estimated to The minimum essential concentration of Se be 85% (Lehman, 1993). to support a culture of D. pulex and D. magna Anderson et al. (2005) highlighted the fact was determined to be 0.1 ppb (Keating and that the food available for herbivorous Dagbusan, 1984). Below this concentration in Cladocera is frequently nutritionally unbal- the environment, Daphnia progressively lost anced and its composition fluctuates. distal antennal segments at molting, mani- Therefore, to support overall homeostasis it is fested cuticle deterioration, and had a short- necessary to release excess substances, espe- ened life span. Elendt (1990) demonstrated cially carbon, which may be done either as that in Daphnia necrotic bands are formed in selective absorption in the gut or the excretion basal segments of antennal rami in the absence of excess substances in an organic form. With of selenium, and that distal parts of antennal reference to carbon, regulation of homeostasis branches are rejected and not restored in sub- of the chemical composition has been investi- sequent molting. Selenium deprivation causes gated in D. magna by Darchambeau et al. alterations in mitochondria and the sarcoplas- (2003). However, the assimilation values mic reticulum, and complete lysis of muscle also depend on environmental conditions. fibrils in antennal muscle cells. This damage According to experimental data obtained by periodically occurs in the absence of Se, a Buikema (1975), the percentage assimilation constituent of glutathione peroxidase, which [i.e. the total energy accumulated during protects cells and their membrane polyunsatu- growth (for young specimens) and used in res- rated lipids against peroxidation. Uptake of piration] in Daphnia is highest at illumination selenium by unfed D. pulex reaches a above 7 foot-candles (fc; approximately

PHYSIOLOGY OF THE CLADOCERA 68 4. NUTRITION

75.3 lx), but is higher with polarized light than feeding on the soft tissues of Daphnia, assimila- with nonpolarized light of the same intensity. tion efficiency was estimated to be 85% The feeding ratio (i.e. food units spent per (Lehman (1993). unit of weight increment) of algal food for Hallam et al. (1990) proposed a model sum- D. magna is about 4 but has been shown exper- marizing the flow of assimilated resources in imentally to be 6 (Gajewskaja, 1945). In the adult daphnid, with special reference to Daphnia and Moina fed on green algae, the per- lipids as a labile energy source (Fig. 4.15). This centage assimilation was 4860% of the con- model has greatly contributed to the organiza- sumed food; the weight gain was 16% in tion of various relevant data available in the Daphnia and 7.6% in Moina (Ostapenya et al., literature. 5 5 1 1968). K1 P/R and K2 P/R T were found to be 22.5% and 49.9% in Daphnia, respectively, and 11% and 23% in Moina, respectively 4.5.1 Digestion Efficiency (Ostapenya et al., 1968), where P is weight gain, R is the ration, and T is the energy Gut Passage Time expenditure for metabolism. Food stays in the cladoceran intestine for Food items are of varying composition and only a few minutes. The intestine has been assimilation by cladocera is dependent on the shown to be evacuated every 7 min in E. lamel- composition of the consumed food. In experi- latus (Smirnov, 1962); every 1119 min in ments on feeding of D. magna with green algae Acantholeberis (Fryer, 1970); every 3135 min in (Scenedesmus), the phosphorus content per DW D. magna, D. longispina, and S. vetulus; and of Daphnia decreased when the algal content every 2024 min in C. quadrangula and M. bra- was low (DeMott et al., 1998). When the ratio chiata (fed on detritus) (Esipova, 1971). of C:P in the food increased from 120 to 900, Cauchie et al. (2000) summarized the data of carbon assimilation declined and the phospho- various authors on gut passage time in rus content in D. magna decreased from 1.47% Daphnia and Bosmina species to be 46 min in to 1.08%. The assimilation of algal food by D. galeata,1525 min in D. longispina, Cladocera differs for different groups of algae 255 min in D. magna, , 260 min in D. pulex, (Schindler, 1971): high values (in μg/h/10) 1940 min in D. pulicaria,56minin D. rosea, were determined for Oscillatoria (blue-green and 510 min in B. longirostris. The only report alga; 5.8), Asterionella (diatom; 6.8), of a longer retention time was for D. schoedleri, Ankistrodesmus (green alga; 7.2), and which has a gut passage time of 135 min. Cryptomonas (Cryptophyceae; 7.7). In chydorids, food passes through the intes- At higher food concentrations, the assimila- tine in about 57 min (Smirnov, 1969, 1971, tion efficiency decreases, as has been shown 1974), while food passes through the intestines for D. magna (Schindler, 1968) and D. galeata of planktonic Daphnia spp. in 2554 min (Urabe and Watanabe, 1991), but the assimila- (Geller, 1975; Pavlutin, 1983; Haney et al., tion efficiency by D. galeata becomes higher 1986; Christofersen, 1988); Murtaugh (1985) with decreasing food concentration, which reported a retention time of 4106 min in affects the carbon balance (Urabe and D. pulicaria. Pavlutin (1983) observed the rate Watanabe, 1991). In addition, the clearance of food evacuation to increase in larger ani- rate decreases with increasing food concentra- mals and at higher food concentrations. tion. Assimilation efficiencies reach c. 50% in Stained material passes through the intestine B. longirostris, D. longispina, C. quadrangula, and of Daphnia in 20 min (Beim and Lavrentieva, C. sphaericus (Lair, 1991). In Bythotrephes 1981).

PHYSIOLOGY OF THE CLADOCERA 4.5 ASSIMILATION 69

Digestion in cladocera is surprisingly efficient. The tonus (or contraction) of the circular mus- With this in mind, Fryer (1970, p. 266) noted: “A cles of the intestine of Daphnia was found by puzzling feature of the Anomopoda is the rapid- Flu¨ckiger (1952) to be increased by the sympatho- ity with which the food passes through the gut. mimetics L-adrenaline bitartrate, D-adrenaline- A further, related, problem ...is the effect of the bitartrate, L-noradrenalinebitartrate, L-ephedrine tremendous dilution of the gut contents brought hydrochloride, and oxyphenylethanomethyla- aboutbythegreatintakeofwater.” mine tartrate. This high efficiency may depend on the fol- As for digestive enzymes, amylases and lowing facts: lipases have been identified in homogenates of Daphnia peptidases (Dehn, 1930; Hasler, 1937; 1. The dense cover of microvilli in the Rodina, 1950). The major proteinases in intestinal lumen (Figs. 4.1 and 4.6) greatly D. magna are trypsins and chymotrypsins increases the gut surface. Quaglia et al. (Agrawal et al., 2005b); these were identified (1976) propose that food is absorbed from in a gut homogenate. Agrawal et al. (2005b) the lumen of the midgut in a digested form also demonstrated that the blue-green alga and liquids are reabsorbed in the Microcystis produces several inhibitors that dif- proctodeum (hindgut); fer in their specificity for these enzymes. 2. The inner surface of the intestine is plicate In Simocephalus, the pH of the anterior part (or folded), which further increases the of the midgut is acidic and it changes to alka- inner surface; line nearer to the anus (Rankin, 1929). In 3. In addition to the huge surface area created Daphnia, the pH of the anterior part of the by microvilli, the efficiency of digestion is midgut is 6.06.2 (Rodina, 1950; Beim and increased through intensive mixing of the Lavrentieva, 1981; Beim et al., 1994), i.e. lower food by peristalsis; than in the external medium, and increases to 4. Food in the intestine is intensively mixed by 7.4 in the hindgut (Rodina, 1950). Secretions of anal water intake; the hepatic ceca of Daphnia and Simocephalus 5. Powerful enzymes are present in the are acidic (Rankin, 1929), and enzyme secre- intestine; and tion in the middle gut of Daphnia is holocri- 6. Dialysis occurs through the peritrophic nous (Schultz and Kennedy, 1976a). membrane. A schematic representation of digestion and In Daphnia, the collected food particles are food transformation in Cladocera (Hallam glued together by salivary gland secretions et al., 1990) is shown in Fig. 4.15. andthensuckedintothegutbyperiodic dilations of the esophagus (Sterba, 1957a). In Daphnia and Simocephalus, the food also 4.5.2 Incomplete Digestion receives secretions from the hepatic caeca. The principal function of the latter is thought It is clear that food is incompletely digested to be secretion of digestive enzymes. by Cladocera. Some of the substances expelled However, hepatic caeca are completely into the environment by Daphnia have been absent in many cladocerans (e.g. in chydor- identified as unsaturated fatty acids (von Elert, ids). The hepatic caeca contract 610 times/ 2000) and are considered to be infochemicals, min in D. magna, but is much less frequent in i.e. biologically active substances that influence S. serrulatus (Rankin, 1929). It is unknown the companion species of algae and other ani- whether enzymes are present in these mals. The release of such substances may be a secretions. result of incomplete resorption.

PHYSIOLOGY OF THE CLADOCERA 70 4. NUTRITION

In a culture of D. magna, the following sub- P-limited diet: Cyclotella (a diatom) or stances were identified as influencing ceno- Scenedesmus (a green alga). Cyclotella was a bet- bium formation in Scenedesmus (von Elert, ter food, as estimated by Daphnia growth, 2000): linoleic acid, α-linolenic acid, myristic owing to its substantially higher content of acid, palmitic acid, palmitoleic acid, stearic 20:5ω3 (both phosphorus saturated and phos- acid, oleic acid, and stearidonic acid. phorus limited) than Scenedesmus under both Considering the great abundance of Cladocera, conditions. D. galeata growth, survival, and such large amounts of released infochemicals reproduction were all higher when fed on may have a considerable influence on the non P-limited Scenedesmus, compared with metabolism and behavior of hydrobionts. P-limited algae (Sundbom and Vrede, 1997). The impact of phosphorus on these biological outcomes was higher than that of ω3 HUFA. 4.5.3 Unbalanced Diet In Bosmina, phosphorus content is thought to be lower, at c. 0.71% DW. Growth and The available food always lacks some com- fecundity were unaffected in Bosmina fed on ponents: nitrogen or phosphorus deficiencies low-phosphorus algae, while in Daphnia these are especially common. This fact partly parameters declined (Schulz and Sterner, explains excessive food consumption by clado- 1999). Thus, under natural conditions Bosmina cera. Characteristics used to assess food qual- are better able to survive a low-phosphorus ity for planktonic Cladocera (e.g. Daphnia) are diet. the C:P ratio (Schulz and Sterner, 2000) and The phosphorus content in Cladocera has the C:N ratio. Factors influencing the former been studied when C:P ratios ranging from ratio have been better investigated than those 140 to 1000 are present in their food (Ferra˜o- affecting the latter. It was noted that Daphnia Filho et al., 2007). The phosphorus content species grow fastest under conditions of phos- changed substantially over this C:P range in phorus-rich food (Acharya et al., 2004) and food in Daphnia ambigua and a hybrid clone of that the phosphorus content of D. magna D. pulex-pulicaria, whereas Ceriodaphnia richardi directly depends on the phosphorus level of and Moina micrura showed tight phosphorus their food (Sterner, 1993; Urabe et al., 1997; homeostasis. The critical algal food quality Becker and Boersma, 2005). According to the required for the propagation of Daphnia spp. latter authors, the phosphorus content in was determined to consist of a C:P ratio of D. magna decreased from c. 16.7 to c. 10 mg/g 225375 (Brett et al., 2000); Anderson and DW as the C:P ratio increased, and enrichment Hessen (2005) indicate that for Daphnia carbon of food with inorganic phosphorus (PO4) sig- is limiting at low levels of available food and nificantly stimulated D. dentifera growth (Elser phosphorus becomes limiting with abundant et al., 2001). food. At a C:P ratio above 230 in scarce seston, The phosphorus content of Daphnia species phosphorus becomes limiting. is directly proportional to the phosphorus con- The consumption of unbalanced food may tent of their food (i.e. it decreases with an lead to the failure of Cladocera cultures increasing C:P ratio in the seston) (DeMott provided with abundant food. When fed et al., 2004) in both natural and experimental exclusively on green protococcaceous algae, conditions. Interestingly, phenotypic variation D. magna accumulated oil drops and stopped prevails over genetic differences among spe- reproducing (Flu¨ ckiger, 1951). The daphnias cies. Mu¨ ller-Navarra (1995b) fed D. galeata on became filled with pink fat droplets, which algae saturated with phosphorus or on were grouped abundantly around the intestine

PHYSIOLOGY OF THE CLADOCERA 4.5 ASSIMILATION 71

(from the first third to the anus), in thoracic Schlechtriem et al. (2006) cultivated D. pulex at limbs, and around nephridia. In such animals, 11C and 22C for 1 month on a HUFA-free the absence of a certain vitally necessary food diet. The conversion of C18 fatty acid precur- ingredient caused reproduction to stop; fur- sors to EPA (20:5ω3) and ARA (20:4ω6) was ther, ovaries could not be discerned, molting observed, and it was concluded that “HUFA occurred very rarely, and they died within such as ARA and EPA are highly conserved 2 days. These daphnias did not use their accu- during starvation” (Schlechtriem et al., 2006, mulated fat; in contrast, those fed normally p. 397). use their fat reserves in the absence of food. The addition of egg yolk or yeast improved conditions but the essential component 4.5.4 The Fate of Ingested Chlorophyll remains unknown. Sterner et al. (1993) fed D. obtusa with This question of the fate of ingested chloro- Scenedesmus containing different levels of nitro- phyll arose rather a long time ago, but it gen and phosphorus (moderately N limited, remains unanswered. Hardy and McDougall severely N limited, and severely P limited) (1894, p. 5) noted that “[i]n the chlorophyll of and found that “no amount of low quality the algae which form so large a portion of the food would support rapid Daphnia growth.” diet of Daphniidae, we have a substance The nitrogen content and N:P ratio in Daphnia whose fate we can to a certain extent trace, were essentially constant despite varying in and we can find that as digestion proceeds the the Scenedesmus. Feeding rates were lower with food mass in the middle region loses its green P-limited than with N-limited Scenedesmus. tint, whereas the fluid contents of the anterior These authors concluded that the mineral become colored a vivid green. Further there is nutritional value of algae may influence the evidence that this dissolved chlorophyll is demographics of herbivorous Cladocera more absorbed, for the striated border of the epithe- than is commonly estimated. In D. pulicaria fed lium becomes colored an intense green and the on N- or P-limited green algae, fecundity was cells charge themselves with yellow pigment reduced compared with controls fed on non- masses.” This is, however, the exterior view. limited algae (Kilham et al., 1997). Indeed, Cladocera consume great quantities If fed solely with starch the functioning of of chlorophyll with algae. Their algal food con- muscles of the antennae and of thoracic limbs tains a lot of chlorophyll: green algae contain is depressed, work of different heart fibers is 1.243.00 g DW/L and blue-green algae con- uncoordinated, a fat body is not seen, and tain 0.684.83 g DW/L (Lavrovskaya, 1965). Daphnia die in about six days (Flu¨ ckiger and The chlorophyll content in lakes may average Flu¨ ck, 1952). Thus, Daphnia is not able to pro- 514 mg/m3 and reach even higher concentra- duce fat directly from starch. Addition of vita- tions (Westlake, 1980; Schulz and Sterner, min B1 (aneurin) to the culture medium of 2000; Brandl et al., 2010a; Brandl et al., 2010b). Daphnia fed solely on starch restored a normal Surprisingly, information on the fate of heart rate (Flu¨ ckiger and Flu¨ ck, 1952), and the chlorophyll during digestion and its further addition of pantothenic acid to a culture of metabolism is scarce, both in the Cladocera lit- Daphnia fed solely on Chlamydomonas increased erature and from experts on photosynthesis the duration of their life by three times and on animal physiology (including rumi- (Fritsch, 1953). nants). For insects, Kuznetsov (1948, p. 111) In Cladocera, HUFA is necessary to support noted: “Very interesting theoretically processes the normal condition of cell membranes. of digestion of chlorophyll and of other food

PHYSIOLOGY OF THE CLADOCERA 72 4. NUTRITION pigments are almost not traced in insects”; chlorophyll or to the creation of green pigment “The question on the origin of chlorophyll in formed from chlorophyll derivatives. hemolymph of insects is very interesting,” It seems that little is actually known, for (p. 12); and “information is yet insufficient for both invertebrates and vertebrates, about the final judgment on so important biological process of chlorophyll digestion. At best, issue” (p. 13) handbooks on animal husbandry note that Daphnids fed on the algae Scenedesmus and ruminates need magnesium and obtain it from Rhodomonas release most of the consumed plants, where a minor fraction of magnesium chlorophyll in their feces; some of it is is bound to chlorophyll. Further studies on the degraded to chlorophyllide a, pheophytin a, fate of chlorophyll in the process of Cladocera and pheophorbide a. It is assumed that either digestion are therefore highly desirable. the pigment or the chloroplasts themselves are poorly assimilated. It has also been shown that Daphniidae convert ingested algal chlorophyll 4.5.5 Chemical Composition of Feces into pheophorbide (Fundel et al., 1998; Little is known about the chemical composi- Pandolfini et al., 2000). At low concentrations tion of cladoceran feces. Steryl chlorin esters of food algae, less pheophorbide a and more (SCEs) are products of the transformation of colorless digestion products were liberated, chlorophyll. The composition of sterols in thus indicating that more pheophorbide a was SCEs within D. magna fecal pellets was studied digested (Fundel et al., 1998). by Soma et al. (2005), who showed that they The levels of pheopigments in the gut contain both sterols formed by metabolism of filter-feeding Cladocera were used by and unaltered sterols from dietary algae. C Gorbunova (pers. comm.) to indicate decreased 27 sterols (except for cholesterol and C sterols, consumption of algae in the presence of mineral 28 major sterols in diatoms) were scarce in these turbidity. It may be added that the decreased SCEs. Cholesterol, probably a product of crus- chlorophyll content in the gut of D. magna, tacean metabolism, was relatively abundant in determined chromatographically, has also been these SCEs. used to indicate reduced feeding in the presence of cypermethrin (Christensen et al., 2006). Fox et al. (1949) believed that Hb formation in Cladocera is not influenced by the presence 4.6 STARVATION of chlorophyll in their food. As a protein, chlorophyll (tetrapyrrole) should undergo the gen- The life span under starvation conditions eral process of digestion. No answer was was summarized by Threlkeld (1976): it was found to questions such as: Into which compo- 46 days for D. magna, 11 days for D. pulex, nents is chlorophyll degraded during diges- 5 days for D. pulex var. pulicaria Forbes, and tion? and Are these components used in the 4 days for M. macrocopa. The mean survival construction of Hb? It would be wasteful if time of starved D. magna was 7.6 days (Elendt, chlorophyll, or partly decomposed chlorophyll, 1989), with a maximum of 10 days (Porter and is simply discarded from the organism: yet, in Orcutt 1980). Fully starving adult D. magna Cladocera this is probably so. Kuznetsov survived for 160246 h, D. galeata for (1948) reviewed the opinions of various 105140 h, neonates of D. magna for 66134 h authors that in herbivorous insects, which con- (longer with a higher maternal lipid content), sume a lot of chlorophyll, the hemolymph and neonates of D. galeata for 60 h (Tessier becomes green due to the permeation of et al., 1983).

PHYSIOLOGY OF THE CLADOCERA 4.6 STARVATION 73

It is clear that these organisms use their In D. pulex, the respiratory quotient (RQ) intrinsic resources in the complete absence decreased from 1.13 to 0.71 over 5 days of star- of food. When maintained in autoclaved vation (Richman, 1958). This decrease in RQ tap water filtered through a 0.22 μm pore demonstrates a change from predominant car- filter, Ceriodaphnia cornuta, M. macrocopa, bohydrate utilization in the metabolism to pro- Diaphanosoma sarsi, and Scapholeberis kingi tein and fat metabolism. could reproduce for three generations, In starved female D. magna, the following although they matured later than did females events occur in midgut enterocytes (Elendt fed with Chlorella (Kumar et al., 2005). In con- and Storch, 1990): depletion of lipid and glyco- trast, Macrothrix spp. were barely able to gen reserves, swelling of mitochondria, reduc- undergo a single generation. tion of the endoplasmic reticulum and of Over 3 days of starvation, D. pulicaria dictyosomes, and a decrease in cell height. weight reduced from c. 8.5 to c. 5.5 μg (DeMott Prolonged starvation of D. magna also results et al., 1997). In starving D. magna, the greatest in a loss of lactate dehydrogenase activity losses were those of DW, proteins, and lipids (anaerobic metabolic activity) in a Daphnia (including those due to the release of young homogenates (Hebert, 1973). In starving formed before starvation), and reductions in Daphnia, there is also a drop in blood concen- total carbohydrate and glycogen occurred dur- tration (Fritsche, 1917). Belayev (1950) inter- ing the first day of starvation (Elendt, 1989). preted the decrease in blood hypertony The triglyceride:total lipid ratio decreased dur- relative to the external water as an indication ing 56 days of starvation from 0.52 to c. 0.15. that food is the source of substances that sup- The changes in the chemical composition of port hypertony. Carotenoids in all tissues are D. pulex caused by starvation are shown in depleted during starvation in Simocephalus Fig. 3.1 (Lemcke and Lampert, 1975). The (Green, 1966a) and the heart rate in starving chemical composition of starved D. magna was D. longispina decreases (Ingle et al., 1937). analyzed in detail by Elendt (1989) and Hessen Hungry daphnias actively filter their food (1990). Hessen (1990) found that the mean particles; it was noted that a period of about phosphorus content of starved Daphnia was 30 min is sufficient to overcome the starvation 1.38% DW; this was unaffected by the quantity effect (Lampert, 1987). At starvation, daphnid of available food. With reference to growth continues for a time as a result of inter- Simocephalus (Green, 1966b), starvation resulted nal stores, and refeeding results in “catch-up” in carotenoids being depleted from all organs. growth (Bradley, Baird, et al., 1991; Bradley, In D. magna starved for 13 days, the beating Perrin, et al., 1991). rate of the thoracic appendages decreased; it Ingle et al. (1937) demonstrated that under increased immediately (from c. 6 Hz to c. 8 Hz) conditions of minimum food D. longispina pro- when food was added (Plath, 1998). In these duce fewer young; when they are again fed organisms, the respiration rate was low and abundantly, they “promptly produce many the addition of food was followed by a three- more young in each brood.” In D. magna, tem- to fourfold increase in the respiration rate and porary starvation has been demonstrated increased swimming activity (Schmoker and experimentally to be followed by immediate Herna´ndez-Leo´n, 2003). By the fourth day cessation of energy allocation to reproduction of starvation, the oxygen consumption of (by ceasing egg production) (Bradley et al., D. obtusa had decreased to 50% of the level of 1991a; Bradley et al., 1991b). This energy allo- well-nourished specimens (Vollenweider, cation is confined to the first half of the instar. 1958). Refeeding with Chlorella results in a recovery

PHYSIOLOGY OF THE CLADOCERA 74 4. NUTRITION of fecundity. Trubetskova and Lampert (1995) can inhibit protease activity in M. macrocopa also found that with starvation, the eggs of (Agrawal et al., 2001). Further, Rohrlack et al. D. magna are fewer and smaller. Of course, the (2004) found that Daphnia feeding on reaction to food shortage (i.e. a decrease in egg Microcystis are unable to shed their old integu- number) is delayed. Makrushin (1966) demon- ment, despite a new integument being strated that in D. pulex and D. longispina ovi- produced. These authors also found that blue- cells decompose if starvation occurs at stage I green algae contain the protease inhibitor or early stage II of their development, but not microviridin J. From the gut of Daphnia,it if starvation occurs later. Stage I was character- penetrates into the blood and disrupts normal ized by Makrushin as the state of the ovary metabolism, which leads to disturbances in when either no fat is present in the cytoplasm molting and interference with normal swim- of ovicells or it is present as tiny droplets; the ming and filtration, which ends fatally. brood pouch may contain eggs that have not Paralytic shellfish toxins are obtained by started cleavage. Such a time lag, dependent Moina mongolica following ingestion of the on the energy reserves of the body, was also dinoflagellate Alexandrium and then are trans- reported by Goulden and Hornig (1980), who ferred to fish that predate on the Moina (Jiang noted that smaller Bosmina, with insufficient et al., 2007). energy reserves, die comparatively sooner. As indicated for Daphnia, the critical time point at which starvation determines the number of 4.8 IMPACT OF XENOBIOTICS ON eggs produced is about halfway through (0.5) DIGESTION the intermolt period; variation in the abundance of food at later stages does not change Large fat cells (i.e. storage cells) in D. magna, the number of eggs (Bradley et al., 1991a; which are especially numerous at the posterior Bradley et al., 1991b). Ebert and Yampolsky curve of the digestive tract, became smaller, (1992) investigated how food shortage leads to contained less glycogen, and their mitochon- the production of fewer eggs in Daphnia. They dria became more spherical following at an found that the number of eggs decreased exposure to 20 μg/L Cd (Bodar et al., 1990b). when the “females were starved at least for 0.6 In solutions of phenol and aluminum sul- of the adult instar duration before egg laying.” fate, pH in the intestine of Daphnia increases In addition, the number of eggs increased from the normal value of 6 up to 8 (Beim et al., when abundant food was given to the 0.6 1994). instar, before the eggs were deposited into the Cadmium is accumulated by C. dubia from brood chamber. their diet (from green algae loaded with cadmium) and from water, but uptake from the diet is slower (Sofyan et al., 2007). Exposure of 4.7 NATURAL TOXICITY D. magna for 48 h to sublethal solutions of CdCl2 or HgCl2 inhibits cellulase, amylase, Cladocera encounter many toxic agents galactosidase, trypsin, and esterase; in contrast, under natural conditions. Blue-green algae exposure to K2Cr2O7 increases activity of these periodically develop in great quantities and enzymes (De Coen and Janssen, 1997). either unfavorably modify the environment or Assimilation of the green alga Selenastrum by are directly toxic. They may be directly toxic D. magna decreased several fold upon expo- 1 or decrease the feeding rate in Daphnia (e.g. sure to 100 μMCd2 (Baillieul and Blust, Rohrlack et al., 2001). Extracts from Microcystis 1999).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 5

Respiration

5.1 ANATOMICAL BACKGROUND conditions and in ventilatory compensation under food-rich conditions. Most gas exchange in cladocera occurs through the body surface (Peters, 1987; Pirow et al., 1999a, 1999b). The gills do not have this 5.2 ENVIRONMENTAL function, since it has been shown that most of BACKGROUND the oxygen necessary is extracted from the feeding current (Pirow et al., 1999a; Seidl et al., Littoral Cladocera frequently live under 2002). Oxygen is then distributed throughout conditions of oxygen deficiency, especially the body and a branch of hemolymph reaches those that bury themselves in the bottom sedi- the brood chamber (Fig. 5.1) (Seidl et al., 2002; ment. In general, pelagic Cladocera suffer less Pirow and Buchen, 2004). Another area of often from oxygen deficiency, except under active respiration is the rectum, during the fre- conditions such as the mass decomposition of quent process of anal water intake. algae or the morning peak of algal respiration. As has already been mentioned, in They are also exposed to diurnal and seasonal Cladocera respiration and feeding are closely changes in the distribution of oxygen concentra- interconnected, with both being performed tion, as well as the decreasing vertical oxygen with the participation of the thoracic limbs. It gradient, which has been well described in the was previously thought that the sites of active context of limnology (see, e.g. Hutchinson, gas exchange were the gills (epipodites) 1957, Ch. 9; Macan, 1963; Dodson, 2005; Kitaev, because it is easily observed that the gill sur- 2007). It should also be remembered that the face is the area of active silver reduction in a oxygen concentration in water decreases with AgNO3 solution. Due to this dual function of increasing temperature. movement of the appendages, the rate of their movement is labile and depends on both the concentration of food particles and the oxygen 5.3 OXYGEN CONSUMPTION concentration (Pirow and Buchen, 2004). According to these authors, hypoxic exposure The oxygen affinity of blood is numerically results in tachycardia under food-free expressed by the partial pressure of oxygen

(A) Ceriodaphnia laticaudata, semisaturation occurs at 0.8 mm Hg of O2, i.e. much lower than that in Daphnia magna (3.1 mm Hg) (Fox, 1945). Different species of Ceriodaphnia can withstand different oxygen limits; C. laticaudata is an example of a species able to live at lower oxygen concentrations (Burgis, 1967). The hemolymph of cladocerans becomes oxygenated while it flows through the ventral part of the carapace and the rostral region via direct diffusion (Pirow, Wollinger, and Paul, 1999b). Winberg (1950) summarized all quantitative data on oxygen consumption by crustaceans P in (B) available at that time. These data included, Perfusion Anterior inter alia, freshwater planktonic cladocerans P and copepods. Winberg concluded that oxygen Ventilation v Diffusion consumption for all crustaceans complies with the equation (Eq. 5.1):

: R 5 0:105W0 81 (5.1)

where R is the rate of oxygen consumption in mg/h at 15C, and W is the weight in g. P a As summarized later by Threlkeld (1976), the P ex rate of oxygen consumption in cladocerans is: Carapace 20.184 lacuna R 5 0.2935w in D. magna; 20.124 Posterior R 5 0.207w (R in µg/day, w in µg) in Peripheral tissue layer Cuticle Haemolymph space Trunk D. pulex var. pulicaria; and 5 20.107 Central tissue cylinder R 0.133w in Simocephalus vetulus.

FIGURE 5.1 Blood flow in Daphnia magna. Pa, oxygen The rate of oxygen consumption is used as partial pressure of the hemolymph entering the trunk; Pex, a measure of intensity of metabolism and, expiratory oxygen partial pressure; Pin, inspiratory oxygen accordingly, of food requirements. partial pressure; Pv, oxygen partial pressure of the hemolymph leaving the trunk. Source: Pirow and Buchen (2004). Oxygen consumption increases with increasing temperature, up to a maximum. For Daphnia middendorffiana, the maximum was that induces half saturation [semisaturation found to be 26C, above which inactivation of (P50), i.e. the formation of 50% of oxyhemoglo- respiratory enzymes started; for Bythotrephes bin at a certain partial pressure of oxygen (mm cederstroemi, the upper limit is 23C—thus, it is Hg)]. The ability of cladocerans to exist at more stenothermic (Yurista, 1999). Oxygen higher or lower concentrations of oxygen gas consumption in S. vetulus, measured using À (O2) is characterized by the property of their Cartesian divers or 100 130 mL bottles, did blood to reach semisaturation at a certain oxy- not change within a pH range of 4.5À9.5 in gen pressure. For example, at 17 Cin immobile specimens, although their general

PHYSIOLOGY OF THE CLADOCERA 5.3 OXYGEN CONSUMPTION 77 oxygen consumption (including movement cladocerans (Daphnia and Simocephalus) (as and filtration) did increase with increasing pH summarized by Smirnov, 1975). Chydorus ovalis À (Ivanova and Klekowski, 1972). S. vetulus consumes 19 21 mg O2/24 h/g wet weight immobilized in the small space in the diver’s (WW) at pH 9 and 23C (Yatsenko, 1928). bulb consumed 29.5% less oxygen than did Information on the respiration of bottom- moving animals (Ivanova and Klekowski, dwelling Cladocera is very scarce. 1972). Further, O’Connor (1950) calculated that Reduced oxygen concentrations induces muscular activity in Daphnia requires 32% of upward swimming in Chydorus sphaericus and their total metabolic energy. Pseudochydorus globosus (Meyers, 1980) at A model of oxygen transport from the envi- decreased light intensities (if not attached to a ronment into tissues has been suggested with substratum). C. sphaericus reacts to higher oxy- reference to D. magna by Moenickes et al. gen levels (2.5 ppm) than does Pseudochydorus (2010). Oxygen consumption correlates with (1.15 ppm). the respiratory electron transport system Tissue oxygenation has been estimated by (ETS); Simciˇ cˇ and Brancelj (1997) reported that NADH fluorescence in the limb muscles of the ETS is localized in the inner mitochondrial Simocephalus (Forasacco and Fontvieille, 2008), membrane and comprises a multienzyme com- using the assumption that NADH content plex containing flavoproteins, metallic pro- positively correlates with oxygen consumption. teins, and cytochromes. This redox system transports electrons from nicotinamide ade- 5.3.2 Respiration in Pelagic Cladocera nine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), and Pelagic species usually enjoy a relatively succinate to O2. Formazan produced in this good oxygen environment (by living at process closely correlates with O2 consump- normoxia). Water flow created by the beating tion; thus, a method of measuring formazan of their thoracic appendages supplies both production spectrophotometrically in homoge- oxygen and food to pelagic species. Beating nates has been found to be extremely sensitive rates of the appendages depend on both the and applied to five species of Daphnia. oxygen concentration and the concentration of available food particles (Pirow and Buchen, 2004). Oxygen tension (pO2) within the Daphnia 5.3.1 Respiration in Littoral À and Bottom-Dwelling Cladocera body is about 4 5 times lower than of the surrounding water (Fox, 1945; Wolvecamp and Benthic cladocera, especially those that bury Waterman, 1960): in D. magna, it is 3.1 mm Hg, themselves in the bottom sediment, live under but in C. laticaudata it is only 0.8 mm Hg. conditions of frequent oxygen deficiency and There have been numerous measurements an unlimited supply of food material. Littoral of oxygen consumption rates in cladocera, chydorids may survive at c. 1.9 mg/L O2 or mainly for Daphnia species. At 20 C, D. longis- À even as low as 0.4 1.7 mg/L O2 (Pacaud, pina consume 19.1 mg O2/24 h/g WW 1939; Bogatova, 1962). For specific types of (Shcherbakov, 1935), i.e. 0.8 mg/h. Under an chydorids, the minimum oxygen concentra- average “normal” temperature, Daphnia con- À µ tions in their environment at 18 22 Cis sume c. 1 LO2 per 1 mg dry weight (DW) per À 1 1.7 mg/L O2 for littoral cladocerans hour (as summarized by Peters, 1987). Oxygen À (Acroperus, Alona, Camptocercus, Eurycercus, and consumption is about 0.2 1.3 mL O2/g WW/h À Pleuroxus) and 0.29 2.43 mg/L O2 for pelagic at 20 CinDaphnia and 0.29 mL O2/g WW/h

PHYSIOLOGY OF THE CLADOCERA 78 5. RESPIRATION in Chydorus (according to Wolvecamp and at O2 concentrations above c. 3 mg/L; below À µ Waterman, 1960), and 0.14 1.18 LO2 per this concentration, the rate of oxygen con- individual (ind.) per day at 16C (according to sumption declined rapidly. Hillbricht-Ilkowska and Karabin, 1970). Paul et al. (1997) and Pirow et al. (2001, Oxygen consumption by D. magna is main- 2004) devised special computerized recording tained at approximately the same level when techniques and have achieved remarkable the oxygen concentration in the water is results in the field of Daphnia respiration. A 4À1.5 mg/L, but it abruptly declines with a special set-up for optophysiological recording further decrease in the external concentration with a video recorder and PC, as well as com- (Skadovskiy, 1955). The respiration of D. magna puterized respirometry, was devised by Paul is higher in feeding animals, lower in animals et al. (1997). For this, Daphnia were placed in a provided with low-food levels, and still lower glass vessel of 0.45 mL volume. The apparatus in fasting specimens (Jensen and Hessen, used by Pirow et al. (2001) consisted of an 2007). Kersting (1978) also demonstrated that inverted microscope equipped with systems D. magna respiration reaches a maximum at a for measuring hemoglobin (Hb) oxygenation, concentration of food algae that is close to the NADH fluorescence (as an indicator of the critical level (i.e. where the food uptake does oxygenation status of tissues), and the move- not increase further and becomes constant). ments of organs. With an excess of food (Chlorella), D. magna res- Pirow et al. (2004) also investigated oxygen piration decreases somewhat (Kersting and transport processes in D. magna using an Leew-Leegwater, 1976). oxygen-sensitive phosphorescence probe, From a quantitative aspect, the relationship Oxyphor R2, injected into the circulatory sys- between the body weight (W) and oxygen contem. Fasting animals were immobilized by sumption (Q) in planktonic cladocera can be gluing their posterior apical spine with histoa- described by the following equation (Eq. 5.2), cryl to a bristle that was then fixed to a cover- and is somewhat below the average level for slip with plastilin. The dye was then all crustaceans (Sushchenya, 1972): microinjected into the circulatory system from thedorsalsideintothespace“directlydown- 5 : 0:803 Q 0 143W (5.2) stream of the heart.” A single daphnid was fixed onto a coverslip and then placed onto For D. pulex, Richman (1958) determined the the bottom of a thermostatted perfusion following relationship between weight and chamber; in this apparatus, the individual oxygen consumption (Eq. 5.3): daphnid was able to move its antennae freely.

Log O2 5 log 0:0014 1 0:881 log W (5.3) Measurements of the distribution of oxygen partial pressure (P) in the hemolymph were The lethal minimum oxygen concentration followed by phosphorescence imaging of the was found to be 0.29 mg/L in D. obtusa and phosphorus range (0À6 kPa). The resulting 0.99 mg/L O2 in D. hyalina (Herbert, 1954). three-dimensional profiles were different for Heisey and Porter (1977) investigated oxy- Hb-rich and Hb-poor specimens. A steep gra- gen consumption and filtering rates in oxygen dient was discovered in Hb-poor specimens, concentrations ranging from air saturation whereas Hb-rich individuals showed flat gra- levels to almost zero. The oxygen consumption dients (Fig. 5.1). in Daphnia galeata mendotae turned out to With increasing temperature, oxygen con- be directly proportional to the oxygen concen- sumption increases but the oxygen content of tration. In D. magna, it was slightly increased water tends to decrease. Oxygen consumption

PHYSIOLOGY OF THE CLADOCERA 5.4 HEMOGLOBIN AND IRON 79 also depends on various other factors, such as 3. By staining Hb blue with benzidine base age (as shown for Simocephalus by Obreshkove (according to the method of Pearse, 1969). and Banta, 1930), illumination (Buikema, 1972), The blue staining was clearly observed to be and vessel size (Zeiss, 1963). The respiratory localized and the crystals produced by meth- rate rapidly increased in previously starved ods (1) and (2) were characteristic of each sub- D. magna provided with increasing concentra- stance. Hb was found in both reddish tion of algae until the “incipient limiting level” specimens and specimens with no visible red- of food concentration was reached (Lampert, dish coloration. 1986). The red color of mud-living Ilyocryptus sor- According to Flu¨ ckiger (1953), sympathomi- didus is caused by Hb, as shown by Fox et al. metics (i.e. L-adrenaline, D-adrenaline, and (1951). If its young are cultivated in well- L-noradrenaline) increase oxygen consumption aerated water, they grow into pale adults. by Daphnia. The presence of carbaminoylcho- It was also demonstrated that bottom-living line chloride (a parasympathomimetic) also and littoral cladocera, such as Ilyocryptus and increases oxygen consumption. C. sphaericus, gain or lose Hb, respectively (Fox, 1955). The presence of Hb in bottom- living cladocera may be related to an oxygen 5.4 HEMOGLOBIN AND IRON deficit in such conditions or even to anoxia. However, some species living on the mud sur- Hb has been reported in various littoral face are not colored red, as was observed e.g. and pelagic Cladocera by many authors. With in Drepanothrix dentata (in Lake Glubokoe, oxygen deficiency, Hb appears in the blood Moscow region). However, the presence and of cladocera and its concentration increases levels of Hb in most bottom-living and inshore over time (Chandler, 1954; Smaridge, 1956; cladocerans remain unexplored. Kobayashi, 1970). 5.4.2 Hemoglobin in Pelagic Cladocera 5.4.1 Hemoglobin in Littoral Cladocera Hb has been found in planktonic Daphnia and Moina, and also in Simocephalus (Fox, 1948; Although Hb is more important in littoral Hoshi, 1957; Hoshi and Nagumo, 1964). The and bottom-living Cladocera than in pelagic maximum Hb absorption in an absorption Cladocera, Hb has mainly been studied in spectrum is different in representatives of dif- pelagic daphnids and in Moina. In littoral chy- ferent species, and even in related species; for dorids, such as Acroperus harpae, Alona affinis, example, it is at 314 nm in D. magna and Eurycercus lamellatus, Picripleuroxus striatus, 418 nm in Ceriodaphnia quadrangula (Czeczuga, Pleuroxus trigonellus, and P. truncatus, Smirnov 1965). (1970) demonstrated the presence of Hb using Generally, Hb is formed when there is an three different methods: oxygen deficit. The Hb content of Daphnia 1. By producing crystals of pyridine decreases before a molt, but increases in the hemochromogen in the bodies of the ovaries at the same time (Fox et al., 1949). chydorids, (according to the method of During an instar (the intermolt period), Hb Hoshi, 1963a, p. 88); passes from the blood into parthenogenetic 2. By producing crystals of hemin (according eggs, as has been shown for Daphnia (Green, to the method of Hoshi (1963b, p. 94); and 1956), and this occurs a short time before

PHYSIOLOGY OF THE CLADOCERA 80 5. RESPIRATION molting. The Hb concentration in Daphnia (Hoshi and Inada, 1973). Curiously, although increases when there is a low oxygen concentra- Hb appears in animals at reduced oxygen con- tion in the environment [shown by Fox, (1955), centrations, inactivation of Hb by carbon mon- Green (1956), and Landon and Stasiak, (1983)]. oxide (CO) did not make daphnias much less The latter authors also demonstrated that the vigorous or decrease their survival in short- Hb concentration in Daphnia is directly propor- term experiments. According to Fox (1948), tional to depth in Arco Lake (Minnesota, USA). Daphnia containing deoxygenated Hb survive In aerated water, the Hb content in Daphnia for a long time: it was therefore thought that decreases. The Hb content of pink Moina Hb takes no part in their respiration. It seems macrocopa was shown to be 1.7 g Hb/100 mL to have only a supplementary role. Long-term blood and 1/15 of this value in pale specimens cultivation, however, revealed that red daph- (Kobayashi, 1981b). Studies on variations of Hb nias do live longer, feed more successfully, content in response to environmental factors, and produce more eggs (Fox, 1948; Fox et al., including oxygen concentration, have been 1951). reviewed by Kobayashi and Hoshi (1984). The According to Kobayashi (1981b), in poorly Hb concentration in D. magna, as determined aerated water the respiration rates of pale by Kobayashi (1981a), ranges from 0.1 to 1.7 g M. macrocopa and of CO-treated red specimens Hb per 100 mL blood. According to later mea- are identical. The lethal oxygen concentration surements, the Hb concentration of D. magna becomes higher in Daphnia at increasing CO ranges from 0.24 to 241 mg Hb/g DW concentrations and is more obvious in pink (Kobayashi and Nezu, 1986). It increased at low Daphnia (Kobayashi and Hoshi, 1980). oxygen concentrations: rapidly in immature Nevertheless, Daphnia containing Hb live lon- specimens and more slowly in larger animals, ger, swim more actively, consume more food, although animals longer than 3 mm did not and produce more parthenogenetic eggs (Fox become red. The lethal oxygen concentration et al., 1951). Kobayashi and Hoshi (1982) believe for pale D. magna is c. 0.22 mL O2/L at 15 C that Hb functions only at low O2 concentra- and c. 0.5 mL O2/L at 30C; for red specimens, tions. In their experiments, all specimens of it is c. 0.1 mL O2/L at 15 C and c. 0.12 mL pale D. magna died at c. 0.5 mL O2/L, whereas O2/L at 30C. Under the same conditions, specimens containing 1À1.5 g Hb/100 mL D. magna males accumulate more Hb than do blood died at c. 0.1 mL O2/L. The critical values females at low oxygen concentrations for C. quadrangula are: 0.9 mL O2/L for pale (Kobayashi, 1970)). Hb is not just a passive specimens and 0.3 mL O2/L for pink specimens chemical, however. In Hb-rich D. magna its con- (Kobayashi, 1983a). Above this level, the rate of tribution to the circulatory transport of oxygen oxygen consumption by C. quadrangula is con- µ to tissues is much greater than in Hb-poor spe- stant: c. 12 LO2/ind./h for pale specimens µ cimens (Ba¨umer et al., 2002). Extra Hb ensures and 11 LO2/ind./h for pink specimens. an adequate oxygen supply to limb muscle tis- Survival may also depend on the former sue in D. magna at moderate oxygen concentra- environment of the tested specimens. For tions in the ambient water (Pirow et al., 2001). example, S. vetulus taken from a pond survive The tissue oxygenation state can be estimated much longer in deoxygenated water than do by NADH fluorescence in muscles. specimens taken from a river (Shkorbatov, Daphnia manage well without Hb; however, 1953). at low oxygen concentrations pale Daphnia Rudiger and Zeis (2011) found an inverse make up for the absence of Hb by increasing relationship between temperature and Hb con- the rate of movement of their thoracic limbs centration in D. longispina from oxygen-rich

PHYSIOLOGY OF THE CLADOCERA 5.4 HEMOGLOBIN AND IRON 81 water, with the seasonal minima of Hb concen- found that the optimum concentration for Hb µ tration coinciding with low food availability formation is 2 g Fe/L (FeCl3.6 H2O). Hb for- and the peak of reproduction. These authors mation is also favored by higher temperatures suggested that in Daphnia Hb functions as both and is not influenced by carbon dioxide levels a respiratory protein and a protein store. (Fox and Phear, 1953). In the body of D. magna, Generally, the role of Hb in cladocera is fac- its distribution was described by Smaridge ultative and these organisms may successfully (1956), who found three forms of iron in live and reproduce without it (e.g. when it is Daphnia tissues: “loosely bound” iron, “firmly inactivated). bound” iron, and iron within heme com- The presence of Hb in fat cells was noted by pounds (Hb and cytochromes). Loosely bound Green (1955); it was later shown that Hb is iron can be stained by, for example, Prussian synthesized in the fat cells and epithelial cells blue and firmly bound iron can be revealed by of epipodites (Goldmann et al., 1999). Hb can treatment with acid alcohol. The iron in Hb also be decomposed in fat cells without yield- and cytochromes may be revealed by treat- ing bile pigments (Green, 1971). Using radioac- ment with, for example, hydrogen peroxide tive iron, Hoshi and Sato (1974) found that or by microincineration. Radioactive ironÀ59 there is a continuous process of Hb synthesis (59Fe) is also incorporated by Daphnia and breakdown in the Daphnia body. Hb (Smaridge, 1956). breakdown is now known to occur in fat cells Loosely bound iron is found in the gut wall, (Smaridge, 1954; Green, 1955), whereas it was gut caeca walls, and fat cells (both as ferrous previously thought that Hb is normally and ferric iron), as well as in ovaries, blood excreted through the shell glands (Fox, 1948). plasma, and appendage walls (ferrous iron). It Populations of Daphnia have also been is assumed that iron is incorporated into newly observed to be colored brown by Hb in synthesized Hb in the fat cells and ovaries. combination with hematin, which contains Some iron may be accumulated in fat cells and three-valent iron in the heme moiety (Fox, then excreted. Iron is also incorporated into 1047À1948; Goodwin, 1955). heme and is present in all tissues, even in The Hb values indicated by these various the lenses of the eyes. Within the bodies of authors therefore depend on various factors D. magna specimens with increasing Hb, iron (e.g. environmental factors and species). is absorbed by the gut wall and is present in newly synthesized Hb in fat cells and the ovaries (Smaridge, 1956). In specimens that are 5.4.3 Iron losing Hb, it is especially present in walls of the gut ceca and fat cells, and in the excretory The iron component of Hb is present in shell glands; Hb is probably broken down in water and more abundantly on the bottom of fat cells. Tazima et al. (1975) also reported that water bodies in both ferrous and ferric forms. in Daphnia ferruginous compounds accumulate Obviously, it can therefore be ingested by cla- in the gut ceca, and that during Hb breakdown docerans directly or in algae. The iron content they can be observed in fat cells and the shell of Hb has been measured as 0.033%, in D. mag- glands. na and 0.317À0.353% in M. macrocopa (Hoshi Iron used for Hb synthesis is absorbed from et al., 1967; Hoshi and Kobayashi, 1971). Hb the midgut and used by fat cells for Hb syn- formation in Daphnia is favored by the pres- thesis; Hb destruction is aided by gut and fat ence of iron in the environment (Fox and cells, and Hb is excreted via the maxillary Phear, 1953; Goodwin, 1960); Dave (1984) glands (as loosely bound ferric iron).

PHYSIOLOGY OF THE CLADOCERA 82 5. RESPIRATION

5.5 EVOLUTION OF CARBON An energy-channeling scheme for Daphnia DIOXIDE AND THE RESPIRATORY was proposed by McCauley et al. (1990), as QUOTIENT shown in Fig. 4.11. Basal metabolism (i.e. metabolism in Daphnia immobilized by

Carbon dioxide (CO2) production by D-tubocurarine) was determined by means of a D. magna is estimated to be 0.03 mg/g air- Cartesian diver (O’Connor, 1950). It was found dried weight by females at 22C and 22% that the expenditure for muscular activities is higher in males (MacArthur and Baillie, 1929). 32% or more of the total metabolism. In In D. pulex, as determined by Richman (1958), D. magna immobilized with uretane, basal À its values are close to those of O2 consumption. metabolism takes 1/4 1/6 of the metabolism Later, CO2 production by D. magna was mea- of an active Daphnia (Postnov and Philippova, sured by Kolupaev (1989) to be 0.38 mg/g 1988). The expenditure for respiration was WW/h in JanuaryÀApril and 0.51 mg/g WW/h determined by Galkovskaya (1970) to 21À44% in MayÀAugust; oxygen consumption was in D. longispina and 32% in Bosmina. Male cla- 0.43 mg/g WW/h and 0.62 mg/g WW/h, docerans have a higher rate of metabolism respectively; and the frequency of thoracic than females (Banta and Brown, 1924). limb beating 299 per min and 346 per min, respectively. According to data published by Kolupaev (1989), the RQ in D. magna is , 1. 5.7 HYPOXIA RQ is the ratio of the carbon dioxide released to the oxygen consumed. The RQ is Hypoxia (low oxygen concentrations) is 1.00 for carbohydrate metabolism, 0.711 for fat commonly experienced by Cladocera. Oxygen metabolism, and 0.781 for protein metabolism deficiency may occur in the early morning fol- (Koshtoyants, 1951). RQ values of , 1 may lowing the prevalence of algal respiration over indicate the transformation of carbohydrate photosynthesis, or when there is a complete into fat accompanied by oxygen binding (e.g. covering of floating Lemna. In the absence of for deposition into eggs) or anoxybiosis (CO2 oxygen, Daphnia, Simocephalus, Scapholeberis, formation during fermentation in an oxygen- Bosmina, and Alona spp. have been shown to free medium). The RQ in well-fed D pulex is remain immobilized for 1À6 h and C. sphaeri- 0.92À1.11 for those of 1.6 mm in length and cus for 18 h (Nikitinsky and Murezowa-Wyss, 0.95À1.24 in larger specimens (Richman, 1958). 1930). The RQ in starved D. pulex decreases to 0.7, There have also been investigations of cla- thus indicating a shift from carbohydrate docera respiration under conditions of low metabolism to the utilization of fat and protein oxygen content, specifically for planktonic (Richman, 1958). Cladocera. It has been shown that during an oxygen shortage Daphnia swim toward regions of higher oxygen concentration (Pardi and Papi, 1961). 5.6 ENERGY BUDGET Rivier (1986) reported the presence of Daphnia with numerous embryos during win- Richman (1958) determined the energy bud- ter in the bottom of ice-covered Lake get of D. pulex (Table 5.1) to comprise expendi- Siverskoe, at a low oxygen content (c. 2 mg/L tures for the growth and production of young, O2). She also found D. longiremis, D. galeata, with the latter using the major part of the D. cristata,andBosmina longirostris reproducing stored energy. under the ice (Rivier, 2012).

PHYSIOLOGY OF THE CLADOCERA 5.7 HYPOXIA 83

TABLE 5.1 Energy Budget of Daphnia pulex at a Moderate Food Concentration

Age Energy Energy for Energy for Growth Energy of Energy of Consumed Growth and Young Respiration Egestion (Cal) (Cal) (Cal) (Cal) (Cal)

Pre-adult 0.469 0.062 0.050 0.050 0.357 Adults after 34 days of growth 5.671 0.071 À 0.791 3.872 (young not included) Adults after 40 days of growth 6.140 À 1.070 0.841 4.229

The low relative value of energy for growth is to be noted in slow-growing adults. Source: Richman (1958).

In D. magna at 1.8 mg/L O2, the following nerve ganglia, and the content of cytochromes parameters decrease in comparison with con- in the tissues also increases (Fox, 1945). trols at higher oxygen concentrations (Homer In addition, oxygen-dependent Hb induc- and Waller, 1983): DW, number of days to first tion takes place in D. magna under hypoxic brood, number of young in the first brood, conditions, thus favoring enhanced oxygen total number of young produced over the 26 transport. At oxygen with a partial pressure of days of the experiment. The median lethal 3 kPa, newly synthesized Hb can be detected À dose (or LD50; after 4 h) is 0.2 0.7 mL O2/L within 18 h and a steady state concentration is for Daphnia and Simocephalus, and 1À1.6 mL O2/L for Leptodora and Bythotrephes (Herbert, 1954). 25 Pink D. magna have been shown to survive for at least 24 h at 5% air saturation while pale ones perish within 1 h at 7% air saturation 20 (Usuki and Yamaguchi, 1979). At low oxygen concentrations, the lactic acid content in Daphnia increases (Fig. 5.2). In D. magna with 15 g/mg protein

embryos, under hypoxic conditions there is a µ higher rate of blood flow through the brood chamber (Seidl et al., 2002). 10 In contrast to D. galeata, oxygen consumption in D. magna rapidly declines at O2 concen- Lactic acid, trations below c. 3 mg/L (Heisey and Porter, 5 1977). An oxygen concentration c. 0.2 mg/L O2 suppresses the formation of large filtering screens in D. magna at a low food concentra- 0 tion, and thus reduces the filtering rate 01 234 56 (Hanazato, 1996). Growth was also retarded. O2-Content, ml/L Hypoxia also frequently induces the appear- FIGURE 5.2 Lactic acid content in pale (open circles) ance of Hb in the body (Gorr et al., 2004; and pink (solid circles) Daphnia at different oxygen concen- Zeiss et al., 2004). In hypoxic Daphnia, the Hb trations over a period of 1 h. Source: Usuki and Yamaguchi content is increased in the blood, muscles, and (1979).

PHYSIOLOGY OF THE CLADOCERA 84 5. RESPIRATION µ reached within 11 days (Zeiss et al., 2003); in 0.019 g N/L/h, in proportion to the O2 the same species, hypoxia-inducible factors concentration. (HIFs) are accumulated and facilitate O2 deliv- Seidl et al. (2005) exposed both the parental ery to hypoxic tissues (Gorr, 2004). generation and progeny of D. magna to Juvenoid hormones contribute to the eleva- 10À19% air saturation. Acclimation to hypoxia tion of Hb levels in D. magna (Gorr et al., involved adjustments to the Hb level and the 2006): at high O2 tension (pO2), these hor- metabolic level: Hb concentration increased by mones induce the robust reactivation of juve- 266% and oxygen affinity increased by 32%. noid response elements, but at low pO2 this Smaller offspring, combined with reduced Hb reaction is inhibited. and metabolic levels, reduced the critical ambi- In addition, in Moina micrura not acclimated ent oxygen tension by about 50%. to hypoxia, the metabolism and the stroke Transgenerational effects were not observed. frequency of their antennae is decreased Zeiss et al. (2009) studied the composition À (Hubareva, 2000a). At 0.7 0.8 mg O2/L, pale of the D. pulex proteome under normoxic (po2 M. micrura may stop filtration but continue 20 kPa) and hypoxic (po2 3 kPa) conditions. swimming, in contrast to red specimens Hypoxia caused a strong inducement of Hb (Svetlichny and Hubareva, 2002b). In D. magna, and carbohydrate-degrading enzymes, i.e. gly- there is no compensatory increase in the beat- colytic enzymes (enolase) and enzymes ing rate of thoracic limbs during hypoxia: it is involved in the degradation of storage and maintained at c. 180 per min (Colmorgen and structural carbohydrates. Paul, 1995). It was later observed that the beat- In hypoxic water, Hb-rich Daphnia carinata ing rate of their appendages does increase preferred a temperature of 19C (vs. 16C for under hypoxic conditions, and that compensa- controls), whereas in normoxic water all the tory tachycardia also occurs, followed by an animals gathered at regions of 23C (Wiggins increase in the Hb concentration (Pirow and and Frappell, 2000). Buchen, 2004). In Daphnia, hypoxic stress and heat (30C) Under conditions of acute hypoxia in caused oscillation in reactive oxygen species, M. micrura, the O:N ratio is 8.5, but this glutathione redox system activity, and HIF-1 increases to 34À40 under oxygen saturation (hypoxia-inducible factor) activity (Becker (Hubareva and Svetlichny, 1998). Under acute et al., 2011). hypoxia, largely anaerobic protein catabolism occurs. During longer periods of hypoxia, active M. micrura became acclimated and use 5.8 ANOXIA whatever oxygen is available for lipid oxidation. M. micrura adaptation to a prolonged There are good reasons for discussing period in low-oxygen environments involves a anaerobiosis in Cladocera. Most of the known greater role for lipids as metabolic substrates species live in the littoral zone and on the bot- (Hubareva, 2000b). Under conditions of acute tom of water bodies, where there is, at least hypoxia (i.e. a decrease in O2 concentration to periodically, a deficiency of oxygen. Many spe- c. 0.5 mg/L), the typical protein-lipid catabo- cies, probably including all Ilyocryptidae, live lism of M. micrura changes completely to the in the surface of mud under permanent condi- anaerobic protein catabolism (Hubareva, tions of very scarce oxygen. 2000a, 2000b; Svetlichny and Hubareva, 2002a, For the upper mud layer of lakes (0À2 cm), 2002b, 2004). Further, the excretion of NH3-N Martynova (2010) reported the presence of O2 µ À drastically decreases from 0.079 g N/L/h to at 0.75 9 mg/L (the bottom water layer), N2 at

PHYSIOLOGY OF THE CLADOCERA 5.9 IMPACT OF XENOBIOTICS ON RESPIRATION 85 À À 42 175 mg/kg WW, CH4 at 1 21 mg/kg, and oxygen concentrations D. magna accumulated À CO2 at 1.5 156 mg/kg. Brand (1946) listed the more lactic acid prior to death (Usuki and findings of various authors: the inhabitants of Yamaguchi, 1979). In Hb-colored Daphnia, the the mud surface of freshwater water bodies lactic acid content is higher at lower oxygen where the oxygen content is close to zero concentrations (Fig. 5.2). include the cladocera Lathonura rectirostris, The responses of D. magna to anoxia were Daphnia pulex, and Simocephalus exspinosus. The studied further by Paul et al. (1998). During lethal minimum oxygen concentration for chy- the first 2 h of anoxia, their anaerobic metabo- À dorids is 0.4 1.7 mg/L O2 (Pacaud, 1939; lism is characterized by L-lactate accumulation Bogatova, 1962); however, in general, informa- in the body (0.36 µmol lactate/g WW/min) tion on anaerobiotic Cladocera is very scarce. and a decrease in pH in the body (metabolic Under severely hypoxic conditions, acidosis). During the first 2 h of anoxia, the C. sphaericus movement in water saturated with heart rate does not change much, but at longer hydrogen was discontinued for 18 h, and in exposures the heart rate decreases. Under sub- Alona for 4 h (Mudretzowa-Wyss, 1933); and sequent conditions of normoxia, the heart rate in nitrogen-saturated water movement ceased in and the body pH return to normal. C. quadrangula for c. 35 min for red specimens In S. vetulus under anaerobic conditions, the and 23 min for pale specimens (Kobayashi glycogen content is 0.5% in the gastrula, 0.67% and Ichikawa, 1987). In the presence of in the nauplius, 0.53% in the released young, hydrogen sulfide (H2S), Daphnia, Simocephalus, and 0.64% in the third instar (Hoshi, 1953). and Bosmina became immobilized within Unlike in aerobic conditions, it does not c. 10À20 sec (Nikitinsky and Mudrezowa-Wyss, increase during postembryonic development. 1930). Hydrogen sulfide is another factor that lim- It has been observed that Chydorus sphaercus its the availability of bottom habitats for does not feed in anoxic water layers (Lair, Cladocera; for example, a low H2S concentra- 1991). As studied by Colmorgen and Paul tion (0.15 mM) causes irreversible immobiliza- (1995) and Paul et al. (1997), during anoxia the tion of C. ovalis (Bryukhatova, 1928). However, frequency and amplitude of the movements of there is a report of a ctenopod-like cladoceran thoracic limbs decreased in D. magna; the heart that lives in the anoxic depths of the Black Sea, continued to beat at a rate similar to normoxia which is saturated with hydrogen sulfide but the stroke volume decreased. According to (Sergeeva, 2004; Korovchinsky and Sergeeva, these authors, during hypoxia the frequency 2008). and amplitude of their thoracic limb movements remained at the usual rate of c. 180 min, but the heart rate increased (i.e. compensatory tachycardia). 5.9 IMPACT OF XENOBIOTICS Thus, under anoxic conditions the metabolism ON RESPIRATION of Cladocera changes radically. Anaerobiosis is generally characterized by the exploitation of car- Information on this subject is very limited. bohydrates in metabolism, suppression of protein Oxygen consumption in Simocephalus is inhibited metabolism (Brand, 1946), and high RQ values by 2.5 mM 2,4-D (Andrushaitis, 1972). In . due to the final stage of CO2 production Simocephalus, the presence of 5mM 2,4- (Koshtoyants, 1951). dichlorophenol-Na in the environment leads to Lactic acid concentration in the body of decreased oxygen consumption (Klekowski and Daphnia is generally very low, but at low Zvirgzds, 1971). At 5À8 mg/L naphthalene, the

PHYSIOLOGY OF THE CLADOCERA 86 5. RESPIRATION

Hb content and oxygen consumption of D. mag- indicating an increase in metabolic expenditure na decreases by about a 25% compared with (Alonzo et al., 2006). controls (Crider et al., 1982). Phenol at a Oxygen consumption decreases in D. magna concentration of 0.1 mg/L reduces the beating in presence of copper and zinc during the first rate of the thoracic limbs in D. magna from 305 hour, then (as determined after 3 h) compensa- beats/min (control) to 204 beats/min (Kolupaev, tory mechanisms are activated (Sladkova and 1988). In contrast, the respiratory demand Kholodkevich, 2012). Therefore, these authors increases in D. magna after a 23Àday exposure suggest that measurements of this effect should to 0.99 mGy/h americium (Am-241), thus be made after the first hour of exposure.

PHYSIOLOGY OF THE CLADOCERA CHAPTER 6

Circulation

6.1 ANATOMICAL BACKGROUND: lateral compartments (He´rouard, 1905; Pirow, BLOOD CELLS Wollinger, and Paul, 1999b). It may be added that the inner membranes of the cladoceran Cladocera possess an open circulatory sys- body need to be better described. tem and a myogenic heart. Heart beats and the The carapace covering the body is double flow of blood (hemolymph) can be easily walled, with the blood flowing into the inter- observed, as Cladocera are semitransparent. In nal carapace lacuna. Leptodora, an additional propulsive organ has The heart is innervated by the cardiac nerve, been described (Gerschler, 1910; Saalfeld, 1936; which reacts to adrenaline (thus increasing the Maynard, 1960), which can easily be found heart rate); however, the central nervous sys- and observed in living specimens in the first tem does not take part in heart regulation, as thoracic limb, at the distal end of the first seg- has been shown by the elimination of its vari- ment: it takes the form of a disk supplied with ous parts (Ermakov, 1936). Later, Ermakov a propulsive muscle. (1937) demonstrated the presence of nerve For Daphnia magna, it has been determined fibers that accelerate and retard the heart rate. that the hemolymph makes up 58% of its wet Cladoceran blood is light yellow, but may weight and 61% of its volume (Kobayashi, also be red due to the presence of hemoglobin 1983b). The heart has lateral ostia for the (Hb). Artemocyanin (biliprotein) has been inflow of hemolymph and a short anterior ves- found in the blood of Daphnia pulex (Peeters sel from which hemolymph is squirted at each et al., 1994). heart beat (Fig. 6.1). Venous and arterial blood Blood cells were originally noted in the flows are separated by a membrane in the area blood flow by early authors (e.g. Gruithuisen, of the heart (Fig. 6.2) (Herrick, 1884). It was 1828; Leydig, 1860, p. 56) and can be easily later found that the whole body space is subdi- observed. Gruithuisen (1828) and many subse- vided by ventral and dorsal membranes into quent authors (e.g. Hardy, 1892) believed that three blood spaces: the ventral lacuna, the dor- Cladocera possess a single type of blood cell. sal lacuna, and the intestinal lacuna. In addi- Cuenot (1891) saw numerous amoebocytes of tion, two vertical membranes divide the size 6À9 μm in the blood of Daphnia schefferi, ventral lacuna and the limbs into the medial Simocephalus vetulus, Chydorus sphaericus, and

Where the blood cells are formed and the process of hemopiesis is unknown. Early authors observed only one type of blood cell in Daphnia (Hardy, 1892; Fritsche, 1917) and in Eurycercus (Smirnov, 1970, 1971). In Eurycercus, the average length of these cells is 15 μm, but in Daphnia they are only 7À8 μmin diameter. The blood cells of Eurycercus are of variable structures and have occasional pseudopods. Closer examination of D. magna revealed two cell types circulating in the hemolymph (Auld et al., 2010): spherical cells (granulo- cytes) and irregular-shaped amoeboid cells (about 9 μm long, and identified as the cells initially described by Metchnikoff (see 1898, Fig. 17.3). The latter cells attack the bacterial parasite Pasteuria ramosa; in parasitized Daphnia, there is a large increase in the number of amoeboid cells.

6.2 BLOOD FLOW FIGURE 6.1 Upper, actual organism. Lower, schematic structure. Upper, blood flow in Eurycercus.1,arterialflow; 2, venous flow; 3, heart; 4, lateral ostium; 5, eddie. Lower, The movement of hemolymph is powered blood flow in Daphnia. (C, heart; cd, dorsal septum; ch, by the heart and by body movements. brood chamber; cp, cloison separating external ventral space Gruithuisen (1828) was probably the first to from the inner space; cv, reflected part of ventral septum; present a general scheme of circulation, within I, gut). Sources: Upper, Smirnov (1971); lower, He´rouard (1905). Simocephalus. Following the movement of blood cells he indicated arterial flows from the Sida crystallina. According to Cuenot, amoebo- heart to the cephalic region, to antennae, over cytes produce pseudopods. He noted local valves, and along the trunk, as well as reverse aggregations of amoebocytes but did not locate venous flow. According to this author, the any lymphatic glands. blood flows along capillary canals. Metchnikoff (1892) also observed a single The time for the complete transit of a blood type of blood cell in Daphnia:hetermedand cell through the circulation of Daphnia is about understood them to be leucocytes. He also 10À20 sec (Dearborn, 1903; Maynard, 1960). observed D. magna hemocytes in the process of The systolic heart volume was determined phagocytosis (Fig. 6.3) (Metchnikoff, 1882, 1884). to be 2.53 nL in D. pulex and 1.28 nL in There are many thousands of hemocytes in Holopedium (Maynard, 1960). Blood flow can Eurycercus, and several hundred in the smaller be clearly seen by the movement of blood cells Acroperus (Smirnov, 1975). According to Jaeger (Fig. 6.1), and the patterns of flow are gener- (1935), the blood cells loaded with fat may set- ally permanent, being separated and directed tle on tissues and turn into cells of the fat by membranes within the body. Herrick (1884) body. reported in Eurycercus the presence of

PHYSIOLOGY OF THE CLADOCERA 6.3 HEART RATE 89

FIGURE 6.2 Eurycercus heart and membranes dividing arterial and venous blood flow. Source: Herrick (1884).

membranes that divide the venous and arterial heart. The systolic volume of the heart in blood flows (venous and arterial sinuses) in D. pulex is 2.53 nL (Maynard, 1960). At its con- the area of the heart (Fig. 6.2). In general, traction in systole, the cranial region of the blood flow is directed forward from the heart heart is first contracted, then the ostial region, (arterial flow), and along the dorsal side of the and then the caudal region, resulting in com- body; in the area of the head it turns and then plete contraction of the heart (Proksova, 1950). passes through the limbs, follows along the In diastole, the cranial region is dilated first, ventral side to the postabdomen, and then and so on, further resulting in a complete wid- returns along the dorsal side of the body and ening of the heart. of the shell, as observed in Daphnia by He´rouard (1905) (Fig. 6.1). In different groups of cladocera, the system of blood flow varies, 6.3 HEART RATE but it has not yet been accurately described. Blood flow in D. magna has been investi- The heart rate can be determined by direct gated in detail by Pirow and Buchen (2004) counting; however, this becomes too difficult (Fig. 5.1). Pirow et al. (2004) also reported at a rate of over 400 beats/min. A special details of oxygen partial pressure throughout device for recording the heart rate was pro- the body of Hb-poor and Hb-rich D. magna, posed by Pre´sig and Ve´ro (1983), based on an and represented the data obtained as a three- optoelectronic circuit that transforms changes dimensional diagram (Fig. 6.4). in light intensity caused by heart beats into In D. pulex and Holopedium, the systole is changes in potential. Numerous studies on about 1.5 times longer than the diastole heart rate in cladocera have been mainly con- (Fig. 6.5) (Storch, 1931). At each contraction, fined to Daphnia, but they have been reported about half of the blood is expelled from the by Smirnov (1965b) for representatives of

PHYSIOLOGY OF THE CLADOCERA 90 6. CIRCULATION

FIGURE 6.3 Phagocytosis in Daphnia. a, spores of Monospora surrounded by leucocytes. Source: Metchnikoff (1892).

FIGURE 6.4 Oxygen partial pressure in the hemolymph throughout the body of Hb-rich 5 and Hb-poor Daphnia magna. Source: Pirow et al. 4 (2004).

(kPa) 2 2 Posterior o P 1

Dorsal

Hb-poor Anterior (Po2amb=5.1 kPa) Hb-rich

(Po2amb=1.7 kPa)

PHYSIOLOGY OF THE CLADOCERA 6.3 HEART RATE 91

Bild 962 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96

2,10

2,00

1,90 0,1 mm 1,80

1,70

1,60 Ostium- Diastole Ostium- Systole Ostium- öffnung verschluβ öffnung

FIGURE 6.5 Diastole and systole in the Daphnia heart. Changes in heart size were measured. Source: Storch (1931).

1 various genera (for 22 species belonging to 16 molting, reproduction; potassium ion (K )- genera from 6 families).The heart rate in litto- induced systolic arrest, and calcium ion 1 ral and pelagic species, as determined by (Ca2 )-induced diastolic arrest; the heart rate direct counting, ranges from 190À320 beats/ also increases with increased body size, up to min at 17À18C in females. The very slow- a certain limit, and the heart rate decreases in moving macrotricid, Ilyocryptus agilis, was a starving Daphnia longispina (Ingle et al., 1937). notable exception: it has a heart rate of only There is a diurnal cycle of heart rate: in 120 beats/min at 20C. Daphnia, the maximal heart rate occurs in late Kimographic recording of the Moinodaphnia afternoon (Tonolli, 1947), which may be linked macleayi heart rate was made by Tonapi et al. to the fact that the heart rate generally (1984): it was 105 beats/min in females with- increases at higher temperatures (Fig. 6.6) out embryos, and 120 in females with (Maynard, 1960). The heart rate also increases embryos, 90 in males, and 120 in males after after feeding (Baylor, 1942; Maynard, 1960), molting (at 22C). This is very slow compared depending on temperature and level of illumi- to most other Cladocera. In D. magna without nation, whereas starvation is followed by a embryos, the heart rate is 350 beats/min: with decreased heart rate. The heart rate also embryos, it is 379 beats/min (Kolupaev, 1989). decreases upon illumination with a beam of Remarkably, the heart rate increases in both light directed at the heart (Schulz, 1928) and females and in males by about 10À20% follow- by ultrasound (up to full arrest) (Nikonov ing every kind of disturbance, i.e. the increase et al. (1970). is emotional, (Smirnov, 1965b). When the dis- Salo (1960) presented data showing that the turbance is discontinued, the former heart rate heart rate in D. magna and D. pulex may be is rapidly restored. Otherwise, the heart rate is equal to, higher than, or lower than the beating stable and only significantly drops just before rate of the limbs. The sum of both parameters death. is far from constant, but there was suggested There are numerous data characterizing the to be a compensatory relationship between the level of heart rate as being dependent on vari- rate of movement of the thoracic limbs and the ous factors. Ermakov (1936) found that the heart rate. In view of this fact, their conclusion heart rate of D. magna changes with starvation, of a compensatory relationship between the

PHYSIOLOGY OF THE CLADOCERA 92 6. CIRCULATION

FIGURE 6.6 Heart rate of female and male 400 Daphnia at different temperatures. Modified from Maynard, 1960. 200

100

0 5 10 15 20 25 28 30 Temperature - °C movement rate of the thoracic limbs and the 6.4 HEART REGULATION heart rate (as suggested by Salo, 1960) seems to be highly questionable and demands further Fischel (1908) described the heart nerve in investigation. However, the existence of com- Daphnia with its ganglia; according to Ermakov pensatory tachycardia has been convincingly (1937), the heart of D. magna is innervated by demonstrated in D. magna under conditions of nerves that accelerate or retard its activity. hypoxia (Paul et al., 1997; Pirow and Buchen, Obreshkove (1942) also reported that the move- 2004) and a lack of food (Pirow and Buchen, ments in the heart and intestine of Daphnia are 2004). The heart rate is below 300 beats/min at accelerated or inhibited through nervous sti- 20 kPa O2 and increases to c. 400 beats/min at muli. However, the heart is myogenic and ace- À 3 7 kPa O2. tylcholine is the inhibitory transmitter Skadovskiy (1955) thought that the heart substance (Postmes et al., 1989); further, an rate in daphnias may serve as an index of met- extract of Daphnia has been reported to contain abolic intensity. It is therefore tempting to acetylcholine-like substances (Artemov and apply the heart rate as an index of metabolic Mitropolitanskaya, 1938). Bekker and Krijsman intensity, assuming that they are quantita- (1951) noted the existence “of a myogenic pace- tively proportional (as was thought also, e.g. maker in the heart of Daphnia, inhibited by by Meijering, 1975). Indeed, the heart rate extracardiac cholinergic nerves,” in the absence increases with increasing temperature (Fig. 6.6). of a neurogenic pacemaker. This view is sup- However, its relationship with the metabolic ported by the experimental evidence described rate is certainly not direct (see also Maynard, below. The influence of various substances on 1960, p. 206) and, in view of the emotional reac- the heart rate has been studied in either intact tions, and the occasional retardation and arrest cladocerans or specially prepared specimens. of the heart in active animals, it can hardly be For standardizing the testing of chemical used for estimating metabolic intensity. Our effects on the heart of D. pulex,Le´vy (1927a) observations convince us that the heart rate is a placed the animal in Ringer solution and made stable parameter, which is mainly changed by two incisions in the dorsal integuments: one in emotional factors, and tends to maintain a con- front of the heart and another behind the stant average until death. heart. In most cases, the heart rate stabilized Despite its variability, the heart rate of after about half an hour. Excess potassium Daphnia has also been suggested as a measure ions in the solution resulted in a rapid intensi- for the estimation of pollution by means of a fication of the heart rate, whereas excess cal- recording device (Kiknadze et al., 1983). cium ions inhibited the heart (Le´vy, 1927b).

PHYSIOLOGY OF THE CLADOCERA 6.4 HEART REGULATION 93

Other observations have been made on Flu¨ ckiger (1952) found that the Daphnia intact specimens. The regulatory influence of heart rate is accelerated by L-adrenaline bitar- various substances on the heart rate had been trate, D-adrenaline bitartrate, L-noradrenaline investigated by the end of the nineteenth cen- bitartrate, L-ephedrine hydrochloride, dihy- tury. According to Pickering (1894), atropine droergotamine methansulfonate, and oxyphe- sulfate (3 mg/mL in water) increases the heart nyl ethanomethylamine tartrate. However, rate of Daphnia, and diastolic stoppage occurs L-adrenaline, D-adrenaline, and L-noradrenaline after about half an hour; caffeine also increases retard the heart rate during the first 5À10 min. the heart rate, and large doses culminate in According to Beim et al. (1970), the D. magna systolic stoppage; muscarine nitrate, veratrine, heart rate is retarded (“with some exceptions”) theobromine, and xanthine do not greatly by adrenaline, aminasin, aprophen, atropine, influence the heart rhythm. dihydroergotoxin, ephedrine, isadrin, noradren- Solutions of pilocarpine (1:100 w/v) and aline, pilocarpine, pituitrin, and strophanthin. ergotamine (1: 10,000) both retard the heart Contradictions may be caused by differences in rate: the action of pilocarpine ends in arrhyth- the concentration tested. The heart rate of mia and diastolic arrest, that of atropine—in Daphnia has also been shown to be retarded by arrhythmia and systolic arrest (Ermakov, 1937). choline (Suomalainen (1939), acetylcholine, Atropine retards the heart rate at a concentra- tetraethyl pyrophosphatase, digitalin, rotenone tion 1:50 (w/v) according to Ermakov (1936, (Bekker and Krijsman, 1951), caffeic acid, pro- 1937), but accelerates it at 2.5:10,000À pranolol (Campbell et al., 2004), ergotamine 6.25:100,000 (w/v), according to Bekker and (Ermakov, 1936, 1937), pilocarpine (Bekker and Krijsman (1951). Obviously, the difference in Krijsman, 1951; Ermakov, 1936, 1937; Bekker these reactions depends on the concentration of and Krijsman, 1951), pituitrin (Hykes, 1926), the tested substance. and an extract of the thymus gland of mammals In D. magna, parasympatheticomimetics (e.g. (Hykes, 1926). Phenobarbital strongly decreases mecholyl) do not slow the heart, whereas car- the heart rate of D. pulex to about 25% at a con- diac tonic drugs (e.g. digitalis) cause slowing of centration as low as 2.9 mg/L (Postmeret al., the heart and its dilatation (Sollman and Webb, 1974). Lactose at 50À200 mM also inhibits 1941). According to these authors, the Daphnia the heart rate and causes severe arrhythmia reactions differ qualitatively in many particular in D. pulex (Campbell et al., 2004). It is ways from those of vertebrates. Baylor (1942) thought that lactose directly affects ion channels observed an inhibitory effect of atropine, acetyl- in the heart, an effect that is reversible within À choline, NaHSO3, and KCl on the heart rate of 3 4h. D. magna, whereas adrenaline had an accelerat- Eserine (physostigmine) produces a toxic ing effect. Baylor (1942) noted a similar regula- effect on the heart of D. magna (Baylor, 1942). tion by acetylcholine and potassium (inhibition) The heart rate of D. pulex increases in the in the Daphnia heart to that of vertebrates. presence of serum from mammals, frog (Rana), The heart rate of Daphnia is also accelerated and carp, and then stops in systole (Vatovec by thyroidine (Hykes, 1926), and by adrenaline and Timet, 1952). Such stimulation is irrespec- (a 1:100,000 solution) (Hykes, 1926; Suomalainen, tive of the sex of the donor animal (Vatovec 1939; Ermakov, 1936, 1937). However, Bekker and Timet, 1955). and Krijsman (1951) found adrenaline to acceler- Postmes et al. (1989) studied the effect on ate the heart at higher concentrations (2:10,000) D. magna of adrenoceptor agonists (1-epineph- but to retard the heart rate at lower concentra- rine-bitartrate, 1-norepinephrine-bitartrate, tions (2:1000,000). 1-phenylephrine HCl, phenylterol HCl) and

PHYSIOLOGY OF THE CLADOCERA 94 6. CIRCULATION adrenoceptor antagonists (DL-metoprolol tartrate increased to c. 70 at a dopamine concentration and DL-propranolol-HCl). It was found that epi- of c. 1 mM (Pen˜alva Arana et al., 2007). nephrine could not be blocked by propranolol Results obtained by Villegas-Navarro et al. 1 (an adrenoceptor antagonist), thus suggesting (2003) suggest the presence of Na -ATPase that the drug action is not meditated by adreno- receptors to verapamil and of adrenergic ceptors. Postmes et al. (1989) found epinephrine receptors in D. magna heart. to inhibit the heart rate, contrary to previous The heart rate of D. magna is decreased by reports by Sollman and Webb (1941), Baylor diphenhydramine (DPHM) and increased in (1942), Bekker and Krijsman (1951). the presence of curcumin (Vaidya et al., 2009). The consistent experimental results on the These authors suggest the following explana- efficiency of cholinolytics, obtained using tions: DPHM may prevent the sympathetic D. magna and rats by Tonkopiy, et al. (1994b), action of histamine, and parasympathetic led these authors to the conclusion that acetylcholine (Ach) may bind (ACh-R) onto Daphnia possess M-cholinoreceptors of a myocardial cells and reduce the heart rate. structure similar to those in mammals: a case Curcumin may antagonize histamine N-methyl of parallelism between remote groups. The transferase and thus prevent histamine cholinolytics tested (amedin, amizil, artro- methylation. Vaidya et al. (2009) think that his- pine, cyclodol, glycine, and spasmolytin) pro- tamine may act as a primary cardiac sympa- vide a protective action following intoxication thetic neurotransmitter. of D. magna by arecoline and armine An estrogenic substance (ethynylestradiol) (Tonkopiy, et al., 1994a). causes a significant decrease (to 1/3 of the nor- Dzialowski et al. (2006) investigated the mal rate) in the heart rate of Daphnia (Walker effect of beta-adrenergic receptor antagonists et al., 1998). on D. magna. The lowest observed effective Tasse and Camougis (1965) were the first to concentration for decreasing the heart rate was investigate the cardiac activity of Daphnia elec- 55 μg/L for propranolol and 3.1 mg/L for trographically. They used three methods: metoprolol. 1. The application of remote electrodes Dopamine in a culture solution was found to revealed a correlation between muscle action produce only a slight effect on the Daphnia heart potential and body movements (Fig. 6.7). rate: from c. 66 beats per 10 sec in the control, it

FIGURE 6.7 Electric activity of Daphnia recorded with remote electrodes (lower trace) correlates with the observed body movements (upper trace). Source: Tasse and Camougis (1965).

PHYSIOLOGY OF THE CLADOCERA 6.5 HEART ARREST 95

2. Metallic electrodes placed on the ventral In a 1/106 dilution of γ-aminobutyric acid abdominal region revealed slow, biphasic (or GABA), the frequency of electrograms waves on which faster, smaller waves were decreased Camougis, 1965). superimposed. The magnitude of the biphasic waves varied from 200 to 500 μV, and their frequency from 1.5 to 4 Hz. There 6.5 HEART ARREST were periods of no electrical activity. 3. Glass capillary electrodes punctured the The heart rate of Cladocera is stable and carapace and were placed in the dorsal drops immediate prior to death. Despite region of the heart. This revealed variations this, the heart may stop for rather a long time in the amount of electrical activity, ranging (from minutes up to an hour) in response from a slow, biphasic wave to a fast to provocation without any detrimental monophasic and larger biphasic wave consequences. (Fig. 6.8). Fundamental frequencies ranged For reasons that are unclear, regular activity from 3 to 9 Hz, averaging at 5.4 Hz, with of the heart is sometimes interrupted for long amplitudes from 100 to 150 μV. Handling of intervals in Eurycercus, Camptocercus, Bosmina, the Daphnia sometimes led to cardiac arrest. Ilyocryptus, and Lathonura (Smirnov, 1965b). In

FIGURE 6.8 Electric activity of the Daphnia heart recorded with capillary microelectrodes in different specimens. Source: Tasse and Camougis, 1965.

PHYSIOLOGY OF THE CLADOCERA 96 6. CIRCULATION

L. rectirostris, after a prolonged interval the event of irritation; for example, in Leptodora heart resumed its activity, without any obvious this happens when it sticks to the surface film reason; the discontinuation of heart beats had and the cells become freely circulating again no negative effect on the animal whatsoever. when the Leptodora is liberated. In Leptodora, In copulating C. sphaericus, the heart rate blood cell adhesion also occurs during ether either remains normal or decreases markedly narcotization (Saalfeld, 1936). in both males and females; in females, the Hardy (1892) believed that blood cells that heart sometimes completely stops. absorb fat may attach to the inner substrata It was observed that if the bend of the and thus participate in the formation of the fat digestive tube of Daphnia is touched with a tissue. There has been no explanation of this fine glass needle, then the heart stops immedi- observation in Cladocera and a mechanism for ately (in systolic phase) and the posterior understanding this process has yet been region of the intestine exhibits intensive peri- outlined. stalsis (Obreshkove, 1942). The period of heart arrest can reach 20 min. After a while, both the heart and intestine return to their normal 6.7 PHAGOCYTOSIS activities. Heart arrest is also caused by touch- ing the dorsal surface at the posterior heart When consumed by D. magna, area. It is thought that nerve endings convey Metschnikowiella bicuspidata (syn. Monospora the inhibitory impulses to the heart. After its bicuspidata, Ascomycetes) spores are liberated arrest, the heart resumes beating, but for sev- from their coat and penetrate the body cavity eral minutes the beats are feeble and irregular. through the wall of the intestine. However, Addition of 0.01À1 μg/L acetylcholine results Metchnikoff (1884) observed that D. magna leu- in immediate normalization of the heart beats. kocytes can surround and destroy these spores In Daphnia treated with 10 μg/L physostigmin by phagocytosis (Fig. 6.3); this process allows prior to heart arrest, the recovery from inhibi- some individuals of Daphnia to survive. tion is rapid and complete (Obreshkove, 1942). However, leukocytes do not respond to The general impression is that Cladocera man- Saprolegnia penetrating the body of D. magna, age well without their heart and do not need it and to some other bacterial parasites. much. Metchnikoff initially described these facts in Events related to the mechanism of heart detail in 1884, and then reported them in 1885 arrest in Cladocera deserve further investigation. and in 1892 in “Lectures on comparative pathology of inflammation.” He also observed (Metchnikoff 1888) phagocytosis of spores of 6.6 ADHESION OF BLOOD CELLS the bacterium Pasteuria ramosa infesting Daphnia. Metchnikoff (1885) had also earlier Blood cells are adhesive and sometimes stay noted Daphnia leucocytes surrounding on the surface of organs. This fact may be wounds. observed especially if a local irritation, injury, Hardy (1892) observed that fat globules and or induction shock, is inflicted—to such an other particles are carried from the gut lumen extent that none may remain in circulation through the gut wall by blood cells, and that (Hardy, 1892). In Acroperus, all hemocytes blood cells may attach to inner substrata, thus sometimes become immovable (Smirnov, contributing to the formation of the fat tissue. 1971). Maynard (1960) indicated that in Phagocytosis was also observed by Daphnia blood cells adhere to tissues in the Metchnikoff in some other invertebrates and

PHYSIOLOGY OF THE CLADOCERA 6.8 IMPACT OF XENOBIOTICS ON HEART RATE 97 warm-blooded animals, and he developed (Smirnova, 1960); 500 mg/L Dikonirt (a herbi- these observations into a theory of general bio- cide containing 2,4-D) caused reduction in the logical and medical importance. Despite the heart rate from 5 beats/sec to 1 beat/sec after convenience of transparent cladocerans for 6 h of exposure (Pre´sig and Ve´ro, 1983). In con- these observations, it seems that no one contin- trast, hydroquinone stimulates heart beats ued with these observations on daphnids or (Kiknadze et al., 1983). their relatives. For a long time, there was As little as 0.1 mg/L phenol depresses the therefore no direct information on phagocyto- heart rate of D. magna from 368 beats/min sis in Cladocera. Moreover, Vonk (1960) and (control) to 254 beats/min (Kolupaev, 1988). A 3 Quaglia et al. (1976) did not observe phagocy- 0.1 lethal concentration, 50% (LC50) of the tosis in the midgut of crustaceans and pyrethroid pesticide, phosphatak, was shown Quaglia et al. (1976) assumed that the food is by Saprykina (1996) to depress the heart rate liquefied within the gut and absorbed by the in Daphnia (in the fourth, fifth, and sixth gen- epithelium. erations), stimulate the beating of thoracic A closer examination of D. magna revealed limbs, and considerably stimulate oxygen the presence of two cell types circulating consumption in the first, second, and fourth within the hemolymph (Auld et al., 2010): generations—by 39%, 49%, and 48%, amoeboid cells (about 9 μm long, and identified respectively. as the cells initially described by Metchnikoff) At a comparatively high concentration of can phagocytosis. They attack Pasteuria, and CuSO4, the thermoresistance of the cardiac there is a large increase in the number of amoe- muscle of D. magna is significantly decreased boid cells in parasitized Daphnia. (Pashkova et al., 1998). It may also be added that an alternating magnetic field with a frequency similar to that 6.8 IMPACT OF XENOBIOTICS ON of D. magna heart beats strongly intensified the HEART RATE heart rate (Usanov et al., 2001b). In an alternating low-intensity magnetic field, the heart The available data indicate that xenobiotics rate of D. magna decreased; at low concentra- generally depress the heart rate of Cladocera tions of phenol, inhibition of the D. magna spp. In D. magna, the addition of CdCl2 is fol- heart rate by a magnetic field is higher than lowed by a noticeable decrease in the heart that by this chemical factor (Usanov et al., rate and the beating rate of thoracic limbs 2001b, 2003).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 7

Excretion

7.1 ANATOMICAL BACKGROUND families (Claus, 1875a, pl. XI; He´rouard, 1905). For example, the convolutions are numerous Although Cladocera possess a functioning in Daphnia and Sida, whereas the gland is organ of secretion, Peters (1987, p. 219) believes shorter in Ceriodaphnia and Moina. It is shortest that excretion occurs mostly through the body in Macrothrix and Acroperus, but all of these surface in Cladocera: “the soluble excreta of have a convoluted efferent duct. Daphnia are released through the general body The maxillary gland of Penilia (a marine surface.” The organ of excretion consists of the species) is very different to those of other cla- paired maxillary glands (shell glands) compris- docerans (Leder, 1915): its duct is short, has no ing the nephridium and its convoluted efferent convolutions, and is dilated in the distal part, ducts (Figs. 1.5 and 7.1). The maxillary gland is producing a kind of urinary bladder (Fig. 7.2). situated in the hemocoel, and is therefore The nephridium of the maxillary gland and exposed to the blood, under the anterior area of the antennal gland (the latter has no channel the carapace, and opens to the outside in the and is probably nonfunctional) are all that anterior part of the body (Claus, 1875a). remain of the coelom in Cladocera. Although Judging by the detailed drawings of Claus, the the transparent maxillary gland is discernible opening to the outside is situated within the without any treatment, coelomic sacs may be anterior part of the brood pouch. A detailed made clearly visible by means of intravital scheme of the structure of the nephridium is staining (Fischel, 1908; Dejdar, 1930; Gicklhorn, given by Gicklhorn (1931a) (Fig. 1.5). 1931a, 1931c) with neutral red, methylene blue, He´rouard (1905) described the various Nile blue sulfate, or Bismarck brown. stages of increasing complexity of the maxillary gland that occurs with age and stated that it is relatively simple in Eurycercus. He also 7.2 THE PROCESS OF EXCRETION noted that the carapace adductor muscles are attached at the inner surface of its convolu- The final products of metabolism are gener- tions. The lengths and arrangements of the ally low molecular weight compounds of nitro- convolutions of this gland are somewhat dif- gen, carbon dioxide (CO2), and water. Like ferent in different representatives of particular other Crustacea, Cladocera are mostly

V applied: the reduction in freezing point was 20.34C for the liquid in the coelomic sac of IV the maxillary gland, 20.05 C for the convoluted duct, and 20.01C for the external water. C These data indicate that the urine excreted by I Daphnia is hypoosmotic to hemolymph and slightly hyperosmotic to the external water. II III Thus, in fresh water, the urine of D. magna is isotonic with the hemolymph but hypertonic to the ambient water. The liquid in the excre- FIGURE 7.1 Nephridium of Daphnia. C, coelomic sac; tory canal is hypotonic to both urine and I-V, its ducts; variously colored by intravital staining. Source: Gicklhorn (1931a). hemolymph; this was interpreted as providing evidence of the reabsorption of salts from the urine and of water being excreted. In the case of D. magna acclimated to water with a salinity of 7m (i.e. 7 parts per 1000), the hemolymph, Vac urine, and liquid in the excretory canal of the D. magna are isotonic to the water.

Ag.

Hb. n 7.2.1 Nitrogen Compounds

oe. In Cladocera, the final products of nitrogen metabolism are principally ammonia derived FIGURE 7.2 Nephridium of Penilia. Ag, efferent duct; from protein metabolism, along with a smaller Hb, urinary bladder; n, nerve; oe, opening; Vac, vacuoles percentage of urea (Fig. 7.3) (Parry, 1960; with liquid. Source: Leder (1915) Wiltshire and Lampert, 1999). Although I failed in my attempt to stain the ammonotelic animals, excreting mainly ammo- coelomic sac of the maxillary gland with nia (Vonk, 1960); at least, this is what occurs freshly prepared Nessler’s reagent in order to under aerobic conditions. What little informa- demonstrate the presence of ammonia, further tion is available on Cladocera excretion relates attempts would be worth making. mainly to daphnids, and occasionally to There are few quantitative estimates of the Bosmina spp. liberation of excretion products by Cladocera. Aladin and Plotnikov (1985) carried out an According to Schmidt (1968), adult D. magna outstanding and unique investigation on the fed on green algae excrete 0.17À0.19 µg concentration, estimated by the temperature N/24 h/µg body N. The excretion of ammonia depression point, of the liquid excreted from nitrogen by D. pulex fed on the green alga the maxillary gland (i.e. urine) of Daphnia Chamydomonas was found to be 0.20 µg magna, in comparison with the ambient water N/individual (ind.)/24 h or 5.11 µg N/mg dry and the hemolymph. This is probably the only weight (DW)/24 h (Jacobsen and Comita, case in which a reduction in freezing tempera- 1976). The mean, steady-state release of ammo- ture has been determined for these three nia by D. magna was determined to be media: the hemolymph, urine, and external 11 nmol/mg DW/h by Gardner and Scavia water. Microcryoscopic techniques were (1981). The mean release of nitrogenous

PHYSIOLOGY OF THE CLADOCERA 7.2 THE PROCESS OF EXCRETION 101

maxillary glands (i.e. shell glands) (Fox, 1948) and iron appears in the shell glands of Daphnia that are losing Hb (Smaridge, 1954). The site of Hb breakdown is the fat body. Amino acids are also excreted to the external environment, as shown with reference to D. magna by Gardner and Miller (1981). The estimated quantity is c. 0.13 nmol amino acids/mg DW/h and c. 21 nmol amino acids/ mg DW/h. They also release ammonia, urea, and phosphorus compounds. In the presence of Daphnia galeata fed on green algae, the concentration of dissolved free amino acids in the environment increases from 1 µMbyup to 3 µM over 2 h (Riemann et al., 1986). Recalculated for carbon, the release rate was FIGURE 7.3 Urea concentration in lake water (Lake c. 0.2 µg C/ind./h, i.e. 12% of that ingested. Scho¨see, Germany) in relation to crustacean zooplankton biomass. Source: Wiltshire and Lampert (1999) 7.2.2 Carbohydrates products by well-fed Daphnia was estimated to In addition to the release of carbon dioxide, µ µ be 0.76 g/mg/h ammonia and 0.36 g/mg/h it has been shown that excess ingested carbon (Wiltshire and Lampert, 1999). In contrast, is excreted by D. magna as dissolved organic µ starved Daphnia liberate 0.45 g/mg/h ammonia carbon (Darchambeau et al., 2003). In the pres- À µ and 0.06 0.1 g/mg/h urea. A total of 6.5 min ence of D. magna, the surrounding water is after feeding was discontinued, D. magna release enriched with dissolved organic carbon, ammonia nitrogen at a rate of 80 nmol/mg/h mostly by excretion following food digestion; and phosphorus at 4.5 nmol/mg/h P (i.e. solu- much lower amounts are liberated from feces ble reactive P) (Scavia and Garner, 1982). (He and Wang, 2006a, 2006b). In other Cladocera (i.e. Bosmina, Ceriodaphnia, Daphnia, and Scapholeberis), the 7.2.3 Phosphorus Compounds excretion rate was determined by Ejsmont- À µ Karabin (1984) to be 0.8 2.6 g/mg DW/h Inorganic phosphorus (PO4-P) is released À µ N-NH4 and 0.26 0.58 g/mg DW/h phospho- into the ambient water by D. magna at a rate of rus. In Daphnia, the excretion rate (as summa- 8.4 ng/ind./h (Rigler, 1961a); phosphatase is rized by Peters, 1987) is c. 1 µg DW/h for also released. The release rate of phosphorus nitrogen and c. 0.5 µg DW/h for phosphorus. by Daphnia spp. is variously estimated to be In addition, the rate of release of ammonia 0.91 µg P/mg DW/h (Peters and Rigler, 1973), nitrogen by Ceriodaphnia reticulata has also 1.1À1.5 µg P/mg DW/h (Olsen and Østgaard, been shown to depend on temperature: 1985), or 0.05À1.5 µg P/mg DW/h (as summa- 1.0 mg/g wet weight (WW)/24 h at 15 C, rized by Yuan Hua Wen, 1994 from data from 1.9 mg/g WW/24 h at 22 C, and 2.4 mg/g various authors obtained using radiotracer or WW/24 h at 27 C (Gophen, 1976). chemical methods). The products of hemoglobin (Hb) decompo- Adult D. galeata release 0.06À0.96 µg P/mg sition are excreted by Cladocera through their C/h into the surrounding water (Vadstein

PHYSIOLOGY OF THE CLADOCERA 102 7. EXCRETION et al., 1995). The excretion rate of phosphorus substances accumulated by Cladocera are for D. galeata was estimated by Pe´rez-Martı´nez unaffected by their metabolism. et al. (1995) to be 6.3% of the total phosphorus/h for adults and 16.2% for juveniles. The specific P release rate in D. pulex decreases as 7.3.1 Accumulation of Inorganic the P:C ratio of its food decreases; there is no Substances further release of phosphorus to the environment below a P:C ratio of 6À8 µg P/mg C If not killed by their high concentrations, (Olsen et al., 1986). D. magna may accumulate xenobiotics, Pe´rez-Martı´nez et al. (1995) also obtained e.g. copper (Cu) or lead (Pb) (Holm-Jensen, quantitative data on phosphorus metabolic 1948). When fed with green algae and Euglena, rates in their three compartment model: the Daphnia selectively accumulated higher con- gut, metabolic pool, and structural pool. centrations of sodium (Na), calcium (Ca), scan- Scavia and McFarland (1982) measured phos- dium (Sc), lanthanum (La), neodymium (Nd), phorus release during the molting cycle of indi- zirconium (Zr), chlorine (Cl), bromine (Br), vidual specimens of D. magna in a specially and nickel (Ni) than were present in Euglena, designed incubation flow cell. The rate of phos- whereas the latter contained higher concentra- phorus release was 6.7 times higher at and after tions of various other elements [including mer- ecdysis than at other phases of the life-cycle. cury (Hg) and arsenic (As)] (Cowgill and Phosphorus excretion rates by natural popu- Burns, 1975). The accumulation of such sub- lations in the Bay of Quinte (Lake Ontario) stances is represent by their bioconcentration were estimated by Peters (1975) to be factor (i.e. accumulation coefficient), which is 2.2À4.8 mg P/m2/day for Daphnia, 0.6À1.2 mg the ratio of the concentration of a pollutant P/m2/day for Diaphanosoma, and 0.6À1.3 mg within the body to its concentration in the P/m2/day for Leptodora. Phosphorus release environment. The bioconcentration factor of by Cladocera has seasonal fluctuations: in D. magna for 54Mn (manganese-54) was 65, Swarze˛dzkie Lake (Poland), a peak of reached after 8 h of exposure to the isotope 10À15 µg P/L/day was reached in June solution, with excretion having started within (Kowalczewska-Madura et al., 2007). 2 h of exposure (Kwassnik et al., 1978). There are numerous studies on the accumulation of various metals and compounds, 7.2.4 Water mostly restricted to daphnids. According to Yu and Wang (2002), in D. magna the assimilation As noted above, Cladocera drink water and efficiency from algal food is 30À77% for cad- surplus water is rapidly excreted. According mium (Cd), up to 44% for chromium (Cr), to Peters (1987), about 8% of the body’s water 24À58% for selenium (Se), and 7À66% for zinc is removed by Daphnia every hour. (Zn). Routes for metal removal into the dissolved phase were different for different metals: excretion was the most important 7.3 BIOACCUMULATION route, molting represented 50À70% of daily OF TOXIC SUBSTANCES Cd efflux and 20À70% of Zn; and the major routes of Cr efflux were via excretion and feces It has been shown that Daphnia accumulate egestion. Substantial amounts of Se were various substances that may be useful, alien, released via the production of offspring. The or harmful to their normal life, and some release of metals has an important impact on

PHYSIOLOGY OF THE CLADOCERA 7.3 BIOACCUMULATION OF TOXIC SUBSTANCES 103 the biogeochemical cycling in lakes. The fol- molting and with the liberation of juveniles lowing data have been obtained for particular (Nilov, 1983). elements. Mercury Arsenic More Hg accumulates in D. magna from the Arsenic from food algae is accumulated by Chlorella it consumes in the form of methyl- Moina predominantly as nonmethylated arse- mercury CH3HgCl than as mercuric chloride nic and dimethyl arsenic compounds (Maeda HgCl2 (Boudou and Ribeyre, 1981, 1983). After et al., 1992). Folt (2005) found that Daphnia 48 h exposure to methylmercury (in food), the accumulate As and Hg, with levels accumu- Hg concentration in D. magna is c. 440 ng/g. lated by Daphnia being 2À3 times higher than The bioconcentration factor of HgCl2 by those in copepods. Ceriodaphnia affinis reaches 2000 in the twentieth generation (7.2 µg/g DW) (Gremiachikh Cadmium and Tomilina, 2010). The Cd content in Holopedium collected from natural lakes in Canada ranged from 0.9 Manganese to 31 µg/g (Yan et al., 1990). Bodar et al. The Mn concentration factor of D. magna is (1990a) determined that D. magna can accumu- 65 and maximum uptake is reached after 8 h late c. 115 ng Cd/mg DW/24 h and can of exposure despite active excretion (Kwassnik develop resistance to Cd in a single generation. et al., 1978). In addition, lethal concentrations of Cd differ for different clones of D. magna. Neptunium Upon exposure to Cd, D. magna respond by reducing their Cd assimilation efficiency and Neptunium (Np) is concentrated by D. magna µ ingestion; in addition, metallothioneins are to 40 g/g in 48 h when present in the medium µ formed in its body during detoxification (Guan at a concentration of 1.23 g/mL (Poston et al., and Wang, 2004a). D. magna juveniles obtain 1990). twice as much Cd from water than from algae (Chlorella) (Barata et al., 2002). The gut ceca are Nickel the main sites of Cd and Ca intake. D. magna exposed to Ni in solution accumu- D. magna sensitivity to Cd increases follow- late it in the F1 generation to a concentration ing irradiation by a millimeter-range low- exceeding 18 times that of untreated controls; intensity electromagnetic field (Gapochka at a Ni concentration of up to 42 µg/L, the gly- et al., 2012). cogen content in the body is noticeably D. magna may accumulate Cd over several decreased (Pane et al., 2004). When fed with generations and recover its biological para- Ni-contaminated algae, D. magna can accumu- meters in Cd-free water over one or two gen- late up to 72 µg Ni/g DW (Evens et al., 2009). erations (Guan and Wang, 2006); therefore, Both Ca and Mg reduce Ni toxicity, but Na they have a high potential for recovery follow- has no effect (Deleebeeck et al., 2008). ing Cd contamination. According to Vandenbrouck et al. (2009, p. 18), in D. magna Ni affects functional gene classes Cesium that are “involved in different metabolic pro- In a solution of 137Cs (cesium-137), D. magna cesses (mainly protein and chitin related pro- accumulate radioactive Cs: the accumulation cesses), cuticular turnover, transport and coefficient reaches 84 at day 5, but decreases at signal transduction.”

PHYSIOLOGY OF THE CLADOCERA 104 7. EXCRETION

Silver Zn-coated metal trays in a mineral culture Silver (Ag) is known to be highly toxic for medium. However, Daphnia perish when fed cladocerans (Rodgers et al., 1997), although the with these algae, which indicates high levels of presence of organic matter alleviates this effect Zn accumulation by the algae. Nevertheless, (Glover et al., 2005b). Ag is incorporated into toxicity may depend on Zn concentration. For D. magna, and not merely adsorbed onto its example, the green alga Pseudokirchneriella was surface (Bianchini et al., 2002a, Bianchini and grown at different Zn levels and accumulated Wood, 2003a, 2003b). Ag accumulation by neo- different Zn concentrations suitable for D. magna nates is higher in the presence of sulfide. feeding (Canli, 2005). When Zn concentrations Acute Ag toxicity in D. magna is proportional in the algae increased from 100 to 220 ng/mg 1 to the whole-body uptake of Na (Bianchini DW, the protein content of D. magna increased µ et al., 2002b). Bianchini and Wood (2003a) from 220 to 300 g/mg DW. determined that Ag causes ionoregulatory dis- For D. magna, the optimum range of Zn is À µ turbance resulting in decreased levels of 300 600 g/L; at these concentrations, Daphnia À µ whole-body Na concentration due to the inhi- contain 212 254 g Zn/g DW (Muyssen and 1 bition of Na uptake; in contrast, whole-body Janssen, 2002). Decreased or increased Zn 2 Cl concentration is unaffected. Exposure to levels within the body occur within 1 day. At 1 1 600 µg Zn/L, Zn concentrations in the body AgNO3 also leads to the inhibition of Na ,K - 1 1 À dependent adenosine triphosphatase (N ,K - fluctuate over 2 3-day intervals, suggesting a ATPase), as a direct result of Ag accumulation role for molting in the elimination of Zn. 1 65 in the body. Inhibition of Na uptake by After 24 h of exposure of Daphnia to Zn chronic Ag exposure is explained by the inhi- (zinc-65), the bioconcentration factor was 1110; 1 µ bition of Na channels at the apical “gill” after 30 days in a solution of 250 g Zn/L, µ membrane (Bianchini and Wood, 2003b). D. magna accumulated c. 800 g/g DW (from Diet-borne Ag or Cu (contained in algae both solution and particles containing Zn) grown in metal-contaminated media) contri- (Nilov, 1980). Zn bioaccumulation is enhanced butes no more to Ceriodaphnia dubia survival or by preexposure to Cd (Guan and Wang, 2004a, reproduction than do the equivalent water- 2004b). borne metals (Kolts et al., 2009). In a solution Increased humic acid chelation of Cu, Cd, and Zn decreases their bioavailability (Winner of AgNO3, D. magna accumulate Ag in their gills and digestive tract, but the accumulation and Gauss, 1986). 110 60 137 is twice as high in the presence of sulfide Radionuclides such as Ag, Co, Cs, 54 (Bianchini et al., 2005). The latter fact was and Mn ingested with food are accumulated explained by the presence of sulfide-bound sil- in D. magna (Adam et al., 2002). ver in the digestive tract. Transfer to clean Table 3.11 shows that bioaccumulation rates water for 5 h leads to a significant decrease in by Cladocera may vary widely. Ag concentration in all body compartments. In a solution of AgNO3, D. magna can accumulate c. 11 ng Ag/mg WW in 24 h (Glover et al., 7.3.2 Accumulation of Organic 2005b). Substances Benzo(a)pyrene Zinc D. magna mainly takes up benzo(a)pyrene (a In laboratory cultures, in the author’s experi- polynuclear aromatic hydrocarbon) from solu- ence, the green alga Scenedesmus flourishes in tion; the presence of particles containing this

PHYSIOLOGY OF THE CLADOCERA 7.5 THE ROUTES OF ELIMINATION OF XENOBIOTICS 105 absorbed chemical decreases its accumulation ingested algae and this concentration is (McCarthy, 1983). If fed with Chlorella contami- maintained over the subsequent 72 h, i.e. no sig- nated with hexachlorobenzene (HCB), D. mag- nificant elimination occurs within 72 h (Joaquim- na can accumulate 1.7 µg HCB/kg DW over Justo and Thome´, 1998). Concentrations reached 6 days (Mun˜os et al., 1996). up to 6 ng/g malathion in Simocephalus vetulus Bioaccumulation of benzo(a)pyrene by after 8 h of exposure (Olvera-Herna´ndez et al., D. magna is reduced by humic substances 2004) and up to c. 0.180 mg/g tetradifon in (McCarthy, 1983; Gourlay et al., 2003); the D. magna after 8 h of exposure (Ferrando et al., effect is highest at pH 6.5 (Kukkonen, 1991). In 1996). addition, the accumulation of dehydroacetic acid from humic water by D. magna is lower than that from a nonhumic water control at a 7.4 TRANSFORMATION pH higher than 6; in contrast, at pH 5.5 and OF XENOBIOTICS lower, the effect is reversed (Kukkonen, 1991). Fluoranthene and pyrene bioaccumulation by Consumed toxic substances may be metabo- D. magna is reduced by the presence of humic lized by Cladocera. Thus, As consumed by acids in the medium (Gourlay et al., 2003). Moina macrocopa fed on algae containing µ Na2HAsO4 accumulates up to 111 g As/g Dichlorodiphenyltrichloroethane DW in the form of inorganic As (75%), mono- Dichlorodiphenyltrichloroethane (DDT) is CH3 (8%), and di-CH3 (16.6%), and is then accumulated by D. magna from ambient water mostly excreted (Maeda et al., 1992). principally through their integuments An example of the transformation of an (Table 3.11) (Crosby and Tucker, 1971). The organic toxicant in the body of a cladoceran is total concentration of DDT in Daphnia may the fate of heptachlor absorbed by D. magna reach over 4.2 g/kg; a large part of the DDT is (Fig. 7.4). Heptachlor is metabolized in the found in the carapace and is removed during daphnid body to 1-hydrochlordene, 1-ketoche- molting. The 100% lethal dose (or LD100) was lordene, and 1-hydroxy-2,3-epoxychlordene, as found to be 1100 ng/mL. well as derivatives such as glucosides and sulfates. (Feroz et al., 1990). Fenvalerate Pyrene consumed by D. magna is trans- Fenvalerate causes a decreases rate of filtra- formed into water-soluble metabolites (Ikenaka tion of Chlamydomonas by D. galeata and et al., 2006). Ceriodaphnia lacustris at sublethal concentrations (i.e. in C. lacustris that already contains 0.01 µg/L) (Day and Kaushik, 1987). Rates of 7.5 THE ROUTES OF ELIMINATION algae assimilation also decrease, especially in OF XENOBIOTICS fenvalerate concentrations .0.1 µg/L. Pollutants can penetrate the body of a cla- Lindane doceran via its carapace or its gut along with According to Hansen (1980), D. magna accu- ingested water and food. They then undergo mulates lindane (a chlorinated hydrocarbon). biodegradation and, especially those that are only slightly biodegradable, are accumulated Polychlorinated Biphenyls (to different extents in different tissues), and D. magna can accumulate c. 8 µg polychlori- are gradually removed. The routes of elimina- nated biphenyls (or PCBs)/g in 24 h from tion are excretion, defecation, molting, and

PHYSIOLOGY OF THE CLADOCERA 106 7. EXCRETION

OH Cl6 Cl6 Cl6 Cl ClO Cl

OH Heptachlor Heptachlor epoxide Heptachlor diol

OH Cl Cl6 6 OH Cl6 OH OH O OH Hydroxychlordene Hydroxyepoxychlordene Trihydroxychlordene

Cl6

O Cl6 O Ketochlordene O Ketoepoxychlordene

FIGURE 7.4 Biotransformation of heptachlor within the Daphnia body. Source: Feroz et al. (1990) transfer to eggs. The actual accumulation is the Se and Zn might depend on the transfer of difference between the intake level and the these metals from mother to offspring. The capacity for elimination via these routes. principal pathway of Cd and Zn elimination According to Carney et al. (1986), Cd from the body of D. magna is excretion to the obtained from solution is concentrated in water, a secondary pathway is transfer to neo- the exoskeleton of D. magna, and ecdysis nates, and the pathways used least are by frees the animal from a considerable part of molting and through feces (Guan and Wang the accumulated Cd. Similarly, 65Zn partly (2004b). accumulates in the exoskeleton and is then Cu is principally accumulated by D. magna removed at molting (Winner and Gauss, 1986). from its food; the Cu efflux rate is 0.20%/day The elimination rate of metals via these at high food concentrations, and excretion routes is little affected by the concentration of accounts for 82À84% of the total Cu loss, Cd, Se, or Zn in the ingested food (Guan and while c. 6.6% is transferred to the offspring Wang, 2004a). However, rapid elimination of within 7 days (Zhao et al., 2009).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 8

Osmotic Regulation

8.1 POTENTIAL ANATOMICAL Different species within a genus may toler- BACKGROUND ate different salinities (such as e.g. Moina sp.). Some species can endure variable salinity Ion exchange in Cladocera is thought to be within rather limited ranges but the presence carried out via the neck organ and the epipo- of salts in the external water is obviously nec- dites of the thoracic limbs. Possible routes of essary. Chydorus ovalis perished in triple- ions through the neck organ of cladocerans distilled water (Winberg, 1933), and ordinary were illustrated by Potts and Durning (1980) distilled water caused high mortality levels in (see Fig. 8.1). On the surface of the epipodite Daphnia magna within 24 h (Stobbart et al., of Daphnia, which is reported to be slightly 1977). The obvious reason for this is distur- alkaline (pH 5 8) (Lavrentjeva and Beim, 1978; bance of their salt metabolism. Beim and Lavrentjeva, 1981; Beim et al., 1994), Total salinity (i.e. total dissolved solids; a special cell membrane, presumed to partici- TDS) of fresh waters, where most Cladocera pate in active ion transport (Kikuchi, 1982), live, is within 100À200 mg/L (see e.g. was identified on the side in contact with the Hutchinson, 1957), reaching c. 1000 mg/L in external medium. bodies of hard water (Kitaev, 2007). The salinity of some inland lakes may be c. 30m and may reach the level of saturated brine. As is well known, oceanic salinity is c. 32m 8.2 ENVIRONMENTAL and may be lower in enclosed seas (16m in BACKGROUND the Black Sea—the conventional boundary between marine and brackish waters). Different species of Cladocera live in fresh It should be noted, however, that both the water, brackish water, and saline inland total salt content and the ionic composition waters, and a few live in seawater. A small of inland saline waters and marine waters number of polyphemid species and one cteno- differ; they may also be different in various pod (Penilia) are oceanic. saline lakes.

Sea water Fresh water FIGURE 8.1 Possible routes of ion move- Na Cl ments through neck organ in fresh and sea water. Solid lines, active transport; broken lines, passive diffusion. Source: Potts and Durning Na Cl (1980).

Na Na Cl Na

8.3 WATER BALANCE: the basal grains, nerves, an antennal ganglion, THE IMPERMEABILITY OF and the brain. The contents of the intestine CHITIN TO WATER were then stained and from there the stain penetrated the nephridia, epipodites, and The general water and salt balance of the finally the intestinal epithelium. (See also cladoceran body is maintained by the process Section 4.3.1.) of osmotic regulation through their water intake, intake of minerals with food, and excre- 8.3.2 Water Drinking tion, and through special channels for the removal of ions. Cladocerans drink water and also take in There are two methods of water intake: some water with their food. Water intake Cladocera take some water in through their was observed by Fox (1952) in Daphnia hyalina integument and they also drink water. The latter (24 swallowing movements/min), Moina bra- seems to be the principal source of water intake. chiata (43 swallowing movements/min), Diaphanosoma brachyurum (29 swallowing movements/min), Penilia avirostris (37 swal- 8.3.1 Impermeability of Integuments lowing movements/min), and Bythotrephes Since chitin is unwettable, cladocera are longimanus (95 swallowing movements/min). almost entirely isolated from their aquatic Water intake through the mouth serves to mix medium; they are connected to the external digestive enzymes with the food and pushes water by drinking, taking in water through the the food through the intestine. It is also anal opening, and partly by osmotic regula- involved in osmotic regulation. tion. Cladocera must excrete any excess water. Some direct permeability of the Daphnia 8.3.3 Anal Water Intake body was measured using intravital staining (trypan blue) by Fonviller and Itkin (1938). Anal water intake is a normal characteristic They found that a 0.01% solution of trypan of Cladocera. Several antiperistaltic contrac- blue stained first of all the esthetascs of the tions of the hindgut follow defecation, and the first antenna, and then penetrated further to evacuated volume is compensated for by anal

PHYSIOLOGY OF THE CLADOCERA 8.4 PROCESS OF OSMOTIC REGULATION 109 water intake. Such water intake has been 3. highly saline continental waters (a few known for a long time in Daphnia (Hardy and species); and McDougall, 1894; Chatton, 1920), Sida, 4. the marine environment (a few species of Leptodora, Bythotrephes (Fox, 1952), Ceriodaphnia, cladocera). Moina, and Bosmina, and observed later in chy- There must therefore be special mechanisms dorids and macrothricids (Fryer, 1970; among the cladocera that enable them to exist Smirnov, 1971), but not in the marine Evadne in such different situations. Species within the and Podon (Fox, 1952). same genus, e.g. Moina, live either in fresh Anal water intake may be conveniently waters or in saline inland waters. Among the observed in a specimen placed in a solution of ctenopods, Penilia avirostris is a marine species, neutral red: after defecation, red liquid fills the whereas its morphological counterpart, Sida posterior gut. Anal intake of stained water was crystallina, is strictly a freshwater species. Their also observed by Chatton (1920), Rankin capacity for osmotic regulation demonstrates (1929), and Fryer (1970). It is possible to see that the physiological background of salinity that after defecation, there generally follow tolerance varies within particular species. several antiperistaltic contractions of the hind- Extensive investigations into osmotic regu- gut. If the fecal particles were not expelled suf- lation, mostly in Cladocera and ostracods, ficiently far away, some are occasionally were made by Aladin (1978À1996). Having drawn back inside with the water (as observed applied microcryoscopic methods, Aladin in Daphnia and Sida). (1981) described three principal types of osmotic regulation in Cladocera: 8.4 PROCESS OF OSMOTIC 1. hyperosmotic regulation—in Sididae (except REGULATION for Penilia), Holopedidae, Daphniidae, Moinidae (except for Moina mongolica), Bosminidae, Chydoridae, and The majority of Cladocera spp. live in fresh Macrothricidae; water. However, some species can prosper in a 2. Hypoosmotic—in Penilia; wide range of salinities, up to highly saline 3. Amphiosmotic—in Aralo-Caspian water bodies (Aladin, 1981; Frey, 1993). Podonidae and M. mongolica. However, it is notable that species that can live successfully in saline lakes do not penetrate marine environments. This may be caused by differences in the salt composition of these two 8.4.1 Freshwater Cladocera environments. The osmotic pressure within In freshwater cladocera, hyperosmotic regu- cladocera is thus supported by the intake and lation maintains the hemolymph hyperosmotic efflux of various substances and of water. to the external medium. They drink water Cladocera can propagate in four different (Fox, 1952), must excrete excess water, and environments that differ radically in their must supplement ions lost through excretion salinity: by obtaining them from food and by reabsorp- 1. freshwater (hundreds of species of tion of salts via the nuchal organ (Aladin, Cladocera); 1991). It has been determined that there is 2. slightly saline water (dozens of species), the sodium and chlorine ion uptake in critical boundary of salinity being 5À8m Simocephalus kept in a solution of sodium chlo- (Khlebovich, 1974); ride; in this way, the hemolymph is

PHYSIOLOGY OF THE CLADOCERA 110 8. OSMOTIC REGULATION maintained at a level hyperosmotic to the which favors normal development of the medium (Nimmo, 1966). embryos. According to Aladin, marine clado- According to Fritsche (1917), the freezing cera swallow water and secrete ions: chlorine temperature (or the freezing point) in D. magna ions in the area of the nuchal organ in podo- can be reduced from 20.20Cto20.61C. It is nids, and from the epipodites of the thoracic higher in younger specimens, and there is limbs in Penilia. At the sites of these organs a some relationship with nutrition and egg pro- special succinate dehydrogenase activity was duction, since it is higher in fed specimens identified, also indicating excretion of salts than in starving ones. It decreases during pro- against a concentration gradient. longed parthenogenesis and is, on average, high in specimens with ephippia. Belayev (1950) found there to be considerable interindi- 8.4.3 Amphiosmotic Regulation vidual variation in temperature depression (Δ) Aladin (1982b) also discovered that Caspian of the blood in Daphnia pulex (0.24À0.45C), and Aral podonids can use hyperosmotic Eurycercus glacialis (0.36À0.39C), and osmotic regulation at salinities below 8m and Bythotrephes longimanus (0.35À0.46C). He con- may exploit hypoosmotic osmotic regulation cluded that D. pulex can regulate its osmotic above 8m. This is a special case of cladocerans pressure if the external salinity is not above 5m. living in continental hypersaline waters. M. mongolica can live in salinities up to 88m (with the salt composition of the Aral Sea). At 8.4.2 Marine Cladocera salinities over 8m, it exploits hypoosmotic osmotic regulation; under 8m, hyperosmotic Marine cladocera (podonids and Penilia) osmotic regulation occurs (Aladin, 1982c, have a hypoosmotic type of osmotic regula- 1983). tion, i.e. they keep their hemolymph hypoosmotic to the external seawater (Aladin, 1979, 1994, 1996). To achieve this, they must continu- 8.4.4 Sodium Exchange in Hyperosmotic ously excrete ions through their epipodites Cladocerans and the nuchal organ (Aladin, 1991). In these cladocerans, increased activity of succinate It has been established that sodium is dehydrogenase was found in the cells of the retained better at pH 3 by an inhabitant of acid nuchal organ, indicating intensive cellular environments (pH 3.4À6.3), Acantholeberis cur- metabolism and active salt excretion against virostris, than by D. magna (pH 6.9À10.2); its the concentration gradient. uptake is reduced in acid waters in both spe- By applying the microcryoscopic method (a cies, but more so in D. magna (Potts and Fryer, version of determination of osmotic pressure 1979). Sodium loss is lower at pH 3 than at pH by the freezing point), Aladin (1978, 1979, 7inA. curvirostris, but is four times greater in 1982a, 1996) found that temperature depres- D. magna (Potts and Fryer, 1979). sion of hemolymph in marine podonids ranges Havas and Likens (1985) studied the effect from 20.5Cto20.76C (depression of seawa- of pH on sodium regulation in D. magna and ter at water salinities above 12m is 20.7Cto Daphnia middendorffiana. The rate of Na loss 21.39C), whereas in Penilia it is 20.72C (that (efflux) at pH 4 and below (in hard water) was of seawater is 20.99C). Aladin also deter- compared with that at pH 8.0: Na uptake was mined that marine cladocera support a lower the same in both. In soft water, Na uptake osmotic concentration within the brood pouch, was inhibited by 69% at pH 4.5 (compared

PHYSIOLOGY OF THE CLADOCERA 8.4 PROCESS OF OSMOTIC REGULATION 111 with the pH 6.5 control), and loss (efflux) 200 g aluminum (Al)/L at pH 4.5 enhances Na increased to 125% of the control at pH 4.5. content in the body of Daphnia and prolongs Thus, there are problems with Na regulation survival. below pH 5.5 in soft water and below pH 4.5 Experimental exposure of D. magna to natu- in hard water. ral organic matter promoted the Na loss from Glover and Wood (2005) found that sodium the daphnid to the water, thus resulting in metabolism in D. magna may be disrupted by reduced whole-body Na levels; this was a acidification and by ionoregulatory toxicants. labile process, dependent on the period of pre- Acidification inhibits Na intake. At low pH, exposure (Glover et al., 2005a). calcium (in the form of CaSO4 and as calcium Na uptake and release in D. magna adults gluconate) inhibits Na uptake by D. magna, and neonates was investigated more recently whereas at higher pH and at high Na concen- by Bianchini and Wood (2008): these processes trations, calcium stimulates Na uptake. turned out to have different mechanisms in Daphnias that are depleted of sodium can adults and in neonates (Fig. 8.2). According to restore their normal content of 26.3 mM/kg these authors, in neonates, a proton-coupled 1 1 WW within 15 h (Stobbart et al., 1977). They Na channel is important for whole-body Na do not accumulate excessive Na. Daphnia galea- uptake at the apical membrane. In contrast, ta mendotae survive better when they have a this membrane does not contribute to whole- 1 higher Na content (at 6À10 mg/g dry weight) body Na uptake in adults: adults possess 1 1 1 (Havens, 1992). At pH 4.5, both the Na content only the Na channel associated with Na /H 1 and survival was reduced. A concentration of exchange. In both cases, protons (H ) for the

FIGURE 8.2 Ion transport in Daphnia magna. Source: Bianchini and Wood (2008).

PHYSIOLOGY OF THE CLADOCERA 112 8. OSMOTIC REGULATION transporters are supplied by carbonic anhy- Homeostasis is supported by both osmoreg- drase. In neonates, at the basolateral mem- ulation and excretion, in cooperation with 1 brane Na is pumped to the extracellular fluid other physiological processes. 1 1 1 1 by a Na ,K-ATPase and a Na /Cl 1 2 exchanger, whereas K and Cl move through 1 2 specific channels. In adults, a Na /Cl 8.5 EFFECT OF XENOBIOTICS ON 1 1 2 exchanger is replaced by a Na /K /2Cl OSMOTIC REGULATION cotransporter. Accordingly, the sensitivity of adults and neonates to osmoregulatory toxi- The following data have been reported. Ni 1 cants is different. is toxic to D. magna due to Mg2 antagonism: 1 exposure to Ni affects Mg2 homeostasis (Pane 21 8.4.5 Turgor et al., 2003). In the body of D. magna,Mg is decreased by 18%, and its uptake also Hydrostatic pressure, or turgor (termed also decreased. No noticeable effect of Ni on the 1 2 the hemocoel pressure by Downing, 1974), Ca2 or Cl balance was observed and there within the cladoceran body is supported in the was no acute (i.e. in short-term experiments) same way as has been described for osmotic toxic effect on oxygen consumption. regulation. Its function is the upkeep of pres- Amiloride (N-amidino-3,5-diamino-6-chloro- sure necessary for mechanical actions. pyrazinecarboxamide hydrochloride) inhibits According to Krogh (1939), turgor requires Na influx in D. magna (Glover and Wood, only a small fraction of osmotic pressure. 2005).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 9

Cell and Tissue Metabolism

9.1 METABOLISM IN TISSUES amylase, phosphoglucomutase (Hebert et al., 1989), aspartate aminotransferase, fumarate Steinberg et al. (2010) measured the protein hydratase, and mannose-6-phosphate isomerase content, antioxidant capacity, internal hydro- (Korıˇ´nek and Hebert, 1996). gen peroxide, total ascorbic acid, and free pro- Berges and Ballantyne (1991) found the fol- line levels (ascorbic acid and proline are both lowing enzymes in whole-body homogenates antioxidants) in Daphnia magna homogenates. of D. magna: citrate synthase (CS), lactate dehy- Enzymes are specific proteins that catalyze drogenase (LDH; for anaerobic metabolic activ- metabolic processes. For Cladocera, numerous ity), pyruvate kinase (PK; for anaerobic individual enzymes have now been discovered. metabolic activity), alanine aminotransferase Those involved in food digestion are found in (ala AT), aspartate aminotransferase (asp AT), the digestive tract of Daphnia: these include pro- glutamate dehydrogenase (GDH), nucleoside teases, lipases, amylases, and cellulase (Hasler, diphosphate kinase (NDPK; an anabolic 1937; and as summarized by Hebert, 1978a and enzyme), and glucose-6-phosphate dehydroge- in Section 4.4). Enzymes involved in various nase (G6Pdh; an anabolic enzyme). metabolic processes have also been found in The acetylcholinesterase (AChE) content in Cladocera homogenates (e.g. Hebert, 1973). In D. magna was determined to be 12.7 mU/mg addition, the following enzymes have been protein (Day and Scott, 1990). The AChE con- used to explore genetic variability in Daphnia tent in Daphnia is inversely proportional to the spp.: alkaline phosphatase, esterase, fumarase, body size; this situation is probably caused by glucose-6-phosphate dehydrogenase, lactate an increase in total protein that is not propor- dehydrogenase, glutamate oxaloacetate trans- tional to substrate hydrolysis (Printes and aminase, leucine aminopeptidase, phosphoglu- Callaham, 2003). AChE activity 2.5 μM/L/ cose isomerase, tetrazolium oxidase (Hebert min/g protein in 1-mm long Daphnia and and Moran, 1980; Hebert and McWalter, 1983; decreases to c. 0.5 μM/L/min/g protein in Hebert et al., 1989; Hebert and Finston, 1996, 3-mm long Daphnia. In this system, parathion 1997; Hebert and Wilson, 2000), malate dehy- (O,O-diethyl-O-p-nitrophenyl phosphorothio- drogenase, xanthine dehydrogenase (Hebert ate) inhibits AChE activity and phenobarbital and McWalter, 1983), aldehyde oxidase, negatively affects the protein.

Moina macrocopa AChE was extracted from catalase activity in comparison with a melanic homogenate supernatant by Martinez-Tabche type. Superoxide dismutase and catalase dem- et al. (1997, 1998), and differences in its activity onstrated diurnal variations in activity, with a relative to control has been used to estimate maximum value at midday. In arctic melanic water quality (Martinez-Tabche et al., 1998). D. tenebrosa, however, diurnal fluctuation is AChE activity is inhibited by lead (Pb), insignificant. sodium dodecylbenzenesulfinate, mixtures Boavida and Heath (1984) confirmed that containing these components, and, especially, phosphatases liberated into the environment crude oil (Martinez-Tabche et al., 1997). are produced by D. magna and not derived D. magna AChE is also inhibited in vivo by at from its algal food. 48-h exposure to the median lethal concentration (LC50) of sodium dichromate and sodium molybdate (Diamantino et al., 2000). Both sub- 9.2 EFFECTS OF XENOBIOTICS ON stances inhibit growth and reproduction, with CYTOLOGY AND METABOLIC sodium dichromate being the more toxic. FACTORS Choline esterase from D. magna hydrolyzes acetyl thiocholine iodide, propionylthiocholine When investigating the lethal effect of solu- iodide, and butirylthiocholine iodide; it is tions of NaCl and HgCl2 on Daphnia, highly sensitive to the organophosphate Simocephalus, and Sida, Beklemishev (1923a, dichlorvos (2,2-dichlorovinyl dimethyl phos- 1923b) found that the effect described a curve, phate; DDVP) (Menzikova, 1988). with either one peak or two or more peaks. Acid phosphatase maintains the level of The latter was observed under conditions inorganic phosphorus in cells. It is found in when some specimens survived and then suf- Ceriodaphnia affinis (Tsvetkov et al., 2012), and fered slower intoxication. This indicates that in D. magna the overall activity of the acid several physiological mechanisms are affected phosphatase complex increases following by the toxic agent. exposure to CdCl2 (Zarubin et al., 1997). The lethal concentrations are different for Alkaline phosphatase (AP) is a potential different substances, and can sometimes be biomarker of phosphorus limitation. The high- very low. It has been shown that 100% of est AP activity in D. magna was found at lower Chydorus sphaericus (Smirnov, 1971, 1974) per- temperatures (10 C), and for every 10 C reduc- ish (i.e. 100% lethal dose, or LD100) within 24 h tion in body temperature from 25 C, AP activ- in a 0.00001% solution of AgNO3 (precipitating ity increases by a factor of 1.67 (Wojewodzic mainly on the epipodites), but they can survive et al., 2011). in a 0.0000003% solution. They also died in a Phenoloxidase activity is increased in well- 0.0005% solution of potassium dichromate, but fed D. magna, and wounding stimulated an survived in a 0.0001% solution; and died in increase in enzyme activity. In addition, clones 0.004% neutralized formalin, but survived in a with higher phenoloxidase activity were more 0.001% solution. In solutions of most other resistant to Pasteuria (Mucklow, 2003). salts tested, death occurred at concentrations Borgeraas and Hessen (2002b) observed of 0.5% or more. diurnal variation in the activity of Daphnia A series of similar measurements was made antioxidant enzymes [catalase, glutathione with D. magna: Ag, Cu, and Hg caused a loss 1 S-transferase (GST), and superoxide dismu- of ions in this Daphnia sp., e.g. of Na ions 1 tase]. Hyaline Daphnia showed higher GST and (Holm-Jensen, 1948). Prior to their death, Na superoxide dismutase activities, but lower concentrations were reduced to one-third of

PHYSIOLOGY OF THE CLADOCERA 9.2 EFFECTS OF XENOBIOTICS ON CYTOLOGY AND METABOLIC FACTORS 115 the normal value. It is thought that this situa- the most intensive assimilation of salts. tion is caused by inhibition of the mechanism Exposure of D. magna to sodium selenate responsible for the active uptake of ions. Toxic (Johnson, 1989) also leads to Ca deposition in effects could be eliminated by the addition of the mitochondria of hepatic ceca. glutathione or (for Ag and Cu) of cysteine. The impact of 3-amino-1,2,4-triazole (a her- D. magna exposure to metals led either to an bicide) on newborn Daphnia was investigated increase or decrease in the protein content of by Schultz and Kennedy (1976a). Young the body: the maximum reduction of total pro- Daphnia are immobilized at a concentration of tein (9%) occurred upon exposure to Ni, and 0.1 mg/L; immobilization of all individuals the maximum increase (40%) upon exposure to occurs after about 22 h. The length of exposure Mg (Biesinger and Cristensen, 1972). required to reach immobility is decreased in In a lake with an elevated content of Cu, Ni, animals approaching ecdysis (molting). In par- and Al, Holopedium had a lower lipid content ticular, mitochondria, especially those of mus- and lipid droplets in the body were smaller by cle cells, are affected. Other effects included 21%, compared with those in an unpolluted tissue swelling, myofilament disarrangement, lake (Arts and Sprules, 1987). In the presence and dissociation of membranes. of CuSO4, the excretion of amino acids to the In D. magna, the toxic effect of phenanthrene external environment by D. magna was notice- and 9,10-phenanthrenequinone (PHQ) (polycy- ably increased (Gardner and Miller, 1981). clic aromatic hydrocarbons) was tested both Mercury is thought to produce soluble albu- with and without Cu (Xie et al., 2006). Copper minates that may penetrate deeper into tissues. (Cu) and PHQ generated reactive oxygen spe- In addition, mercuric chloride (HgCl2) has cies, and this process involved mitochondria. proven to be highly toxic to Simocephalus: the The proposed pathways for Cu cycling and the actual killing rates were determined at concen- roles of Cu are shown in Fig. 3.9. In living trations ranging from 0.5 M to 50 μM D. magna cells, Cu1 or superoxide dismutase (Breukelman, 1932). may reduce superoxide radicals to hydrogen 1 In Daphnia,Cu2 and triphenyltin chloride peroxide and cause oxidative damage (Xie cause chromosomal aberrations in cells of et al., 2006). the intestines, and especially in cells of the embryos (Filenko and Lazareva, 1989). At chronic levels of exposure, Zn accumulates 9.2.1 Disturbances to Enzyme Activity mainly in cellular organelles and in heat-stable protein fractions in D. magna (Wang and Guan, Exposure of D. magna to metals (Biesinger 2010). and Cristensen, 1972) leads to either an Gut diverticula (hepatic ceca) are the organs increase or a decrease in glutamic oxaloacetic that are principally attacked by xenobiotics. In transmutase activity within the body: the max- 1 D. magna exposed to 12À52 μgCd2 /L, the gut imum decrease (26%) occurred upon nickel diverticula became shrunken and paralyzed, exposure, and the maximum increase (100%) and Cd granules were found in the mitochon- upon exposure to Mn (a 65% increase occurred dria and microvilli of the distorted ceca following exposure to Mg). (Griffiths, 1980). Sublethal solutions of CoCl2, Exposure of D. magna to xenobiotics inhibits NiCl2,Al2(SO4)3, and Cd(NO3)2 cause degener- enzyme activity: copper (II) chloride (CuCl2) ation of the hepatic ceca in D. magna (Luzgin, inhibition of succinate dehydrogenase activity 1982a). Degradation starts from the apex of the occurs long before a lethal effect is observed cecum, thus indicating that this is the site of (Luzgin, 1982b); parathion (at concentrations

PHYSIOLOGY OF THE CLADOCERA 116 9. CELL AND TISSUE METABOLISM of 0.05À5 μg/L) (Dortland, 1978); and the sur- concentration is reduced by treatment with acri- factants dodecylbenzyl sulfonate and sodium dine, 1,10-phenathroline, benzo(a)quinoline, dodecyl sulfonate inhibit AChE (the enzyme phenantridine, and phenazine (N-heterocyclic that hydrolyzes acetylcholine, a neurotransmit- polyaromatic hydrocarbons) (Feldmanova´ et al., ter) (Guilhermino et al., 2000). 2006). Anticholinesterase compounds are present in Following an initial 10-h exposure, mala- waste water and, being physiologically active, thion decreased both protein content and represent dangers for animal and human health. AChE activity in Simocephalus vetulus; lipid They are released into the environment as final content was reduced after 24 h exposure, but waste products from agricultural, medical, and lipid peroxidation levels increased over the military enterprises or as a result of accidents. whole exposure period (4À50 h) (Olvera- Tonkopiy et al. (1993) investigated the choliner- Herna´ndez et al., 2004). gic system of D. magna by applying various Methoprene (a juvenile hormone agonist) is anticholinesterase compounds, including revers- toxic to endocrine-related processes, i.e. ible inhibitors, organophosphates, and carba- growth, molts, and fecundity, in D. magna mates. Anticholinesterase compounds increased (Olmstead and LeBlanc, 2001a, 2001b). the toxic effect of the myorelaxant ditilin. PK and malate dehydrogenase were inhib- These authors demonstrated that central ited after a 7-day exposure of D. magna to m-cholinolytics reduce the toxicity of armine 0.05 mg/L 3,4-dichloroaniline (Morgado and and aminostigmine. Soares, 1995). However, after exposure for Cholinesterases from D. magna are notice- 14À21 days, the activities of these enzymes ably inhibited by zinc (Zn) (Diamantino et al., were stimulated relative to control. D. magna 2003). Sturm and Hansen (1999) studied cho- steroid hydroxylase is differentially modulated linesterase (ChE) inhibition in D. magna by by exposure to the toxicants phenobarbital, parathion, dichlorvos, and aldicarb [2-methyl- β-naphthoflavone, piperonyl butoxide, and 2(methylthio)propioaldehyde-O-(methylcarba- malathion (Baldwin and LeBlanc, 1994b). moyl)oxime]. The presence of pesticides D. magna exposure to 811 μg/L di-2-ethylhexyl decreases ChE in a dose-dependent manner: phthalate for 21 days resulted in decreased parathion is efficient at 0.1 μg/L, dichlorvos glycogen content (Knowles et al., 1987). at 1 μg/L, and aldicarb at 100 μg/L. A polycyclic aromatic hydrocarbon, pyrene AChE and carboxylesterase (CbE) inhibition is transformed in D. magna with the participa- by organophosphorus pesticides (malathion tion of cytochrome P450 monooxygenase and chlorpyrifos) and carbamate pesticides (Akkanen and Kukkonen, 2003), whereas the (carbofuran) was assessed in D. magna by presence of piperonyl butoxide inhibits pyrene Barata et al. (2004). CbE is more sensitive than biotransformation. AChE to organophosphorus pesticides, but both Exposure of D. magna to 60 μg/L chlorde- are equally sensitive to carbofuran. Mortality cone for 7 days resulted in a reduction in RNA increased at low levels of AChE inhibition by levels from 20 μgtoc.9μg per individual carbofuran, while upon exposure to organo- (McKee and Knowles, 1986); the DNA concen- phosphorus pesticides mortality increased tration decreased from c. 0.6 μg to c. 0.2 μg per when the level of inhibition was . 50%. individual, respectively; and ADP and ATP Glutathione is a thiol-containing tripeptide concentrations decreased after longer exposure (γ-glutamyl-cysteinyl-glycine), which functions times. as a reducing agent in cells (Elliott and Elliott, Enzyme activation by xenobiotics may also 1997). It is found in D. magna cells and its occur under certain conditions. Carbohydrases

PHYSIOLOGY OF THE CLADOCERA 9.3 DETOXIFICATION 117

(estimated by amylolytic activity and saccha- were induced by Cu. The most responsive bio- rase activity) in D. magna were activated by markers of oxidative stress were glutathione the herbicide Roundup (glyphosate) in vitro at peroxidase, catalase, and GST (the key antioxi- concentrations up to 50 mg/L (Filippov et al., dant enzymes). 2010). Upon exposure to Roundup, the overall D. magna lactate dehydrogenase activity is proteolytic activity of D. magna increases and noticeably inhibited by Zn (Diamantino et al., the overall amylolytic activity decreases 2001). D. magna β-galactosidase activity is (Papchenkova et al., 2009); this was interpreted decreased in the presence of fluoranthene (a to indicate an increased role for proteins in phototoxic polycyclic aromatic hydrocarbon) metabolism at sublethal concentrations of following ultraviolet illumination (Hatch and Roundup. No adaptation was observed over Burton, 1999). four generations. The activity of D. magna heme peroxidase is The impact of cadmium (Cd) on delta- significantly increased in kerosene-contaminated aminolevulinic acid (ALA-D) synthesis, an groundwater (Connon et al., 2003). early step in the biosynthesis of heme (porphy- In addition, the inhibition of Daphnia rin), was tested by Berglind (1985). D. magna enzymes by pollutants, seen as reduced fluo- was exposed to cadmium (as CdCl2.2.5 H2O) in rescence, has been suggested as a sensitive tox- concentrations of 0, 0.1, 0.2, 0.4, and 1.6 μg Cd/ icity test (Kubitz et al., 1995). L: ALA-D concentration fluctuated around the The toxicity of strophanthin for D. magna, control values. However, after 16 days the determined by LD50, was found to be different hemoglobin (Hb) content decreased to 31À80% in different seasons, being highest in JanuaryÀ of the control. Further experiments (Berglind, February and lowest in OctoberÀNovember 1986) were made to test the effect of Cd (at (Rautenberg, 1953). concentrations of 0, 0.2, and 2.0 μg/L) and Daphnia exhibit a three-phase attraction or other heavy metals, both separately and in repulsion effect in response to coumarin or cop- combinations. Pb (in concentrations of 0, 0.26, per (II) sulfate (CuSO4) (Heintz, 1964): the ani- and 260 μg/L) was added in the form of lead mals are repulsed at low and high acetate Pb(CH3COO)2 and Zn (0, 020, and concentrations, and attracted at intermediate μ 200 g/L) as zinc sulfate (ZnSO4.7 H2O). ALA-D concentrations. Maximum attraction occurred activity was enhanced by Cd and by at 10 ng/L for coumarin and 600 mg/L for 1 high concentrations of phosphorus (P) Zn, CuSO4. but inhibited by Pb, Pb 1 Cd, Pb 1 Zn, and Pb 1 Cd 1 Zn. Stimulation by Cd was abolished byZn.Hbcontentdidnotdecrease,evenat 9.3 DETOXIFICATION 260 μg/L Pb. The effect of 48-h menadione, paraquat, Cladocera liberation from a toxic substance endosulfan, Cd, or Cu exposure on D. magna may be a result of depurination in a clean enzymes varied (Barata et al., 2005). A low environment or targeted metabolic processes. response of antioxidant enzymes to menadione GSTs, the enzymes that participate in chemi- and endosulfan was observed to correlate cal detoxification, have been isolated from with increasing levels of lipid peroxidation; D. magna (LeBlanc et al., 1988; Baldwin and antioxidant activities were enhanced by para- LeBlanc, 1996). D. magna GSTs are inhibited by quat; there was a low antioxidant enzyme 1,4-benzoquinone and 2,4-dichlorophenoxyacetic response to Cd; and high levels of both antiox- acid (Dierickx, 1987a). The presence of five major idant enzyme activities and lipid peroxidation GSTs has been demonstrated in D. magna,ashas

PHYSIOLOGY OF THE CLADOCERA 118 9. CELL AND TISSUE METABOLISM detoxification of 1-chloro-2,4-dinitrobenzene weight of c. 10 kDa are found in Daphnia. (CDNB) (Dierickx, 1987b). Agents that inhibit or It was found that over 80% of the Cd burden increase the GST content either increase or in the body of D. pulicaria grown in Cd- decrease CDNB toxicity, respectively. containing water is bound by this 10-kDa Detoxification of tannins, which are thought protein (Gingrich et al., 1984). A similar per- to be ingested with algal food, was studied in centage (up to 75%) of the Cd burden in the Daphnia and Simocephalus and found to be car- body of D. magna was also determined to be ried out by special enzymes (cytochromes bound to metallothioneins; increased Cd in P450, esterases, and GSTs) (Ray et al., 2000). daphnids induces subcellular reorganization of Pentachlorophenol is metabolized in essential metals (Cu and Zn), and a higher D. magna by sulfate conjugation; it is then content of these metals in the soluble cellular excreted at a rate of 2.65 nmol/g/h (Kukkonen fraction (Fraysse et al., 2006). and Oikari, 1988). Cytochrome P450 enzymes may contribute In benzo(a)pyrene depurated from D. mag- to detoxification and acclimation of D. magna na, 0.1 of its initial quantity remained after to chronic toxaphene exposure, as cytochrome 40 h (McCarthy, 1983). P450 inhibition by piperonyl butoxide led to a Metallothioneins (special soluble proteins, decline in growth rate, fecundity, and survival the proteins of detoxification) with a molecular (Kashian, 2004).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 10

Growth and Molting

10.1 GROWTH 10.1.1 Life Span The life span of Cladocera species can Increases in the body length of Cladocera reach several months, for example, up to 182 principally occur between molts. For example, days in planktonic cladocerans (Fritsch, 1953). within 10 sec after molting a Daphnia magna In ctenopods, the maximum life duration is was observed to increase in length from between 19 and 74 days, as summarized by 1.3 mm to 1.6 mm (Green, 1956a). Korovchinsky (2004). Newborn Cladocerans grow rapidly. Their growth during prereproductive instars is lin- In culture, chydorids live for about 3 months. For example, Pleuroxus denticulatus lives for 121 ear, and then it becomes slower, especially in days at 15C (Shan, 1969), and Alona costata species that bear two parthenogenetic eggs. lives for up to 140 days, frequently producing Their growth slows down upon reaching parthenogenetic broods (Smirnov, 1965a, 1971), maturity. In mature chydorids, for example, and then dies under the same culture and feed- increments in length become very small; in ing conditions. Much shorter life spans have contrast, in mature daphnids slow growth con- been recorded by various authors for Moina tinues (Zaffagnini, 1964; Smirnov, 1971). macrocopa Information on Cladocera growth was summa- : a maximum of 14 days (Terao and Tanaka, 1928a), 11 days (Razlutskii, 1992), and rized by Frey and Hann (1985). In general, an average longevity of 12.5 days (Chuah et al., growth is accelerated at higher temperatures 2007). The same species lived for up to 15 days and food concentrations but is retarded at in experiments carried out by Garcia et al. lower temperatures, low food concentrations, (2004), but in the studies of Makrushin moderate illumination of 301 lx, and illumina- (Makrushin, 2011; Voronin and Makrushin, tion with green light (Buikema, 1972). Yerba 2006) its life span was only up to 9 days. For and Herna´ndez-Leo´n (2004) identified a signif- Moina rectirostris Moina brachiata icant relationship between somatic growth and (syn. ) the maximum life span was determined to be 28 days in aminoacyl-tRNA synthetase activity, both in culture (Razlutskii, 1992). terms of protein and dry weight.

Naturally, the longevity of any species 40 million heart beats (Meijering and Redfern, depends on its living conditions, including 1962). About 8000 heart beats occur between food and temperature. The life span of Daphnia molting and the liberation of eggs into the spp. decreases under conditions of high tem- brood pouch, according to Meijering (1960). perature (Armitage and Landau, 1982) and Fritsch (1953) recorded that D. magna males oxygen deficit (MacArthur and Baillie, 1929; live for about 30 days and have 19.1 million Zhukova, 1955). At comparatively low tem- heart beats. peratures (810C), which slow down meta- Extreme environmental conditions can bolic processes, the life span of D. magna is reduce life span. For example, the mean life greatly increased (as determined by span of the low-saline species Diaphanosoma MacArthur and Baillie, 1929): it is 25 days at celebensis, decreased from 24 to 5 days at a 28 C, 42 days at 18 C, 88 days at 10 C, and 108 salinity of 30 PSU (practical salinity units) days at 8C. Female Daphnia longispina can live (Achuthankutty et al., 2000). for up to c. 66 days (1600 h) at 20C, but for only 234 days at 5C, whereas male D. longispina lived up to 50 and 170 days, respectively, at 10.1.2 Mortality the same temperatures (Munro and White, 1975). It has been shown that other subopti- Gainutdinov et al. (1997b) found that the mum conditions may also prolong the life presence of sugars in the surrounding water, span of Daphnia (Ingle, 1933). Limited food which is controlled by algae, modifies D. mag- contributes to increased life span in Daphnia na mortality by a mechanism involving sen- (Ingle et al., 1937; Hirosaki, 1953; Skadovskiy, sory cells, amplification of the signal, and 1955; Sterba, 1956b; Steinberg et al., 2010) and modification of neuroendocrine units in the Simocephalus (Hirosaki, 1953) spp. Ingle et al. nervous system. (1937) demonstrated that D. longispina live for Mortality unrelated to predation and related 40% longer under conditions of minimum food to an unidentified infection that caused a 10% compared to well-fed specimens. Starved daily loss of Daphnia hyalina was reported for Daphnia produced fewer young, but the total Lake Constance (Germany) (Gries and Gu¨ de, number of young produced is nearly identical 1999). in poorly fed (which live longer) and well-fed (with a shorter life span) Daphnia. The life span of D. magna is prolonged by heparin (Schechter, 1950). Interestingly, vita- 10.2 MODIFICATION OF FORM min K reduces the life span of D. magna at a concentration of 400 μg/L, whereas it is some- There is sometimes a significant difference what stimulatory at a concentration of about in the body forms of adults and juveniles of 200 μg/L (Schechter, 1950). The longest life the same species, e.g. in some macrothricids. span of D. longispina was recorded when cal- Adults of the same species of Cladocera are cium pantothenate was added to the culture also variable in form; striking examples are medium (Ingle et al., 1937). species of Daphnia and Bosmina, which show Meijering (1958) suggested measuring the numerous phenotypes. This morphological life span in heart beats; D. magna reached 47 plasticity affects the general form, the body million heart beats at 30 instars (about 65 size, and the development of dorsal and poste- days). In another communication, 50% of those rior spines. Previous reports have frequently that survived were reported to have had about investigated these morphological changes, but

PHYSIOLOGY OF THE CLADOCERA 10.2 MODIFICATION OF FORM 121 the physiological prerequisites for such modifi- of water), 50% of D. pulex perished within 6 h, cations of form are currently unknown. and 50% of Eurycercus lamellatus died within 7 h (Smirnov, 1971). Daphnia can perish within a day in shaken or intensively aerated cultures 10.2.1 Mechanical Damage and (Harvey, 1972). Regeneration Specimens (e.g. of Bosmina, Daphnia,or Ceriodaphnia) with obvious signs of injury and Mechanical Damage wound healing, caused for example by preda- Cladocera living on substrata are well pro- tory cyclopoids (Li and Li, 1979), are fre- tected from mechanical damage by their com- quently found in Cladocera samples (Kerfoot, paratively thick carapaces, and their body 1975; Murtaugh, 1981). The proportion of form is modified to enable their penetration of damaged prey may be used as a measure of various littoral obstacles. The thickest carapace predation pressure but these abnormalities is probably that of Pseudochydorus globosus, can also stem from disturbances in embryonic μ which can be up to 12 m (Fryer, 1968). In development. addition, broken structures are frequently present, as well as wounds inflicted by Regeneration predators. In a case of a wound inflicted on a Daphnia as a hole in the valve and followed by regener- Turbulence ation, a decreased hole size was seen in succes- With respect to modification of the general sive instars (Anderson and Brown, 1930; Daphnia form, according to Hutchinson (1953, Anderson, 1933). Within a few hours after an p. 155), turbulence provides “a continual ran- injury, the edge of the wound is surrounded dom stimulation of the sense organs.” by a brown material, which is assumed to be Hutchinson continues (1953, p. 156) that “there clotted blood with oxidized tyrosine. Over the are an enormous number of interconnected course of successive instars, the wound area internal things.” However, the physiological decreases until it is completely closed. mechanisms by which external turbulence Anderson and Brown (1930) have shown that modifies the body form are currently unknown. during wound healing in D. magna, chitin With moderate turbulence, the neonates of secretion starts 60% of the way through the Daphnia cucullata developed large cephalic hel- intermolt period. mets at their older age (Hrbacek, 1959; Laforsch and Tollrian, 2004a). Clot Formation Ermakov (1927) collected a sample of Formation of a blood clot was observed by D. pulex and D. magna in which about 5% of Ermakov (1927, 1929) at sites where an the specimens had damaged antennae or antenna or the shell had been dissected. Some anomalous regenerated structures. In Sida blood initially flows from the wound, but then (females), the percentage of damaged speci- a clot is formed and hemorrhage stops. Most mens can reach 25% (Ermakov, 1929) or even cladocerans can survive such operations. 30% (Korovchinsky, 2004). Wave beating kills A lost appendage is usually regenerated, a noticeable fraction of Chydorus, Daphnia, and although not completely. It has been found Bosmina (Manuilova, 1955). She also recorded experimentally that missing (amputated) seg- that the ratio of living to dead Chydorus sphaer- ments of an antenna may not regenerate; nev- icus reaches 2.9:1 after storms (Manuilova, ertheless, the setae do (Fig. 10.1) (Agar, 1930). 1956). In a shaker (at 200 shakes/min in 60 mL Remarkably, regeneration either restores the

PHYSIOLOGY OF THE CLADOCERA 122 10. GROWTH AND MOLTING

2 3 5 4

7 8 9 10 11 12

FIGURE 10.1 Antenna regeneration in Daphnia over successive instars. Source: Agar (1930).

normal structure or may produce hypermor- which constrains the growth and reproduction phoses, hypomorphoses (submorphoses), tera- of Cladocera. tomorphoses, or dichotomy (as defined by The lowest calcium threshold for the growth Ermakov, 1927, 1929). These facts are impor- of D. pulex juveniles was measured as c. tant for understanding the morphological 1.5 mg/L (Riessen et al., 2011). Early juveniles (morphogenetic) potential of body structures. of D. magna (Hessen et al., 2000a) and Daphnia Strictly speaking, there is no hard boundary galeata (Rukke, 2002) have especially high cal- between variation and teratology (i.e. forma- cium requirements. tion of abnormal structures). According to Shimkevich (1909, 1925), the Chemomorphoses trends shown by anomalies and teratologies The term chemomorphosis was suggested to may be exploited by normal morphogenesis. describe morphological variation caused by Teratologies may easily be reproduced experi- exogenous chemical agents (Hebert and mentally and the events that occur during Grewe, 1985). It has been repeatedly reported subsequent moltings observed. In addition, that the formation of spined morphs of experimental extirpation of the eye has mainly Daphnia (i.e. those with neck teeth) is caused been successful (see Chapter 13, section 13.4.3). by the presence of a predator (Chaoborus) and its excreted kairomones (Havel, 1985; 10.2.2 Chemical Growth Factors Hanazato, 1991; Hanazato and Ooi, 1992; Hunter and Pyle, 2004). Both females and Somatic growth is constrained by food males of Daphnia develop larger protective hel- availability, including the availability of mets or dorsal denticles on their shells in polyunsaturated fatty acids (Schlotz and medium enriched with an extract derived from Martin-Creuzburg, 2011). Cyanobacteria are predators (Chaoborus larvae or Anisops) characterized by a low content of eicosapentae- (Grant and Bayly, 1981; Krueger and Dodson, noic acid (20:5ω3) (Mu¨ ller-Navarra et al., 2000) 1981; Hebert and Grewe, 1985; Spitze, 1992; and a low sterol content (von Elert et al., 2002), Hanazato, 1990; Hanazato and Ooi, 1992;

PHYSIOLOGY OF THE CLADOCERA 10.3 IMPACT OF XENOBIOTICS ON MORPHOLOGY 123

Hanazato, 1995; Repka and Pihlajamaa, 1996). species of Daphnia in water from crowded cul- They also develop a cephalic spine and an tures of the same or another species. elongated tail spine (Brancelj et al. 1996). A reduction in body size and changes to the The morphology of various Daphnia spp. carapace morphology have also been observed, has been shown to change under the influence including reduction in the tail spine length of chemicals (kairomones) liberated into the (in D. lumholtzi and D. ambigua). Kairomones environment by predators (e.g. invertebrates or physical factors seem to either induce or and fish) (Black, 1993; Larsson and Dodson, inhibit the proliferation of tissues. Thus, identi- 1993; Tollrian, 1990, 1993, 1994, 1995). In fication of the chemical controlling factor(s) is D. pulex grown in the presence of predators, the next priority. characteristics of their life cycle are also chan- However, identification of the chemical ged (Black, 1993), for example, including the nature of this factor is easier than elucidating comparatively rapid growth of juveniles. the actual pathway through which it effects Generally effects include elongation of both changes in morphology. It is much more diffi- cephalic helmets and the tail spine. This reac- cult to find out how proliferation is channeled tion also causes an increased rigidity of to produce the modified structure. Barry Daphnia carapace (Laforsche et al., 2004). (2002) applied substances to D. pulex that dif- While working with Daphnia, Jacobs (1980) ferentially affect neurotransmission, including concluded that there are specific growth deter- those that either enhance (nicotine and physo- minants that preferentially act on mitotic rates stigmine) or inhibit (atropine) cholinergic within the Cladocera helmet. Helmet cells are transmission; or stimulate (cis-4-aminocrotonic not supplied with nerve fibers, and their acid, diazepam, and muscimol,) or antagonize growth determinants are carried in the hemo- (bicuculline, picrotoxin, and SR95531) the lymph. Later, Beaton and Hebert (1994, 1997) action of γ-aminobutyric acid (or GABA). identified polyploid cells in the cephalic epi- Neckteeth development was enhanced by phy- dermis of Daphnia; the DNA content in these sostigmine and picrotoxin but suppressed by cells is higher than in the thoracic regions, and atropine. It is thought that these compounds mitotic activity is concentrated around them. influence the cells that release hormones Thus, these cells are assumed to be develop- responsible for the development of neckteeth. mental control centers that govern the shape of Khlebovich and Degtyarev (2005) assumed the head. that two alternative hereditary programs, Morphological reactions to chemicals start corresponding to the typical and defensive in embryos that have shed the third mem- (long-spined) forms, are present in D. pulex,as brane; these have liberated chemosensilla that actinomycin D inhibits the transformation can detect chemicals (Laforsch and Tollrian, between these forms. 2004b). However, morphological changes have been observed in the third instar in D. cucullata and D. pulex, but not in three other species. 10.3 IMPACT OF XENOBIOTICS ON A reverse morphogenetic process was dis- MORPHOLOGY covered by Riessen (1984): loss of helmets was observed in generations of helmeted D. retro- Inorganic and organic xenobiotics cause curva produced in the absence of predators deformities of the carapace, the formation of and with abundant food. The opposite process, abnormal setae on antenna, the disappearance i.e. a reduction in tail spine length, was also of antennal segments, and the abortion of eggs observed by Burns (2000), who cultivated nine and embryos (Fig. 10.2) (Shcherban, 1986).

PHYSIOLOGY OF THE CLADOCERA 124 10. GROWTH AND MOLTING

FIGURE 10.2 Daphnia antenna deformation in a toxic environment. Left, control; right, antenna deformed by a toxic environment. Source: Shcherban (1980).

D. magna growth is inhibited by the verte- development (Trofimova, 1979). In addition, brate antiandrogen, cyproterone acetate; this Laurox-9 causes teratogenic changes in Daphnia compound has no effect on molting and devel- progeny such as deformation of the carapace, opmental parameters at concentrations up to reduction in the number of segments in the 5 mM (which are nontoxic to Daphnia) branches of their antennae, and an increased (LeBlanc and McLachlan, 1999). number of setae on their segments (Shcherban, Copper (Cu) and nickel (Ni) reduced the 1980). induction of neck teeth in D. pulex in the pres- The effects of the insecticide carbamyl and ence of a Chaoborus kairomone (Hunter and the Chaoborus kairomone on the formation of Pyle, 2004); in contrast to Cu, 200 μg/L Ni neck teeth in D. pulex are thought to be syner- decreased the length of neck teeth but gistic (Hanazato and Dodson, 1992). Carbaryl increased their number. Later, Mirza and Pyle inhibits the formation of antipredator morpho- (2009) found that D. pulex neonates from logical features in Bosmina longirostris mothers exposed to a kairomone 1 Cu had (Sakamoto et al., 2009). fewer and shorter neck teeth than those from In D. magna, embryonic abnormalities are mothers exposed to kairomone alone. also caused by fenarimol (an agricultural fun- Zinc (Zn) insufficiency leads to “an increased gicide) via its antiecdysteroidal activity (Mu demand on the animal’s pool of available sele- and LeBlanc, 2002). Malformations of the cara- nium” in Daphnia, resulting in cuticle deteriora- pace and swimming setae are caused by a mix- tion (resembling senescent specimens) and ture of fluxetine and clofibric acid (Flaherty depressed reproduction. The situation is and Dodson, 2005). improved by the addition of 5 ppb (parts per Following exposure to tributyltin chloride, billion) Se (Keating and Caffrey, 1989). D. magna large fat cells (storage cells), which In D. magna, Banlen solution (a herbicide are especially numerous at the posterior curve mixture of 2-methoxi-4-chlorphenoxiacetic acid of the digestive tract, become smaller, glyco- and 2-methoxi-3,6-dichlorbenzoic acid) at a con- gen is lost, rough endoplasmic reticulum centration of 1.33 g/L produced a high percent- became less abundant and more vesicular, and age of juveniles with a deformed carapace, their mitochondria are modified (Bodar et al., obviously due to disturbance in embryonic 1990b).

PHYSIOLOGY OF THE CLADOCERA 10.4 MOLTING 125

10.4 MOLTING removed by movements of the postabdomen, but some stay in inaccessible places until the In Cladocera, molting and growth are inher- next molting. As molts occur every few days, ently interconnected. Periodical molting is they can be considered an instrument of sanita- interconnected with periodic physiological tion for the animal’s surface. Green (1974) processes. The integuments of freshly molted counted an increasing number of attached epi- specimens are soft and thin. bionts during the intermolt period. He also dis- At molting, all integuments of the body, covered that some Peritricha leave Daphnia prior including those of its minutest parts, are to molting, probably due to changes in the removed. Thus, exuvia may provide a conve- cuticula. nient material for the investigation of Molting (e.g. in Daphnia) is accompanied by morphology. a drain of calcium (Ca), P, and carbon (C) Molts may be numerous, reaching e.g. 48 in (Hessen and Rukke 2000a). In D. magna during Acroperus (Smirnov, 1965a). The number of one molt cycle (from one episode of neonate molts is likely to be different in different liberation and molting to the next ones) the cladocerans. content of potassium (K) and Zn decreased but In Anomopods, an embryo undergoes a molt that of Ca and iron (Fe) did not (Yukleevskikh, just after leaving the brood pouch (Kotov, 2000). 1997). After this follow two molts (and accord- Chitobiase (N-acetyl-β-D-glucosaminidase) ingly two instars) prior to maturation in female takes part in exoskeleton degradation and chydorids and Bosmina, and up to six or more recycling, as shown in D. magna. There is some in daphnids and Eurycercus (Frey and Hann, flux of chitobiase from the old to the new cuti- 1985); and two to six molts in ctenopods (and cle. Chitobiase activity varies significantly over even up to 10 in Sida) (Korovchinsky, 2004). In the molt cycle, with a fivefold increase 6 h or D. magna, five or more prereproductive molts less before the next molt (Espie and Roff, have been observed (Anderson, 1932). 1995). During molting, chitobiase is released Juveniles are liberated from the brood with the molting fluid into the aqueous envi- chamber a few hours before molting, and eggs ronment. Duchet et al. (2011) found a positive are released to the brood chamber a few min- correlation between chitobiase activity in the utes after molting (Rammner, 1929; Shan, water and the number of Daphnia neonates 1969). However, molting also occurs in the produced during the observation period. It absence of eggs or when eggs within the brood was therefore assumed that chitobiase activity pouch decompose (Rammner, 1929). may be used to assess the condition of In some benthic forms, such as Ilyocryptus, the population without the destruction of Monospilus, and Oxyurella, valves are retained specimens. during molting in parthenogenetic specimens. Molting is controlled by neurosecretion. However, prior to bisexual reproduction, the Bosch de Aguilar (1969, 1972) concluded that the accumulated valves are discarded. Daphnia esophageal group (see Section 13.2) of the ante- obtusa grown on severely phosphorus (P)- rior region of the nervous system produces a fac- limited green algae (Scenedesmus) are often tor that inhibits molting. Martin-Creuzburg et al. observed with a postmolting exuvium attached (2007) measured ecdysteroid levels during a to their posterior end (Sterner et al., 1993). complete molt cycle in D. magna. Free ecdyster- Cladocerans are attacked by various epibionts oids predominated in whole-body extracts: their and endoparasites. Some of the epibionts are concentration increased sharply in the early

PHYSIOLOGY OF THE CLADOCERA 126 10. GROWTH AND MOLTING premolt stage, followed by a sharp decline prior (1955, p. 211) observed that “molting delimits to ecdysis. It was concluded that molting is the size of arthropods—abundant small forms probably induced by 20-hydroxyecdysone. Only are created—and in this way they adapt to small amounts of ecdysteroids are found in exploit the smallest life spaces and, accord- newly deposited eggs. ingly, undergo extreme adaptation to the It is notable that treatment of D. magna with highly restricted life possibilities of the ‘empty ecdysteroids (ecdysone and 20-hydroxyecdy- cavities’ in nature that they fill” [translated sone)atconcentrationsofupto5μMinthecul- from Russian]. ture medium led to unsuccessful exuviation (Bodar et al., 1990c). When tested on D. magna, β estrogens (i.e. 17 -estradiol, diethylstilbestrol, 10.4.1 Impact of Xenobiotics on Molting and 4-nonylphenol) also inhibited molts, whereas bisphenol A did not (Baldwin et al., 1995). Some xenobiotics can interfere with the In general, molting does not depend on illu- hormone-regulated molting process in arthro- mination or darkness, although illumination pods by acting as antagonists of endogenous with green light accelerates molting (Buikema, ecdysteroids. The exposure of D. magna to 1972). 20 μg Cd/L for 8 days led to a decrease in the In D. magna, the heart rate increases before whole-body ecdysterol concentration from c. molting, decreases after molting, and then 200 to 70 pg ecd equivalent/mg dry weight increases again within several minutes (Bodar et al., 1990b); this was probably the rea- (Meijering and von Reden, 1962). An intermolt son for their unsuccessful exuviation. pigmentation cycle (of carotenoid protein) of When exposed to PCB 29 (2,4,5-trichloribi- fat cells in Simocephalus was reported by Green phenyl), arochlor 1242, and diethyl phthalate, (1966b); a sequence of development of the fat D. magna takes longer than the controls to body and the ovaries (Sterba, 1956a) also fol- complete molting (Zou and Fingerman, 1997). lows the intermolt cycle (Fig. 4.13). Exposure of D. magna to a solution containing More experimental work on metabolic 20-hydroxyecdysone or ponasterone caused events, such as on respiration, during the molt- incomplete ecdysis, followed by premature ing cycle is necessary. An example of such a death (Baldwin et al., 2001). study is the uptake of 85Sr (strontium-85) after molting, which is lost at every molting (Fig. 3.10) in D. magna: this was recorded over several moltings (Marshall et al., 1964). 10.5 SENESCENCE Under natural conditions, Daphnia feeding on blue-green algae (Microcystis) were unable There is only circumstantial evidence con- to shed their old integument although a new cerning senescence in Cladocera. Meijering one had already been produced (Rohrlack (1958a, 1958b, 1962b) suggested measuring et al., 2004). These authors found that blue- biotic time in heart beats, and found that mor- green algae contain the protease inhibitor tality increases at the age of 18 million heart microviridin J. From the gut of Daphnia,it beats for D. magna. There are morphological penetrates into the blood and disrupts normal signs of senescence as well as physiological metabolism, which leads to a disturbance in signs, e.g. decreased heart rate and decreased molting, and thus of swimming and filtration, fecundity (Dudycha, 2003). Dudycha demon- and is followed by death. Cladocera undergo strated that such events may be different in periodic molting. Phylogenetically, Livanov different species of Daphnia. In senile females,

PHYSIOLOGY OF THE CLADOCERA 10.6 IMPACT OF XENOBIOTICS ON LIFE SPAN 127 growth is inhibited and may become negative, senescence program involving the mitochon- as noted by Frey and Hann (1985). dria (Anisimov et al., 2008). In a 55 nM In moribund D. magna, the heart rate solution, the opposite effect was observed, decreases (MacArthur and Baillie, 1929), and obviously due to toxicity. in old age fat within the fat body is depleted, the midgut epithelium is progressively degraded, the muscles degrade, and general activity decreases (Schulze-Robbecke, 1951). 10.6 IMPACT OF XENOBIOTICS As observed in D. magna, old males may ON LIFE SPAN become sterile (Meijering, 1962a). According to Skadovskiy (1955), if well-fed daphnias are Cladocera live in a highly dynamic solution given limited food, then their fecundity containing a great many inorganic and organic decreases by 2.5 times and they manifest signs substances, including xenobiotics. There are of senescence (e.g. decreased heart rate). numerous factual data on mortality rates at Meijering and Redfern (1962) have named increasing concentrations of xenobiotics, and the period before death in D. magna the debile only some are discussed here. D. magna mor- phase. It was noted that moribund D. magna tality was used as a parameter for the assess- lose sodium (Na) (Stobbart et al., 1977). ment of water quality and bottom sediments Makrushin (3011) observed and confirmed his- by Romanenko et al. (2011). tologically that natural death occurs in A 100% lethal dose (or LD100) of Cd com- 1 M. macrocopa before they lose the ability to prised a 2-day exposure to 2 mg/L Cd2 reproduce, whereas senescent Moina sink to (Mardarevich et al., 2001); and a median lethal the bottom as their antennae stop beating. dose (or LD50) was provided by a 2-day expo- With reference to D. magna, the hypothesis sure to a lead (Pb) ion concentration of 1 mg/L was proposed by Gainutdinov et al. (1997a) (Mardarevich et al., 2001). that death results from a program of elimina- The acute toxicity of 17 differently substi- tion of the organism that is regulated by tuted benzaldehydes to D. magna was studied changes in the environment, perceived via by Jin et al. (1998). The bioreactive toxicity of thermo- and chemoreceptors and resulting these compounds was quantitatively depen- from the integrative processing of sensory dent on the nature of the substituted groups. information. The larvicides spinosad and diflubenzuron In Ceriodaphnia affinis cultivated in a 5.5 nM significantly affect D. magna and D. pulex life solution of the antioxidant SkQ1 [10-(60- spans; D. magna is more severely affected than plastoquinonyl)decyltriphenylphosphonium], D. pulex by diflubenzuron (Duchet et al., 2011). longevity increased due to interruption of the

PHYSIOLOGY OF THE CLADOCERA CHAPTER 11

Reproduction

11.1 ANATOMICAL BACKGROUND 11.2 CYCLICITY

Female Cladocera have paired ovaries and Although they may be quite long, periods males have paired testes, along both sides of of parthenogenetic reproduction are inter- the abdomen. In the ovary, there are several rupted by gamogenetic (bisexual) reproduc- four-cell groups: one cell develops into an egg, tion, which results in the formation of dormant while the others are nurse cells that are eggs that serve for the latent period of the life resorbed during egg formation (Weismann, cycle, termed diapause. The period of partheno- 1877a, 1877b). The process of oogenesis was genetic reproduction along with the following investigated more recently by Makrushin period of gamogenetic reproduction was (1966À1980). termed a cycle by Weismann (1880). If there is Spermatogenesis was described by only one cycle per year, the population is Wingstrand (1978). The sperm of different spe- called monocyclic; if there is more than one cies were found to be significantly different in cycle, then it is called dicyclic, tricyclic,orpoly- form, sometimes even in representatives of the cyclic. When there is no period of bisexual same genus. reproduction, it is called acyclic. Males emerge Parthenogenetic females either lay many from eggs that show no external differences eggs into the brood pouch (i.e. they are poly- from the usual parthenogenetic eggs (Banta embryonic) or normally lay only two eggs and Brown, 1924). (most chydorids). In polyembryonic species, According to Vereshchagin (1912), this the number of eggs is not fixed and may cyclicity depends on the latitude (i.e. on the decrease if the food supply is insufficient. length of the ice-free period), but varies in dif- Cladocera are one of the rare groups of ani- ferent species. Frey (1982) considered the gen- mals, together with aphids, that reproduce eration change further and noted that in the principally through parthenogenesis. With the tropics gamogenesis is rather irregular and is exception of Leptodora, they have no larval unrelated to any particular season. He also stages in the free-living period of their life noted that in the northern United States, as in cycle. Europe, gamogenesis occurs prior to winter for

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00011-2 129 © 2014 Elsevier Inc. All rights reserved. 130 11. REPRODUCTION most species. Further south, gamogenesis in the shell; in anomopods) or without a special “northern species” becomes less intensive and case (in ctenopods). tends to occur in spring, whereas in “tropical The oocytes growing in the ovaries derive species” gamogenesis occurs irregularly. their fat from the fat body, and thus the con- Under laboratory conditions, the period of tent of fat in the fat body is inversely propor- parthenogenetic reproduction, i.e. when only tional to the development of the oocytes diploid females are produced, may be almost (Sterba, 1956a). Fat reserves (especially trigly- indefinitely long. Continuous parthenogenetic cerol) are transferred to the ovaries late in each reproduction of some Cladocera spp. has been intermolt period (Tessier et al., 1983). observed in the laboratory for up to 13 years Although Cladocera seem to be able to (Banta, 1925). In culture, over 700 uninter- reproduce through an almost indefinitely long rupted parthenogenetic generations have been number of parthenogenetic generations, it has recorded for Daphnia pulex and Moina macrocopa been shown that inbreeding D. longispina after (Banta and Brown, 1929). the 279th, 278th, 442nd, and 570th generation Part of Daphnia population may pass into results in an increasingly lower hatching rate gamogenesis while the rest of it continues par- of sexual eggs and the production of dwarfs, thenogenetic reproduction, as shown, for exam- sterile individuals, specimens that produce ple, with reference to Daphnia longispina by nonviable parthenogenetic eggs, fewer young, Manuilova (1959). In addition, parthenogenetic and of mainly or all-male young (Banta and reproduction may be resumed after the forma- Wood, 1937). tion of gamogenetic eggs and ephippia, as Addition to the culture medium of gonado- has been shown in Daphnia (Sklayrova, 1938; tropin, oxytocin, prefison, or prednisolone Green, 1956a; Makrushin, 1968) and Moina modified the life cycle parameters of Daphnia (Makrushin, 1968). For Daphnia, Simocephalus, magna (Vinokurova, 1977). Following the addi- and Moina, the same female may, in the same tion of gonadotropin, oxytocin, or prednisolone, brood, produce (1) parthenogenetic females, Daphnia reach maturity earlier. Embryogenesis (2) parthenogenetic females and males, or (3) is accelerated by gonadotropin, whereas prefi- parthenogenetic females, gamogenetic females, son retards maturation and embryogenesis. and males (Papanicolau, 1910; Agar, 1920).

11.3.1 Fecundity 11.3 PARTHENOGENETIC Cladocera may produce dozens of litters REPRODUCTION during their lifetime; thus, one specimen may potentially produce large numbers of progeny. Parthenogenetic reproduction yields prog- According to Wojnarovich (1958, 1959), this eny that immediately start an active life of can be up to 15 litters in D. magna, totaling up their own upon liberation from the parental to 1055 juveniles. For one specimen of D. mag- brood pouch. There are no free-living larval na, Shpet (1968) recorded the potential prog- stages in Cladocera, except in Leptodora. After eny to be 106 individuals. Moina species are an indefinite number of parthenogenetic gen- even more prolific. erations, a cladoceran species may pass into In polyembryonic cladocerans, the number gamogenetic (bisexual) reproduction, during of parthenogenetic eggs produced is directly which dormant eggs are produced, either sur- proportional to body size (Green, 1954), varies rounded by an ephippium (a reinforced part of with season, and depends on environmental

PHYSIOLOGY OF THE CLADOCERA 11.3 PARTHENOGENETIC REPRODUCTION 131 conditions. Dormant eggs are less numerous: of embryos was also noted for Daphnia, Evadne, one is present in ephippium of chydorids, one and Podonevadne by Rivier (1974). Moreover, or two in daphnids, one or two in Moina, and Ramult (1926) found variations in the vitality up to 11 in Eurycercus lamellatus (Smirnov, of eggs within the same brood following expo- 1971). sure to solutions of mineral salts. Reproduction and molting in Cladocera are Dependence of Fecundity on Food profoundly influenced by the photoperiod Abundance: Lag Effect (Parker, 1966). It is thought that the photo- Cladocera species have either a few large par- period influences production of a correspond- thenogenetic eggs in a single brood (Aloninae ing neurohumor. It was reported that marine and most Chydorinae) or numerous small eggs. representatives of Polyphemoidea (Pseudevadne The diversity of life histories within Cladocera, and Evadne) release neonates during total dark- with respect to predation and food resources, ness, i.e. between 10 PM and 4 AM (Onbe´, 2002). was analyzed by Lynch (1989). Egg production in D. magna is reduced at Polyembryonic families include the low concentrations of calcium (Ca) (Hessen Daphniidae, Moinidae, Sididae, Eurycercidae, and Rukke, 2000a, 2000b). and some of the Macrothricidae. In polyembryonic daphnids, the number of eggs per indi- Effect of Resource Quantity vidual is highly variable and may reach c. 40, Planktonic Cladocera exist under conditions depending on environmental conditions. For of fluctuating food stocks due to factors includ- Simocephalus, the number of eggs in a single ing the periodic development of algae. When female may reach 40, but is 20 on average food is in short supply, they produce smaller (as (Green, 1966a). shown e.g. for D. pulex by Lynch, 1989) and Except for Archepleuroxus, chydorids bear fewer eggs. Tessier and Consolatti (1991) also two parthenogenetic eggs or a single latent observed that the quantity and weight of egg. When they have inadequate food, chydor- Daphnia neonates depend on the level of avail- ids produce one parthenogenetic egg or none able food and the C:N ratio of the food: they at all. A very rare exception was observed in called this “adaptive plasticity in reproduction.” Kosino Lakes (Moscow): two Chydorus sphaeri- For reactions to starvation, see also Chapter 4, cus females contained three parthenogenetic section 4.6. eggs (instead of the usual two), a phenomenon never previously seen during decades of work Reproductive Constraint at Low Food with thousands of samples from every conti- Concentrations nent. A still rarer case was reported by Under unfavorable low-food conditions, Michael and Frey (1984.p. 94) for the usually D. pulex produces fewer eggs e.g. two, or even two-egged Disparalona rostrata: “quite a num- one (Pyatakov, 1956). When there is an ber of D. rostrata females were carrying from extremely large reduction in the number of 4 to 10 small parthenogenetic eggs. This is the eggs and the formation of only one egg during first time in our examination of many thou- the intermolt period, the ovaries function alter- sands of parthenogenetic females from many nately. The fewer eggs produced are also different species.” larger. The clutch size is smaller when food The size of juveniles within the same brood supply is limited, as shown for Daphnia (e.g. may vary, as noted with reference to by Gliwicz and Noavida, 1996). As shown Simocephalus (Shkorbatov, 1953) and D. magna under experimental conditions by Gliwicz and (Green, 1956a). Nonsynchronous development Guisande (1992), when food is scarce, Daphnia

PHYSIOLOGY OF THE CLADOCERA 132 11. REPRODUCTION produce small clutches of comparatively large food availability to the females (Trubetskova eggs; in contrast, eggs produced with abun- and Lampert, 1995). The eggs are very strong dant food are larger, and offspring hatched and the egg envelopes of Simocephalus have from larger eggs can survive for longer during been shown to withstand centrifugation at periods of starvation. 1058 3 g (Hoshi, 1950a). The size (volume) of parthenogenetic eggs Effect of Resource Quality varies within the same species and depends on Vitamins have been shown to affect repro- the season and the length of the females (Green, duction. In D. pulex, the number of progeny 1956a). In Bosmina longirostris, Kerfoot (1974) was highest at a vitamin B12 concentration of found that the females carry small parthenoge- 0.75 g/L; when deprived of vitamin B12,they netic eggs in spring-summer; in late fall, larger did not produce viable progeny (Keating, 1985). eggs are produced that contain double the yolk Following the addition of pantothenic acid to volume and produce larger young. This cycle is, food consisting solely of Chlamydomonas,thelife however, not strongly associated with nutri- span of Daphnia increased by a factor of three tional conditions. Egg size in D. magna was larg- (Fritsch, 1953). est under conditions of low food availability and small at starvation level in the study of Trubetskova and Lampert (1995). 11.3.2 Impact of Environmental Factors The chemical composition of Daphnia hyalina eggs 9.3% DW of nitrogen (N), 53.6% DW of car- The most intensive M. macrocopa reproduc- bon (C), and 1.2 % DW of phosphorus (P) tion was recorded at 28 C by Terao and (Baudouin and Ravera, 1972). The P content of Tanaka (1928b). Within the temperature range D. magna eggs did not depend on its level in to which they were exposed, the highest fecun- foodandvariedonlyslightly,withinc. dity was measured at .30 CinLatonopsis cf. 14À14.6 mg P/g DW, with large variations in the australis and at 20À30 CinDiaphanosoma bra- C:P ratio (Becker and Boersma, 2005). chyurum (Chaparro-Herrera et al., 2011). The color of parthenogenetic eggs is brown- Effect of Xenobiotics on Fecundity ish, but may vary within the same species. Eggs may be green (“ova plerumque viridis”) Since the first publications on toxicological (Mu¨ ller, 1785, p. 81; Sars, 1862), greenish-blue, experiments, numerous data have been reported orange, or brown in Daphnia (Konovalov and on fecundity decreasing upon exposure to xeno- Konovalova, 1955) and blue or violet in Moina biotics. For example, following exposure to (Papanicolau, 1910). Clearly depending on the À 21 25 50 nM Cd , D. magna fecundity decreased type of food available, parthenogenetic eggs of from c. 6.8 to 2 neonates/female, and at 100 nM 21 Acroperus harpae may be gray, green, orange, or Cd , it declined to zero (Baillieul and Blust, violet, all of which develop into viable 1999). progeny. Hemoglobin (Hb) is passed into developing 11.3.3 The Physiology of oocytes in the Daphnia ovary, and the transfer Parthenogenetic Eggs and Embryos is especially intensive in the last few hours prior to egg laying (Dresel, 1948). Two possi- The physiological traits of eggs and their early ble explanations for this were suggested by developmental stages will be described next. Dresel: either the Hb molecules can pass The density of D. magna eggs is about through the cell membranes or they must be 0.37 mg dry weight (DW)/mm3, irrespective of broken and rapidly resynthesized in the

PHYSIOLOGY OF THE CLADOCERA 11.3 PARTHENOGENETIC REPRODUCTION 133 oocytes. Before parthenogenetic eggs are laid, animals and at 30C for embryos from red ani- they receive Hb from the blood at the end of mals (Kobayashi et al., 1987). each instar, as shown for Daphnia by Green After being laid into the brood pouch, the (1965b); the parthenogenetic eggs of parthenogenetic eggs of most cladocera Simocephalus also contain Hb (Hoshi, 1957). develop using the resources contained within The Hb content in eggs is 0.17 mg Hb/g DW the egg, independent of the maternal metabo- from pale D. magna and 0.53 mg Hb/g DW lism. The eggs may, therefore, be cultivated in from red D. magna (Kobayashi and Nezu, a watch glass (Krogh, 1939; Kobayashi et al., 1986). During their development, the Hb con- 1987; Sobral et al., 2001). They develop well if tent of embryos decreases (Fox, 1948), but removed no earlier than the first hour after embryos with their Hb blocked by carbon transfer to the brood chamber (Krogh, 1939). monoxide (CO) remain normally active (Fox, However, monoids and polyphemids are an 1948). The Hb content is higher in early exception (Weissman, 1877a, 1887b) in that embryos of D. magna than in the hemolymph Polyphemus females produce small eggs and pos- of adults, and in an anoxic environment a lon- sess placenta (the original German term is ger time is required for Hb deoxygenation in Na¨hrboden) to supply nutrition for the eggs devel- early embryos than in adults (Kobayashi and oping in the brood chamber. Patt (1947) presented Takahashi, 1993). The Hb content in a single cytological evidence of this process (Fig. 11.1). D. magna embryo was measured as 0.12 μg A kind of placenta is also present in Moina. (Kobayashi et al., 1987). Moreover, the oxygen In other Cladocera, parthenogenetic eggs affinity of Hb in D. magna embryos is higher may be successfully cultivated in water, out- than in adults (Hoshi et al., 1977). side the brood chamber; this has been The carotenoid content of cladocera eggs demonstrated for Simocephalus, Scapholeberis, depends on the quality of the food consumed Eurycercus (Rammner, 1933; Hoshi, 1951b), by the adult (Green, 1957). Developing eggs are Daphnia (Obreshkove and Fraser, 1940a, colored green or blue by carotenoid protein; in 1940b), Ceriodaphnia reticulata (Shuba and contrast, in fully developed embryos that are Costa, 1972), and Chydorus (Green, 1961b). about to be released, the fat globules become As shown for Simocephalus vetulus, lipid orange colored due to the release of carotenoid oxidation predominates in early embryos, from the bound protein, as was observed by whereas free-living individuals rely on carbo- Green (1957) in many littoral and planktonic hydrate oxidation (Hoshi, 1951). species. About half of the mother’s carotenoids The sources of material for the development are transferred to the eggs of each brood of cladoceran eggs are the yolk and oil. In a (Herring, 1968). This pigment is not utilized by developing egg, the initial large fat globule the developing eggs or embryos and its pres- diminishes in size and splits (Green, 1957). ence does not enhance either the viability or fer- Later in the developed embryo, the fat is dis- tility of the eggs. Carotenoids have never been tributed as small globules in the fat cells. observed in Leptodora eggs (Green, 1957). Relatively larger eggs convey to the embryos a The hatchability of D. magna eggs is best greater relative amount of triacylglycerol (100%) at an oxygen tension c. 15 torr (approx- that is left over from embryonic development imately 2000 Pa), and it dropped at c. 12 torr (Goulden et al., 1987). (approximately 1600 Pa). Hatchability was also The egg membrane is slightly permeable to highest at temperatures of 10À27C and water (Krogh, 1939). The osmotic pressure in par- abruptly decreased at 30C; 50% hatchability thenogenetic and fertilized winter eggs was recorded at 29C for embryos from pale is 57À72 mM and reaches 216 mM within

PHYSIOLOGY OF THE CLADOCERA 134 11. REPRODUCTION

Nb. b.c. Nb. of about 5m; during embryogenesis, this does not Nb. b.c. hyp. b.c. bl.s. decrease below 4m (Aladin and Valdivia Vilar, ov. ov. ov. 1987). When they start feeding, hypertonia abruptly increases because the total osmotic concentration of the hemolymph initially depends gut on the amount of ingested salts. 12 3 b.c. The respiration rates of D. magna eggs and emb early embryos are about a third of those of Nb. neonates and adults (Glazier, 1991). Thus, in hyp. brooding females they take a small proportion of the total oxygen consumption. It was Nb. reported in Daphnia embryos that oxygen consumption per unit weight increases at later F.c. 45 6developmental stages, in contrast to the postembryonic period (Eremova, 1991). Dorsal blood sinus The peak of RNA concentration occurs at the last stages of D. pulex embryo development and G.v. is followed by a gradual decrease during juvenile development (Gorokhova and Kyle, 2002), whereas the DNA concentration is highest in the nu. early stages of postnatal development. In their early stages, Simocephalus embryo development is mainly due to fat metabolism; at m.f. later stages, close to the emergence of juveniles, the respiratory quotient (RQ) shifts toward predominantly carbohydrate (i.e. glycogen) metabolism (Hoshi, 1950b, 1951): at the gastrula stage, Direction of secretion o.g. the RQ is 0.74À0.8; in an embryo liberated from v. the egg’s envelope, the RQ is 0.88; and in s.p. newborn juveniles, it is 0.99. In the embryo, gly- ch. cogen is firmly bound to protein and fat; the gly- 7 Brood chamber cogen content increases in the organs of the developing embryo and then drops in there- FIGURE 11.1 Placenta of Polyphemus during develop- À leased young (Hoshi, 1951a). Hoshi believed that ment. 1, a female; 2 6, development stages; 7, a typical free-living S. vetulus young predominantly use Na¨hrboden cell; b.c., brood chamber; bl.s., dorsal blood sinus; ch., hypodermis and chitin lining brood chamber; carbohydrate as a metabolic substrate. emb., embryo; f.c., marginal fold cells of Na¨hrboden; G. In Simocephalus, the longest survival under v., Golgi bodies; hyp., hypodermis cell; m.f., mitochon- anaerobic conditions occurs during gastrula, dria; Nb., Na¨hrboden; nu., nucleus; o.g., osmiophilic suggesting that anaerobic metabolism predo- bodies; Ov., ovary; s.p., secretion plate; v., secretion vac- minates in this period (Hoshi, 1957). uole. Source: Patt (1947). It has been suggested that Ceriodaphnia eggs can delay hatching in the presence of chemical 50À80 h (Krogh, 1939). In Cladocera with an cues from predators (Blaustein, 1997). open brood chamber, the total osmotic concentra- Finally, it may be added that D. pulex parthe- tion of fluid in the embryo is supported at a level nogenetic eggs are reported to have remained

PHYSIOLOGY OF THE CLADOCERA 11.3 PARTHENOGENETIC REPRODUCTION 135 viable (at least some of them) and to have Maternal exposure of D. magna to the fungi- hatched after passing through the digestive tract cide propiconazole at 0.25 mg/L led to devel- of a fish (Carassius auratus) (Samchyshyna, 2002). opmental arrest at later stages of embryonic maturation, and thus to embryonic abnormalities and death (Kast-Hutcheson et al., 2001). Effects of Extreme Limits of Environmental Methyl farnesoate, pyriproxyfen, and fenoxy- Factors and Xenobiotics on Eggs and carb [ethyl 2-(4-phenoxyphenoxy) ethylcarba- Embryos mate; a juvenile hormone analog] all disrupt At the extreme limits of temperature, ecdysteroid-regulated aspects of embryo devel- Cladocera do not reproduce. During several opment in D. magna; exposure of ecdysteroid- weeks at 5C, no reproduction occurred in responsive cells from D. magna embryos to C. sphaericus and Pleuroxus denticulatus (Keen, 20-hydroxyecdysterone reduces cell prolifera- 1971). Food limitation results in decreased tion (Mu and LeBlanc, 2004). fecundity and interrupted reproduction. The insecticide tanrek blocks the oocyte A pH above 10, which often occurs in lakes, development in D. magna and is directly toxic is detrimental for Daphnia galeata egg develop- to newborns and adults (Papchenkova, 2012). ment (Vijverberg et al., 1996). Hanazato and Dodson (1992) considered the The effect of xenobiotics has mainly been effect of the Chaoborus kairomone and the tested in adult cladocerans. However, it has insecticide carbamyl on D. pulex neckteeth for- been shown, with reference to D. magna, that mation to be synergic. eggs and embryos are sensitive to cadmium (Cd), copper (Cu), dodecyl benzyl sulfonate, 11.3.4 Abortion of Eggs and 3,4-dichloroaniline (Sobral et al., 2001). Mercury (Hg) at concentrations of 0.32 μg/L A considerable proportion of the parthenoge- and higher is highly toxic to embryos, as netic eggs within a brood may be nonviable. In shown with reference to D. magna (Khangarot D. cucullata and D. galeata,20À35% of females and Das, 2009). The number of D. magna carried a number of nondeveloping eggs and the embryos was also affected by 15 aniline deri- maximum proportion in such females was 70% vatives in a dose-dependent manner, although (Boersma and Vijverberg, 1995). These authors no morphological abnormalities were induced noted that “it might be a more common phenom- (Abe et al., 2001). The total number of off- enon than is supposed” and mention nutritional spring was slightly decreased in D. magna deficiency as a possible cause. Threlkeld (1979, exposed to 17α-ethynylestradiol and norethin- p. 611) noted that “[i]t is unclear how wide- drone (Goto and Hiromi, 2003). In D. magna, spread such egg abortion is.” According to more 4-nonylphenol can be directly toxic and recent evidence, this is a widespread and quanti- embryotoxic, depending on its concentration tatively significant event under normal, natural (LeBlanc et al., 2000): this substance interferes conditions. In addition, some of the embryos with the metabolic elimination of testosterone. may be resorbed during development. Exposure of maternal Daphnia to testosterone In Daphnia, egg mortality and the abortion of caused abnormalities in the development of embryos seem to be caused by nutritional defi- their embryos. However, 4-nonylphenol- ciency (Gajewskaja, 1949; Boersma and induced abnormalities are different from those Vijverberg, 1995). In D. rosea,undernaturalcon- caused by testosterone. The concentration ditions eggs degeneration can reach 30% of their threshold for the induction of developmental total number when there is an inadequate con- abnormalities is rather low (c. 44 μg/L). centration of algal food (Redfield, 1981).

PHYSIOLOGY OF THE CLADOCERA 136 11. REPRODUCTION

Resorption of embryos under extreme conditions beginning of 1994, although some experimen- has been observed in D. pulex, D. pamirensis, tal work was done earlier by Dehn (1950, Evadne,andPodonevadne (Rivier, 1974), and 1955). Dehn (1955) was probably the first to Burgis (1967) recorded the presence of “sterile” draw attention to fat in food as a factor con- (nondeveloping) eggs in Ceriodaphnia.For trolling cladoceran population dynamics. Bythotrephes, this has been demonstrated to be a While experimenting with Moina, she found natural process, not necessarily related to water that higher quantities of fat, which occur in pollution (Rivier, 2005). declining algal populations, stimulate the In daphnids, parthenogenetic eggs have appearance of males. Dehn (1950, 1955) also been shown to develop successfully outside found that feeding Moina with whole or the brood chamber (Obreshkove and Fraser, defatted yeast with an addition of an extract 1940a, 1940b); therefore, when liberated into (containing ergosterin) resulted in the produc- the brood chamber, they do not receive any tion of 30% males, as well as females contain- nutrition from the female. Egg mortality must ing resting eggs. therefore be caused by inadequate food during the period of egg formation. Egg abortion is aggravated by extreme or Female Hormones toxic conditions. Lesnikov (1967) noted Daphnia Estrogenic effects on D. magna were deter- pressing eggs out from the brood pouch fol- mined using the model estrogen diethylstilbes- lowing a prolonged exposure to phenols or oil. trol (DES; a nonsteroid vertebrate estrogen) A noticeable percentage of eggs were also (Baldwin et al., 1995). Chronic exposure to aborted by D. magna in the presence of a 0.50 mg/L DES reduced molting frequency decomposing mass of the blue-green alga and the fecundity of second-generation daph- Microcystis (Barros et al., 2000) or the surfac- nids. Over a period of 21 days, 4-nonylphenol tant, laurox-9 (Shcherban, 1980), In addition, treatment decreased the number of offspring 3,4-dichloroaniline caused abortion or resorp- in the first and second generations, whereas tion of eggs by Daphnia (Ivanova et al., 1989). It DES had a similar effect on the second genera- has also been shown that Daphnia can lose eggs tion only. Exposure of female D. magna to DES as a result of postabdomenal movements that and to methoprene (an insect juvenile hormone clean blue-green algae from their limbs analog) stimulated development of the abdom- (Bednarska and Slusarczyk,´ 2011). inal process (a characteristic of females) (Olmstead and LeBlanc, 2000). 11.3.5 Sex Hormones The number of young produced by Daphnia doubled in a solution containing gonadotropin Sex hormones are derivatives of steroids (i.e. the urine of nongravid cows, at 0.3À0.7 (i.e. they are steroid hormones): these include International Units (IU)/mL in the culture the male hormones, testosterone and andros- medium) in comparison with medium containing terone, and the female hormones, estron (pro- gonadotropin inactivated by heating at 68 Cfor gesterone) and estradiol. Both growth and 1h(Cehoviˇ c,´ 1954a, 1954b). reproduction are steroid hormone-dependent The normal content of glucocorticoids in processes. Although the presence of sex hor- D. magna is 8.4À12.7 pmol/g hydrocortisone and mones may be assumed from general biologi- 6.9À10.1 pmol/g corticosterone (Polunina, 1999); cal considerations, the first papers to directly when estradiol was added to the culture medium, demonstrate the presence and role of sex hor- these values changed to 8.3À22.1 pmol/g and mones in daphnids were only published at the 14À20 pmol/g, respectively.

PHYSIOLOGY OF THE CLADOCERA 11.3 PARTHENOGENETIC REPRODUCTION 137

Hydrocortisone at 0.25 mg/L increases the testosterone glucose conjugates is elevated, D. magna life span, as well as its heart rate and thus indicating an alteration to their steroid the number of eggs in the brood pouch, com- metabolism (Baldwin et al., 1995). In D. magna, pared with controls (Kudikin, 2001). Fecundity the elimination of several of the glucose- is also increased by progesterone. conjugated testosterone metabolites also decreased in proportion to the pentachlorophe- Male Hormones nol concentration; sulfate-conjugated metabo- The biotransformation of testosterone in lites also decreased (Parks and Le Blanc, 1996). D. magna was investigated by Baldwin and Testosterone metabolism in D. magna is LeBlanc (1994a) (Fig. 11.2). Numerous metabo- also modified by xenobiotics. Nonylphenyl- lites are produced at phase I of the biotransfor- polyethylene glycol at 5.0 mg/L inhibits meta- mation: polar metabolites are preferentially bolic elimination of testosterone in D. magna excreted, whereas nonpolar ones are preferen- (Baldwin et al., 1997). Interference of testoster- tially retained. Testosterone and phase I meta- one metabolism in D. magna by the xenobiotic 4- bolites are excreted in glucose-conjugated nonylphenol was also studied by Baldwin et al. forms and 2α-hydroxytestosterone as a sulfate (1997). The presence of 25 μg/L or 100 μg/L 4- conjugate. Nonpolar phase I metabolites of tes- nonylphenol in solution disrupted the testoster- tosterone are not sulfate conjugated. These one metabolic pathway, resulting in the elimina- results demonstrate that Daphnia can convert tion of testosterone and the accumulation of polycyclic compounds into multiple polar and androgenic derivatives. Gibble and Baer (2003) nonpolar metabolites. also reported that 100 μg/L 4-nonylphenol in Under conditions of chronic exposure to solution modifies the formation of secondary 0.50 mg/L DES the rate of elimination of morphological characteristics in D. magna. hydroxylated metabolites of testosterone in Testosterone metabolism is enhanced by tri- D. magna is significantly reduced and that of butyltin at concentrations approaching lethal

RETENTION FIGURE 11.2 Testosterone metabolism by Daphnia magna. Source: Baldwin and LeBlanc (1994b). Testosterone Nonpolar metabolites

TESTOSTERONE

Hydroxy Glucose Sulfate Glucose Sulfate Glucose

PHYSIOLOGY OF THE CLADOCERA 138 11. REPRODUCTION levels (c. 2.5 μg/L); this was estimated by the development of embryos in D. magna takes two elevated production of hydroxylated, reduced/ molt cycles: during the first cycle, an ovicell dehydrogenated, and glucose-conjugated testos- develops in the ovary; following molting, the terone metabolites (Oberdo¨rster et al., 1998). eggs are transferred to the brood chamber, where Exposure of D. magna to 1.3 μg/L tributyltin they develop into embryos; and these are increases the rate of elimination of oxido- released just prior to the next molt. The beginning reduced, hydroxylated, and glucose-conjugated of the intermolt period in adult females is indi- testosterone derivatives (LeBlanc and cated by the presence of a molted exoskeleton. McLachlan, 2000). These authors showed that The production of males may be influenced tributyltin causes modifications to the metabo- by various substances: 20-hydoxyecdysone lism (Fig. 11.3)thatresultinanincreasedpro- (a crustacean hormone) at concentrations of 1 or duction of oxido-reduced derivatives that are 10 μg/L increased production of all-male broods preferentially retained D. magna tissues. in D. pulex, but a concentration of 100 μg/L It has been shown experimentally that oxy- reversed the effect (Peterson et al., 2001). tocin increases the fecundity of D. magna According to Banta and Brown (1924), in (Polunina, 1999; Nikitina and Polunina, 2000). M. macrocopa the critical period in which eggs Quantitative differences in the concentrations within the ovary may be induced to develop of hydrocortisone and corticosterone in the into either females or males is confined to body of D. magna were determined to be both 2À4 h before the eggs leave the ovary. Agents seasonal and caused by exogenous oxytocin, that reduce the maternal metabolism induce prednisolone, testosterone, and estradiol the production of males. The critical time men- (Nikitina and Polunina, 2000). tioned above is the period when the spindle is Olmstead and LeBlanc (2002) discovered a formed during embryogenesis. hormone controlling the formation of males in The exposure of sensitive ovarian eggs to Daphnia.ExposureofD. magna cultures to solu- 400 nM MF results in the production of 100% tions of methyl farnesoate (MF) at concentrations males, whereas exposure to 52 nM MF pro- greater than 30 nM stimulated the production of duces mixed broods. Similar to MF, the expo- males. MF is produced by the mandibular organ sure of D. magna to pyriproxyfen or in crustaceans. The sensitive period in ovarian methoprene causes D. magna oocytes to egg development occurs after 12À24 h. The develop into males (Olmstead and LeBlanc,

Glucosylandrostanediol FIGURE 11.3 Metabolic pathways of testosterone in D. magna that are stimulated by β Androstanediol tributyltin exposure (shown in bold). DHT, 17 - GlucosyIDHT Glucosylandrostenedione hydroxy-5α-androstan-3-one. Source: LeBlanc and McLachlan (2000). DHT Androstenedione

Testosterone

6β-Hydroxytestosterone Glucosyltestosterone

Glucosy1-6β-Hydroxytestosterone

PHYSIOLOGY OF THE CLADOCERA 11.3 PARTHENOGENETIC REPRODUCTION 139

2003). Tatarazako et al. (2003) exposed males had not been previously observed. D. magna females less than 24 h old to MF, Following this method, Sinev and La-Orsri methoprene, two phenoxyphenoxy derivatives Sanoamuang (2011) exposed females of tropi- (pyriproxyfen and fenoxycarb), and juvenile cal chydorids to 100 nM MF and obtained hormone III; all resulted in the progeny being males, which are usually rare in nature. dominated by males (Fig. 11.4). Exposure to The induction of male formation by exter- pyripoxyfen and fenoxycarb at a concentration nally added substances indicates the possibil- of 330 ng/L resulted in the production of ity that stimulation by substances that almost all males. Other substances cause the accumulate seasonally in natural water affects production of males only at higher concentra- the appearance of males. tions (over 3.7 μg/L). Xenobiotics may also induce the formation In Moina and Ceriodaphnia spp., male neo- of males. The production of males increased in nates appeared following treatment with D. pulicaria in the presence of atrazine (at fenoxycarb (Oda et al., 2005a). In D. magna, 2À2.3 ppb; Fig. 11.5) (Dodson et al., 1999), and they appear when the mothers are exposed to in Moina and Ceriodaphnia in the presence of juvenile hormones and the insecticides kino- fenoxycarb (Shigeto Oda et al., 2005). prene, hydroprene, epofenonane, and fenoxycarb (Oda et al., 2005b). Exposure of Daphnia Natural Factors that Influence the to MF also results in the formation of males Formation of Males and to an increased Hb content in the body Various factors that cause the onset of gamo- (Rider et al., 2005). Clones that produced males genesis also lead to the formation of males also showed increased Hb production, (listed in Section 11.4). The formation of males is whereas non-male-producing clones produced induced by increased temperature (Fries, 1964), no Hb in response to the hormone. The a decrease in day length to 12 h (Stross and Hill, authors therefore concluded that both physio- 1965), and crowding. Males are produced by logical processes are regulated by the same Moina following food shortages (Stuart and signaling pathway. Cooper, 1932), and males and gamogenetic Kim et al. (2006) obtained males in Daphnia females are produced by Moina in a medium and Bosmina species following MF treatment, supplemented with metabolites that accumulate despite the fact that, in three of their species, in their culture (Orlov and Cherepanov, 1986).

FIGURE 11.4 Increased proportion of males is caused by exposure to various hormonal substances. Source: Tatarazako et al. (2003).

PHYSIOLOGY OF THE CLADOCERA 140 11. REPRODUCTION

dependent on biologically active substances; for example, the exposure of males to androsterone (a steroid vertebrate androgen) stimulated development of the first antennae (Olmstead and LeBlanc, 2000). A combination of food deprivation and crowding also resulted in the production of males and ephippia by D. magna (Olmstead and LeBlanc, 2001b), while treatment with methoprene resulted in the production of males and inhibited the production of dormant (resting) eggs.

FIGURE 11.5 Increase in male formation in the pres- 11.4 GAMOGENETIC ence of atrazine. Source: Dodson et al. (1999) REPRODUCTION: DIAPAUSE

At the onset of unfavorable conditions, Fat is known to be accumulated by algae Cladocera form males and gamogenetic under conditions of depleted nutrients and by females, and switch to bisexual reproduction. senescent algal cultures. Thus, two processes However, male-producing and non-male- seem to occur in nature: (1) the accumulation producing females may coexist, e.g. as noted of fat by cladocerans due to feeding on fat-rich for D. longispina by Manuilova (1959) and for algae and (2) stimulation of male production D. pulex by Innes (1997). This suggests the by the algal fat. presence of different clones within a single population of a species, as shown by Carvalho and Hughes (1983) by differential photoperi- Formation of Males Following Exposure odic induction of ephippia formation in to Chemical Compounds D. magna (Fig. 11.6). Very early studies by Banta and Brown The onset of bisexual reproduction may be (1930) found that chloreton, phenylurethane, caused by different factors (see, e.g. Spaak, and potassium cyanide (KCN) retard the 1995): food shortage (Tauson, 1931), changes in development of M. macrocopa and increase the the food quality (fat accumulation by declining percentage of males in their progeny. algal populations) (Dehn, 1948, 1950, 1955), Substances that disrupt the endocrine system photoperiod (Buikema, 1968), crowding (Banta have been shown to also perturb sex determi- and Brown, 1929), and temperature are factors nation. Due to these effects, an increased ratio that are frequently mentioned, with explana- of males was produced in D. magna that were tions provided for each. exposed to solutions of dichlorodiphenyltri- Gamogenetic reproduction was observed chloroethane (or DDT), methoxychlor, and to be caused by changes in the photoperiod 4-nonylphenol (Baer and Owens, 1999). The (in Daphnia) (Stross, 1965, 1969c) and by the premature appearance of males may also lead accumulation of metabolic products in the to reduced formation of ephippia. The devel- medium (in Moina brachiata and M. macrocopa, opment of morphological traits that are charac- but only by those from the same species) teristic of a particular sex was shown to be (Lopatina and Zadereev, 2007). In Scapholeberis

PHYSIOLOGY OF THE CLADOCERA 11.4 GAMOGENETIC REPRODUCTION: DIAPAUSE 141

FIGURE 11.6 Differential induction 12.0 of photoperiodic ephippia formation by different Daphnia magna clones. Source: Carvalho and Hughes (1983).

11.83

11.66 –1

11.50 Hours of daylight (24 h) Hours of daylight

11.33

11.16

11.0 1234567 Scale : Clone 25 % Ephippial females 0 armata, ephippial females appeared under con- likely to be a result of the combined action of ditions of a short photoperiod (11.4 h light; vs. several factors. It has been shown experimen- 15.5 h light) (Berner et al., 1991), usually after tally for D. magna that formation of gamoge- one or more parthenogenetic broods. This is netic eggs is induced by a combination of

PHYSIOLOGY OF THE CLADOCERA 142 11. REPRODUCTION three factors: food limitation, short-day photo- ephippial females, and males within a single period, and chemically induced crowding brood (Papanicolau, 1910; Agar, 1920; Orlov (Kleiven et al., 1992). and Cherepanov, 1986; Zadereev et al., 1998). Latent eggs are contained in the ephippium A single female D. pulex may produce only in anomopods or are liberated in ctenopods. females, only males, or a mixture of both sexes The ephippium takes various forms in differ- (Innes, 1997). Cladocerans may therefore pass ent species. In Lathonura and Alonopsis,it not only from the production of parthenoge- consists of an upper-posterior part of a netic eggs to the production of latent eggs in shell, which is reinforced by special glands ephippia but may also backtrack from ephip- (Fig. 11.4) (discovered by Makrushin, 1970). In pia formation to formation of parthenogenetic Daphnia, the same species and a clone may eggs, as reported for Moina and Daphnia produce both buoyant and non-buoyant ephip- (Makrushin, 1969). pia, as shown in D. pulicaria (Ca´ceres et al., Ephippia formation has been experimentally 2007). induced in D. magna at low food levels, high Protective melanistic coloration of ephippia culture densities, and short-day photoperiods varies from 0.5% to 96.5% of its surface, as (12 h:12 h light:dark periods) (Carvalho and shown for D. pulicaria (Gerrish and Ca´ceres, Hughes, 1983). D. schoedleri switches to gamo- 2003), and is caused by genetic and environ- genesis when there is an increased content mental components. In most daphnids, ephip- of neurosecretory hormones in its body pia with resting eggs mainly float on the water (Alekseev, 2010). In energetic terms, the forma- surface (Makrushin et al., 1990), whereas in tion of an ephippial clutch is equivalent to lay- other anomopods they sink or are glued to ing eight to nine parthenogenetic eggs underwater surfaces (Fryer, 1972; Makrushin (Zadereev et al., 1998). et al., 1990). In macrothricids, at least, the Gynandromorphism should also be dis- ephippia are attached to substrata, thus ensur- cussed. The presence of individuals with mixed ing “continuity of an already established col- female and male features (gynandromorphic) ony” (Fryer, 1972, p. 79). has been reported for daphnids, chydorids, and Within the same species, the sequence of par- Leptodora (Frey, 1965). Experimentally, the for- thenogenetic and bisexual reproduction may dif- mation of gynandromorphs was caused in fer in different water bodies. Manuilova (1951) D. magna by MF treatment (Olmstead and notes that D. longispina in ponds (of the LeBlanc, 2007): especially high numbers of Yazhelbitsy Fish Farm, Russia) showed a grad- gynandromorphs were produced at concentra- ual increase in bisexual reproduction from its tions of c. 12 μg/L, which is intermediate initiation until the middle of July, when parthe- between the concentrations that produce 100% nogenetic reproduction was completely discon- females and those that produce 100% of males. tinued. In other ponds, the same species The presence of hybrids between species is reproduced both parthenogenetically and bisex- a rather common occurrence (Agar, 1920; ually throughout the summer. The latter was Spaak, 1995, 2000; Flo¨ssner, 2000; alsothecaseforC. reticulata in ponds of the Korovchinsky and Boikova, 2009). Naturally Yazhelbitsy Fish Farm, in contrast to those in occurring hybridization was recorded in Lake Kolomenskoe (Tverskaya Oblast, Russia). Simocephalus by Hann (1987) and in Daphnia It has also been observed that a female of by Hobæk et al. (2004). Shan (1971) experi- Simocephalus, Daphnia,orMoina may produce mentally demonstrated the possibility of parthenogenetic females, parthenogenetic crossing Pleuroxus denticulatus and females and males, or parthenogenetic females, P. procurvus.

PHYSIOLOGY OF THE CLADOCERA 11.4 GAMOGENETIC REPRODUCTION: DIAPAUSE 143

11.4.1 The Physiology of Males of the same species by contacting their antennules with the surface of the female ephippial Almost all of facts summarized so far have shell, which has various different sculptural concerned females. However, the size and characteristics in different species. The grasp- behavior of males is different, which implies ing antennules of Moina males are also some- physiological differences. Unfortunately, data what different in different species. The same on the physiology of males are very scarce. explanation was suggested for Bosmina by The life span of males is considerably shorter Kerfoot and Peterson (1980). than that of females because, as shown with reference to D. magna (MacArthur and Baillie, 1929), they are more sensitive to temperature By Chemical Sensing changes. Daphnia males have a higher beat fre- The attraction of males by glycoproteins quency of their thoracic appendages than do was demonstrated in Daphnia obtusa and females (Pen˜alva-Arana et al., 2007), a higher Ceriodaphnia dubia by Carmona and Snell oxygen consumption (Vollenweider, 1958), (1995). The attractant (pheromone) was present higher heart rates (Fig. 6.8) (MacArthur and in the area around the ovaries, where it Baillie, 1929; Maynard, 1960), and a higher Hb exceeded the background concentration by content (Kobayashi, 1970). two or more times. According to Carmona and Data on the growth of males is comparatively Snell, after the male attaches to the female’s limited; it was briefly summarized by Frey and valve, it then introduces its first antenna and Hann (1985). In chydorid males, there are two touches the ovary’s surface with its sensory immature instars and one mature instar (Frey papillae. More than one type of glycoprotein and Hann, 1985). After their third (mature) was found to be present at this site, thus indi- instar, Eurycercus longirostris males live for up to cating a potential mechanism for thee recogni- 3 weeks (Hann, 1980). In D. magna, males live tion of conspecific females by males. almost as long as females—up to 45 days at 28C, and longer at lower temperatures (MacArthur and Baillie, 1929). When exposed to 11.4.2 The Physiology of Dormant Eggs 10À50 mM NaOH or 1À10 mM KCN, the sur- À The following notes are clearly incomplete; vival time of Daphnia males is 75 85% that of however, they characterize the current state of females (MacArthur and Baillie, 1929). investigations in this field. Resting (dormant) Males have a strong drive for attachment to eggs are produced by all cladocera. In the females, as described e.g. by Van Damme and absence of males, gamogenetic eggs are usu- Dumont (2006). In the presence of females, ally resorbed, either in the ovary or in the C. sphaericus males attach not only to females but ephippium (Zhukova, 1955). In the absence of also to other males. Sometimes, males (in the males, empty ephippia may be produced. presence of females) attach to several other The same female may form several ephip- males, making chains of two to three individuals pia, one after another, as has been observed in (Smirnov, 1971). Moina (Murakami, 1961) and Eurycercus. After There are several ways by which males can gamogenesis, parthenogenetic reproduction recognize conspecific females. may be resumed (Sklayrova, 1938; Green, 1956a). By Touch The term pseudosexual reproduction has been In Moina, Goulden (1966, 1968) reported applied to Daphnia, some races of which pro- that males recognize the gamogenetic female duce ephippial eggs and ephippia in the

PHYSIOLOGY OF THE CLADOCERA 144 11. REPRODUCTION absence of males that develop without fertili- from sediments that had been dry for 13 years zation (Banta, 1925; Schrader, 1925). Such races (Crispim and Watanabe, 2001). Much longer have been called thelytokous, with the ephippial periods of viability have also been reported: eggs being known as pseudosexual eggs. resting eggs of Daphnia have been known to During their formation in Acroperus, Alona remain viable for more than a century affinis,andPleuroxus truncatus, resting eggs are (Caćeres, 1998; Kawasaki et al., 2004b) and enveloped in a special membrane formed by even in some populations for longer than 200 the paired protoephippial gland (described by years (Caćeres, 1998). Resting eggs remain via- Makrushin, 1972). In Lathonura (Macrothricidae) ble after periods of drying, freezing, and expo- and chydorids, Makrushin (1970) discovered sure to the digestive system of predators glands that contribute to the formation of (Kawasaki et al., 2004b). For example, latent ephippia (Fig. 11.7). These glands accumulate a Alona guttata eggs remained viable after pass- secretion during the formation of the resting ing through the digestive tract of a duck eggs, which is maximal at molting. In (Proctor, 1964). Lathonura, the gland surrounds the inner sur- The respiration rate of the resting eggs (i.e. face of the hindgut, whereas in Acroperus it is of the developing embryos within resting μ situated in the dorsal surface of the trunk, eggs) was determined to be c. 2.5 gO2/mg inside the brood pouch. DW/h in Leptodora kindtii at 4C and increased μ Resting eggs are well protected from to c. 6 gO2/mg DW/h at 6 C; in Bythotrephes μ mechanical damage. In D. pulex, the envelope longimanus, the respiration was c. 0.5 gO2/ of resting eggs is 2.2 μm thick, whereas the mg DW/h and did not change within a tem- outer wall of nonresistant eggs is only 0.35 μm perature range of 2.4À6C (Andrew and (Seidman and Larsen, 1979). The shell compo- Herzig, 1984). sition of resting eggs of Daphnia includes crystalline calcium phosphate and magnetic Drying material; their distinctive chemical composi- Resting eggs may survive desiccation; it may tion and honeycomb structure are thought to be recalled that this fact was used by Sent-Iler ensure survival under harsh conditions (1860) and by Sars (1886, 1888) for rearing living (Kawasaki et al., 2004b). The distribution of specimens from dry lake sediment. This efficient elements over the integument of D. magna method remains in current use (Van Damme ephippia was found not to be homogeneous and Dumont, 2010). (Kawasaki et al., 2004b): P, Ca, and K were The number of resting eggs per unit area of present mostly over the embryos, whereas sul- the bottom of a water body may be very high, fur (S) is distributed over the eggshell. but is rarely actually counted. In Daphnia,the Ephippia formation in Daphnia has been number (per m2) of diapause eggs may reach shown to depend on illumination, not, how- 25,000 (D. pulicaria in Oneida Lake, New York) or ever, on its intensity or polarization (Buikema, 80,000 (D. galeata mendotae)(Caćeres, 1998); num- 1968), but rather on a short-day photoperiod, bers of Moina ephippia may reach 600,000 (data in combination with the population density from Japan) (Murakami, 1961). In the author’s (Stross, 1969a, 1969b, 1987; Stross and Hill, experience, floating ephippia of Moina that were 1968). It has also been observed that D. magna driven by wind to one side of a pond (in the does not form dormant eggs in complete dark- park of the Agricultural Academy, Moscow) ness (Slusarczyk´ et al., 2005). could be collected in unlimited quantities (using Resting eggs may survive in ephippia for 8-L pails). However, after long periods of desic- many years. Living Cladocera were obtained cation, the percentage of hatching eggs was

PHYSIOLOGY OF THE CLADOCERA 11.4 GAMOGENETIC REPRODUCTION: DIAPAUSE 145

FIGURE 11.7 Glands that participate in ephippia formation in Lathonura and Alonopsis. Upper left, Alonopsis elon- gatus female prior to molting. Latent egg is outside the optical section. Upper right and bottom right, Lathonura gamogenetic female. Bottom left, Lathonura parthenogenetic female. 1, secretory gland; 2, gland cells; 3, brood chamber; 4, ovary; 5, gut; 6, claws; 7, bases of setae natatoriae; 8, gut contents; 9, seta natatoria; 10, anus. Source: Makrushin (1970).

observed to decrease, as was shown in D. pulex with light of wavelength 350À475 μmand by Midzuno et al. (1960) and in Moina by energy of 2 3 106 ergs/cm2 (2 kJ/m2), with a Murakami (1961). wavelength of 410 μmbeingthemostefficient Dried and rehydrated ephippial eggs of (Davison, 1969). For 100% activation, resting D. pulex start developing when illuminated eggs had to remain in the dark for at least

PHYSIOLOGY OF THE CLADOCERA 146 11. REPRODUCTION

5 days. Drying turned out to be unnecessary for wet-dry cycles, and illumination was also the development of resting eggs of Streblocerus necessary for hatching (Lu et al., 2000). serricaudatus (Fryer, 1972). Makrushin (1978, 1980) attributes the ability Cryoprotectants of resting eggs to withstand desiccation to sev- Dormant eggs contain more glycerol and eral properties of the yolk: its homogeneity, moreHsp60(aheatshockprotein)compared fine structure, and absence of vacuoles. Since withsubitaneouseggs(Pauwelsetal.,2007). living progeny may emerge from latent eggs of Glycerol is involved in energy storage and is various ages, generations at different periods also a cryoprotectant. The heat shock protein within a biological community may be mixed. is thought to assist in maintaining structural integrity and inhibiting cellular metabolism. Hoshi (1957) noted that sexual eggs contain Freezing no Hb. Under European temperate conditions, Dormant D. magna eggs contain approxi- latent eggs hatch after ice thawing within a mately double the amount of trehalose than rather brief period (Ca´ceres, 1998). Diapause their asexual eggs, which increases their toler- eggs may withstand long-term freezing. ance of freezing (Putman et al., 2011). Latent daphnid eggs can withstand freezing Hatching of a resting egg occurs due to for 2 years and those of Bosmina obtusirostris rupture of the outer, inelastic membrane (cho- can withstand repeated freezing (Makrushin rion) by the increased size of the developing et al., 1990). embryo and subsequent uptake of water Resting eggs, at least those of mid-latitude through the embryonic membranes (as Palearctic cladocera, can withstand freezing observed in Daphnia by Selvaraj, 1979), or due down to about 250C, judging by observations to the osmotic uptake of water, which made in an area with this average minimum stretches the elastic embryonic membranes winter temperature (Yakutia in Siberia, and breaks the outer inelastic membrane Russia); the diversity of such fauna in this (Davison, 1969; Fryer, 1996). The swelling is region is no lower than that of warmer osmotic and can be retarded by sucrose solu- European areas. In addition, mud samples col- tions (Davison, 1969). lected in West Greenland and kept frozen at Resting eggs always produce females, as 218C for 18 years produced Chydorus, Alona, has been shown e.g. in Pleuroxus denticulatus Macrothrix, and Daphnia (Meijering, 2003). by Shan (1969). The hatching of resting eggs Freshwater Cladocera ephippia can also depends on environmental conditions; for withstand seawater, but under such conditions example, illumination favors the hatching of do not hatch and their resting period is dormant eggs. P. denticulatus ephippial eggs extended (Meijering, 1970). storedinwaterinthedarkfor84dayswere A certain period of freezing is thought to induced by fluorescent illumination to hatch be necessary before the start of ephippia within 6À15 days; earlier times occurred at development. Illumination is also necessary longer photoperiods (Shan, 1970). The hatch- for this process. Both factors seem to be dif- ing yield decreases at higher light intensities: ferent for different species and clones (Stross, in the light, up to 50% of Daphnia ephippial 1966). The hatching rate of ephippial eggs of eggs hatched within 100 days, and only Moina mongolica increased with incubation for 0À2% in the dark. Moreover, several weeks up to 62 days under conditions of alternative of storage at room temperature was

PHYSIOLOGY OF THE CLADOCERA 11.5 IMPACT OF XENOBIOTICS ON REPRODUCTION 147 necessary before latent eggs could respond the hatchability of resting eggs of D. curviros- to light (Pancella and Stross, 1963) and pre- tris (Angeler et al., 2005). vious desiccation reduced the hatching rate. Light perception by resting eggs is thought to involve carotenoids (Davison, 1969). Exposure to decreased temperature and 11.5 IMPACT OF XENOBIOTICS 12 h:12 h light:dark cycles resulted in the best ON REPRODUCTION hatching rates in Daphnia carinata (Tsitsilas and Barry, 2002). Moreover, it was shown that 11.5.1 Inhibitory Effects more resting eggs of Acroperus and Alona rectangula hatch at 10À20C than at higher tem- Generally, xenobiotics inhibit reproduction peratures; and a photoperiod of 16 h light/day and reduce the number of progeny in is generally more favorable (Vandekerkhove, Cladocera. Therefore, measurements of Declerck, Brendonk, et al., 2005). However, D. magna numbers have been used for the these authors found no such preference, in assessment of water quality and bottom sedi- other species, e.g. C. reticulata and Daphnia ments (Romanenko et al., 2011). parvula. Cadmium (as cadmium chloride; CdCl2) Hatching of Pleuroxus denticulatus resting levels as low as 0.01 mg/L negatively influence eggs occurs earlier at a higher illumination the growth of M. macrocopa and Macrothrix tri- levels and longer photoperiods; however, their serialis populations (Garcia et al., 2004.). In hatching rate decreased under continuous illu- M. mongolica, algae (Chlorella) preexposed to mination at . 600 foot-candles (fc; approxi- Cd caused a reduction in the number of neo- mately 6458 lx) (Kuo-cheng Shan, 1970). nates, which increased in subsequent broods; Resting eggs removed from dark ephippia this effect was also caused by the lowest Cd 2 hatched earlier. However, a period of dor- concentration tested, 8.5 3 10 21 g Cd per mancy and drying is not always necessary for Chlorella cell (Wang et al., 2010). the further development of resting eggs. In In contrast, Cu incorporation into food M. macrocopa, the majority of resting eggs Chlorella did not cause a decreased brood size started development without drying within for the first brood of M. mongolica; however, a 2À24 days of their formation (Wood, 1932). It decrease was observed in all subsequent was then shown experimentally that dormant broods (Wang et al., 2007). (sexual) eggs of D. longispina, as well as those Ni reduced the reproduction rate of D. magna of M. macrocopa, do not need drying for further (a decrease of 33% in the total number of off- development (Wood and Banta, 1937). These spring per female) and reduced the number of authors believed that altered osmotic concen- juveniles in the first brood by 21À33% (Evens tration of the medium is responsible for the et al., 2009). Moreover, D. carinata maturation initiation of development of dormant eggs in was delayed by the presence of lanthanum (La) D. longispina. at levels below the lethal concentration (Barry Failure of dormant eggs of Daphnia to hatch and Meehan, 2000). might be caused by degradation of biochemi- The number D. pulex progeny decreased in cal components that are critical for responding the presence of cobalt (at 5 μg Co/L; provided to hatching stimuli and renewal of their devel- in algal food) but was restored by the opment (Vanvlasselaer and De Meester, 2008). addition of vitamin B12 (Keating, 1985). Xenobiotics (as has been shown with refer- The highest number of progeny was reached À μ ence to the fire retardant FireTrol 934) decrease at 0.75 1.00 g/L vitamin B12.

PHYSIOLOGY OF THE CLADOCERA 148 11. REPRODUCTION

At concentrations of 0.04À2.66 g/L, (Gershkovich et al., 2012), tested at 1 μg/L Cr, Banlen (a herbicide, consisting of 2-methyl- 0.02À0.002 mg/L, and 0.03À3 μg/L, respec- 4-chlorophenoxyacetic acid and 2-methoxy- tively. At 1 μg/L Cr, the average life span 3,6-dichlorobenzoic acid) generally increased decreased compared with control. In addition, the number of eggs per brood in D. magna, ethanol concentrations of 0.125% increased the but led to a high percentage of aborted eggs reproduction rate of Chydorus ovalis compared in subsequent generations (Trofimova, 1979). with controls without alcohol (Yatsenko, 1928). μ At a concentration of 0.66 g/L, this process It was also shown that 25 g/L ZnCl2 was not accompanied by the production of increased the number of Moina irrasa progeny deformed juveniles over six broods (Zou, 1997) and chronic expo- M. macrocopa fecundity was reduced by 97% sure to 36 g/L fluoxetine increased fecundity in the presence of 2 μg/L endosulfan (an insec- in D. magna. ticide) (Chuah et al., 2007). Saponin is lethal for D. magna at 5 mg/L À D. magna withstood exposure to 0.1 1000 pg/L [100% lethal dose (LD100) in 2 h] (Naberezhnyi 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but and Gorbatenkiy, 1973). At lower concentra- its reproduction rate decreased after 8 days (Wu tions, it leads to a decrease in life span. At et al., 2001). 0.2 mg/L and 2 mg/L, it initially has a stimu- Low concentrations of anionic surfactants latory effect, i.e. there are more numerous (alkyl benzosulfonate, alkyl monosulfate, and progeny than in the control. sulfanole chloride) caused abortion of eggs and Cadmium chloride increased the total acid embryos in D magna, and sometimes resorption phosphatase activity in D. magna, although the of their gonads (Shcherban, 1979). These surfac- activity of individual molecular forms of tants are more toxic than are surfactants based this enzyme either increased or decreased on polymers. At superphosphate concentra- (Tsvetkov et al., 1997). tions of . 0.5 g/L, Daphnia embryos develop Although brief, this section is important but cannot break their egg integuments and because it describes how pollutants that are therefore perish (Sklayrova, 1938). Exposure initially toxic and inhibitory may be diluted to acetylcholinesterase-inhibiting pesticides and can then stimulate various vital processes. (dimethoate and pirimicarb) caused reproduc- (See also Chapter 10, section 10.5, concerning tive damage to D. magna, thus decreasing the Ceriodaphnia). number of offspring (Andersen et al., 2006).

11.5.3 Transgenerational Transfer 11.5.2 Stimulatory Effects of Xenobiotics In contrast to their effects at high concentra- The transgenerational transfer methylmer- tions, low concentrations of various sub- cury was studied in D. magna by Tsui and stances may stimulate vital functions or Wang (2004a). It takes 2.5À3 days for methyl- increase life span. This phenomenon is known mercury assimilated in the gut to be trans- as hormesis. With reference to Ceriodaphnia affi- ferred to eggs in the brood chamber. D. magna nis, it was shown that its life span and fecun- quickly acclimates to Hg, but animals that dity increases at low concentrations of recover from Hg stress are more vulnerable to potassium dichromate (K2Cr2O7), ethanol, or Hg toxicity (Tsui and Wang, 2005). the antioxidant SkQ1 [(10-(2,5-dihydroxy-3, For selenium (Se), it was found that about À 4-dimethylphenyl)decyl)triphenylphosphonium] 19 24% of Se in the F0 generation is transferred

PHYSIOLOGY OF THE CLADOCERA 11.6 PARASITES AND PARASITIC CASTRATION 149 maternally via reproduction to the F1 genera- As observed by Metchnikoff [1892 (1947)], tion (Lam and Wen-Xiong Wang, 2006); for Cu, spores of Metschnikowiella bicuspidata (syn. c. 6.5% of the mother’s Cu is transferred to the Monospora bicuspidata, Ascomycetes) that are offspring within 7 days (Zhao et al., 2009). consumed by D. magna are liberated from their coat and penetrate into the body cavity through the wall of the intestine, where they 11.6 PARASITES AND PARASITIC are surrounded, ingested, and destroyed by CASTRATION leukocytes (phagocytosis). Microsporidia that are developing inside Cladocera are attacked by both predators and the body, especially in the ovary, decrease parasites. Cladocerans parasites were noted by fecundity in Cladocera (Green, 1974; Voronin, numerous early authors, such as Leydig (1860). 1986; Voronin and Makrushin, 2006), resulting Frequent and diverse Daphnia diseases caused in a general decline in the population. They by bacteria, sporozoa, fungi, and Saprolegnia have been found in Alonella, Bosmina, were noted by Metchnikoff (1892). Parasites and Chydorus, Daphnia, Ilyocryptus, Moina, Pleuroxus epibionts of Cladocera have been investigated, truncatus, Scapholeberis, Sida, and Simocephalus. and extensive information is summarized by In the summer of 1985, up to 97% of B. longir- Green (1974). Endoparasites comprise bacteria, ostris in the Volga Delta were infested with fungi, Sarcodina, Plasmosporidia (Subclass Coelosporidium (Gorbunov et al., 1995). A simi- Microsporidia), Cestoda, and Nematoda (Green, lar infestation of Bosmina was observed in the 1974). Heavy infestations of both epibionts and Rybinsk Reservoir (Russia) in the 1960s. endoparasites decrease cladocera egg produc- D. magna infestation with the microspori- tion. Epibionts are shed with each molting. dium Octosporea bayeri induced a higher pro- Metchnikoff (1888) described Daphnia that portion of males in the progeny, and less perished due to infestation by the bacterium sperm was produced in parasitized adult Pasteuria ramosa. Sometimes, phagocytes ingest males (Roth et al., 2008). spores of this bacterium; this was described in Infestation of eggs by Aphanomyces daphniae detail, and it was confirmed that Daphnia egg (Saprolegniaceae) in the brood chamber production discontinued after infestation of D. magna has also been reported (Stazi (Ebert et al., 1996; Auld et al., 2010). et al., 1994).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 12

Locomotion

12.1 ANATOMICAL BACKGROUND Organs involved in locomotion, as well as in the movements of the inner organs, are Cladocera muscles do not form large com- set in motion by muscles; exoskeletal struc- pact masses and are mainly discernible indi- tures, in combination with the muscles, form vidually. The general structure of the muscular mechanical tools for the various vital actions system was studied in planktonic Daphnia by of the animal and its component parts. The Binder (1929, 1931) (Fig. 1.3). Since these early attachment of muscles to the integuments in reports, no other investigators have attempted Daphnia pulex was investigated histologically detailed, general investigations in this critically by Schultz and Kennedy (1977), who also stud- important field. Abundant, but incomplete, data ied Daphnia tendons and showed that they are has been published by Fryer (1963, 1968) for located within muscle cells and not within the some chydorids, macrothricids (Fryer, 1974), and epidermis. daphnids (Fryer, 1991) (see Figs. 1.1 and 1.2). Thus, the system of locomotion clearly dif- Muscle fibers comprise the cross-striated fers between crawling and swimming species. skeletal muscles, the dilatators of the esopha- The former represent the ancestral muscular gus and anus, and the constrictors of the rec- system, but the available data for crawling spe- tum; the longitudinal muscles of the gut cies are mostly fragmentary (Fryer, 1963, 1968, consist of smooth fibers (Binder, 1929). 1974). Littoral cladocerans have diverse forms: The largest cladoceran muscles are the dor- globular, lenticular, and elongated. In one case sal muscles and the three longitudinal ventral (Graptoleberis), lateral compression is combined muscles that extend along each side of the with dorsoventral flattening and elongation. body from the head to the postabdomen. The Most of these species crawl and only occasion- lowest ventral muscle rises at the posterior ally swim, which probably influences their and is attached in the upper zone of the hydrodynamic properties. In contrast to littoral abdominal region in Daphnia (according to species, the pelagic species are laterally com- Binder, 1931; Green, 1956c) and Eurycercus pressed and are mostly not in contact with the (Green, 1956c), and to the postabdomen in substrata. chydorids; the uppermost ventral muscle

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00012-4 151 © 2014 Elsevier Inc. All rights reserved. 152 12. LOCOMOTION descends and is attached in the postabdomen. small and large clumps of organic debris and The crossing over of these muscles allows the there may be both fine-grained and coarse- possibility of postabdomenal movements in a grained sediments of organic and mineral medial plane. material on the bottom of water bodies. From the lateral viewpoint, three large Cladocera may stick to the surface film of antennal (paired) muscles can be seen in the water, from which they are not generally able daphnian head: the two anterior muscles are to free themselves. The only reported exception the antennal abductors, and the third muscle is was observed by Green (1956c): Camptocercus the antennal levator (Binder, 1931). There is lilljeborgi, which possesses a unique system also a small flexor antennula that is well repre- comprising a long, reinforced abdomen and a sented e.g. in chydorids but reduced in postabdomen containing specialized muscles, Daphnia females. can rotate its postabdomen outside the shell The labrum is set in motion by levator labri; and thus free itself from the surface film. there are also other small muscles within the Pelagic Cladocera usually live their entire labrum. In addition, the adductor muscle of life cycle without making any connection to a the carapace extends from valve to valve substrate. The aquatic environment is viscid under the gut. (Zaret, 1980) and may be characterized by the In crawling species, movement is princi- following parameters (Eq. 12.1). pally achieved by the action of muscles of the Reynolds number ðReÞ 5 ρua=η 5 ua=v (12.1) thoracic limbs, postabdomen, and antennae, but in pelagic, free-swimming species this is where done by means of the antennal muscles. ρ is the water density (51 g/cm3); The muscle system has been studied in a u is the flow rate in relation to the body; few species, but variations among different a is the length of a swiming animal; families have not been reported; in general, η is the water viscosity (50.010 ps at 20C); this is rarely commented upon. No detailed and comparative investigation has been made in v is kinematic viscosity (5η/ρ; for water, differently specialized species. Investigations v 5 η cm2/sec. of transformations in the muscle system and For cladocera, length “a” may be assumed relevant skeletal structures in different genera to be 0.1 cm. are therefore highly desirable. Such studies For small chydorids, “u” may be taken to be would contribute to our understanding of fun- 5 mm/sec. damental issues regarding the phylogeny and mode of life of Cladocera. Under these conditions, for most chydorids, Re 5 0.5 cm/sec 3 0.1 cm/0.01 cm2/sec 5 5 (which is much lower than for rapidly swimming and larger fish). 12.2 ENVIRONMENTAL BACKGROUND It may be assumed that for all Cladocera the Re ranges from 0.1 to 100. At slow swimming Littoral Cladocera live among various sub- velocities, Cladocera movement depends on strata that offer all types of surfaces. the viscosity of the aquatic environment, Frequently, there are filamentous algae of vari- which in its turn directly depends on tempera- ous diameters commensurate with the crawl- ture. Little is known about the hydrodynamics ing appendages of chydorids. There are also of small moving objects, such as cladocera; this

PHYSIOLOGY OF THE CLADOCERA 12.3 MOVEMENT TRAJECTORIES 153 is therefore a promising field of investigation debris and algal filaments) movements. The (as noted by Fryer, 1968, p. 347). reasons for such tortuous movement have not been studied, although Scourfield (1905, p. 2) suggested that such movements may depend 12.3 MOVEMENT TRAJECTORIES on the absence of landmarks from which the animal might take a bearing: “It is the diffi- Cladocera are slightly heavier than water culty of keeping a straight course owing to the and sink if they make no effort to swim. It has want of fixed points or datum lines, for both been shown experimentally that living Daphnia horizontal and vertical directions, from which sink more slowly than predicted by Stokes’ bearings may be taken.” law, and more slowly than anesthetized speci- Crawling is performed by means of thoracic mens (Dodson and Ramcharan, 1991; Gorski limb I and is aided by the postabdomen. In and Dodson, 1996). The difference may be due most Aloninae (Chydoridae), their antennae to their form and to the feeding current pro- also participate in crawling and are used when duced by the Daphnia. A potential hydrostatic such species occasionally switch to swimming. role for oil drops that are situated at certain In Chydorinae (Chydoridae), the antennae are places within the body is evident but has not pressed to the body while crawling and are been especially studied in Cladocera. only used while swimming. Crawling Cladocera Pelagic Cladocera and most of the littoral only move forward (never backward). species are always moving. Benthic species of The gaits of crawling Cladocera have not the genus Ilyocryptus, on the other hand, either been investigated. move slowly or do not crawl for long periods, and some species may be immobile for long periods e.g. Lathonura (before suddenly jump- 12.3.2 Pelagic Cladocera ing) and Drepanothrix (both belonging to the family Macrothricidae). Scapholeberis stays, at The trajectories of planktonic cladocerans least for some of the time, attached to the sur- are mainly linear and consist of sinking and face film. refloating (“hop-and-sink” behavior), as well The movement of Cladocera species is as a progressing vector (Lochhead, 1961). rarely unidirectional, and their tracks are Changes in Daphnia swimming speed in mostly complicated and tortuous; however, response to light intensity was discussed by until recently, methods for recording three- Ringelberg (1964). However, swimming behav- dimensional movement had not been devel- ior may be variable and individualistic. Fryer oped (Uttieri et al., 2005). (1991, p. 14) notes: “Even large, heavily built species, such as Daphnia magna, do not merely 12.3.1 Littoral Cladocera ‘hop’ essentially in the vertical plane, but can swim horizontally, dive steeply and rapidly Littoral cladocerans exploit various kinds of head first, pursue a meandering course that movement. Particular species may crawl and can be changed with great rapidity, swim with occasionally start swimming. Species living on the body inclined to one side, and orientate the bottom may run rapidly or swim along tor- and navigate with great precision.” tuous trajectories, or they may be slow or even The observed swimming behavior of immobile for long periods. Fryer (1968) Daphnia spp. in three-dimensional (3D) space observed crawling (running on and between is strongly affected by the light level, food substrata) and scrambling (among clumps of availability, and the volume of an observation

PHYSIOLOGY OF THE CLADOCERA 154 12. LOCOMOTION

FIGURE 12.1 Trajectories of (A) (B) mating Daphnia. A, male unsuc- cessfully pursuing female. B, male successfully grasping female C, two males pursuing one female. Source: Brewer (1998).

1mm

(C)

10 mm

1mm chamber (Dodson et al., 1997). In the dark, adductor) to the carapace length in seven Daphnia spp. swam slowly and exposure to Daphnia species and found it to vary from 0.15 infochemicals from fish changed neither their (in D. nivalis) to 0.33 (in D. projecta). He also distribution pattern nor swimming speed noted that the force of the stroke depends on (Larsson et al., 2000). Using video recording the cross section of the muscle. The next step and computer analysis of their motion, in evaluating the swimming capacity involved O’Keefe et al. (1998) obtained digitized 3D measuring the stroke rate of the antennae. video records of Daphnia movement. Large dif- Hebert (1978b, p. 318) also correctly ferences were observed in the swimming highlighted the usefulness of this measure- speeds of individuals belonging to different ment for understanding swimming in clones of the same species. Uttieri et al. (2004) Cladocera: “a thorough investigation of loco- noted that a random component is predomi- motion in Daphnia, with particular regard to nant in D. pulex movements. the relationship between muscle structure and Daphnia trajectories were recorded using a propulsion in media with varying viscosities, digital image analysis system (Baillieul and would be well worthwhile.” Blust, 1999) or a Critter-Spy, i.e. a high resolution 3D recording system (Seurat et al., 2004), 12.3.3 Swimming Parameters while a 2D representation of the movements of mating Daphnia was obtained by Brewer (1998) Movement may be measured as absolute (see Fig. 12.1). units or in body lengths. Hebert (1978b) seems to have been the first to relate the swimming ability of Daphnia to Littoral Species the size of the antennal muscles, and made The swimming rate of small chydorids is special measurements. He pointed out the rela- 2.47 mm/sec and is 17 mm/sec for 2-mm tionship between their swimming capacity and long Eurycercus lamellatus (Smirnov, 1971, the size of their antennal muscles. He also cal- 1975). As expressed in the number of body culated the ratio of the length of the first length per second, it is slowest in Paralona antennal abductor (which he terms the pigra (syn. Chydorus piger)(54.5) and fastest in

PHYSIOLOGY OF THE CLADOCERA 12.4 MUSCLE PHYSIOLOGY 155

Alonella exigua (547), but is much slower in Under conditions of scarce food, hungry Alonella excisa (519) (Fryer, 1968). In most Daphnia show a tendency to wander, com- littoral chydorids (with a body length of pared to satiated ones; this reaction starts after 0.31 mm), the swimming speed is from 10 to a delay, when the intestine becomes empty 20 body lengths/sec (Fryer, 1968); the rate of (Szlauer, 1962). It has also been observed antennal strokes in chydorids is 252406/min that the movement of D. obtusa grown on (Smirnov, 1971). severely phosphorus (P)-limited green algae (Scenedesmus) is comparatively sluggish Pelagic Species (Sterner et al., 1993). The rate of antennal beats in Daphnia ranges from 75 to 250/min (McMahon and Rigler, Trajectories 1963; Fryer, 1991) but is variable and is usually The behavior of littoral Cladocera is interrupted by periods of rest (Fryer, 1991, extremely diverse; there are sluggish ones with p. 12). In Bosmina longirostris, the stroke rate prolonged immobility and to rapid ones that varies within 540 Hz (Zaret and Kerfoot, run along various trajectories. The various tra- 1980). It is thought that the antennal rate jectories of Cladocera, especially those of the increases in gravid D. magna due to both its littoral species, have been little studied and increased weight and the necessity of counter- make a promising field of investigation. balancing the displaced center of gravity (Fox Pelagic cladocerans e.g. Daphnia, exhibit and Mitchell, 1953; Lochhead, 1961). In swim- more strictly defined movements, which nor- ming Ceriodaphnia reticulata, the beat rate of mally consists of sinking, refloating, and mov- their antennae is c. 1 every 2 sec (Seely and ing forward. The swimming tracks of Bosmina Lutnesky, 1998). and Polyphemus, and their turning rates, were According to Kerfoot et al. (1980), the speed compared in a study by Young and Taylor of Cladocera varies from 0.01 to 1.0 cm/sec. (1990). The tracks of Polyphemus were mostly The swimming speed of planktonic Daphnia oriented orthogonally to those of Bosmina, thus longispina is 1.4 cm/sec at a current speed of providing them with a greater probability of 6 cm/sec (Luferova, 1962). At a moderate cur- encountering prey. Kerfoot (1978) filmed the rent speed, most Daphnia swam against the trajectories and akinesis (dead-man response) current, with a swimming speed of 0.6 cm/sec. of Bosmina when attacked by Cyclops Szlauer (1965) observed that the swimming (Fig. 14.1). speeds of adult and young Daphnia are different, which leads to their different distributions. 12.4 MUSCLE PHYSIOLOGY In Ceriodaphnia quadrangula, the swimming speed in pale and red specimens was about Information about the physiology of cladoc- 43 cm/min at a low oxygen concentration eran muscles is scarce. Sollman and Webb and 25C; increasing oxygen concentration was (1941) exposed striated (i.e. voluntary) muscles followed by an increase in swimming distance. of D. magna to mecholyl, pilocarpine, and phy- In red specimens, when the hemoglobin (Hb) sostigmine and observed little or no effect. was blocked by carbon monoxide, the swim- Epinephrine and nicotine stimulated muscular ming activity increased at increasing oxygen activity in Daphnia, but curare first increased concentrations in almost the same way as and eventually arrested all movements. These occurred in pale specimens (Kobayashi and authors also applied electrodes directly to the Yoshida, 1986). shell of Daphnia and gave them a single electric

PHYSIOLOGY OF THE CLADOCERA 156 12. LOCOMOTION shock. This resulted in a single contraction of transformed into a special region, the postabdo- their postabdomen and antennae, and a few men, which is bent at 90180 to the body axis. beats of their thoracic limbs. The postabdomen is the principal lever that Flu¨ckiger (1952) observed the action of the pushes the animal (thus liberating it from obsta- swimming antennae muscles of Daphnia to be cles) and cleans its food-collecting apparatus; it retarded by L-adrenaline bitartrate, D-adrenaline is especially well developed in bottom-living bitartrate, L-noradrenaline bitartrate, L-ephedrine cladocerans, particularly in chydorids. hydrochloride, dihydroergotamine methanesul- Concerning the major joint of Eurycercus, Fryer fonate, and oxyphenylethanomethylamine tar- (1963, p. 341) noted that “[e]nergy losses at trate. However, no clear activity was observed joints are reduced to the absolute minimum as in the muscles of their thoracic limbs. only a single ‘joint’ is involved” (i.e. between A swarm of Daphnia emits a weak electric the postabdomen and the rest of the body). To field, originating from their muscular activi- understand the action of these levers, both their ties, which can be perceived by fish (Freund exoskeletal structure and the muscles that con- et al., 2002). trol their action should be considered. The The biomechanics of cladoceran movements resulting effort depends on where the muscles comprise levers, joints, and methods of muscle are attached and the length of the postabdo- attachment to the skeleton. men. Camptocercus lilljeborgi, with its very long postabdomen, makes a unique, extreme case. Green (1956c) observed that its postabdomen 12.4.1 Joints may move outside the shell and liberate the Movement of the Cladocera, as well as some animal from obstacles, including from adhesion aspects of their food handling, is related to the to the surface film of the water. Green also dis- structure of their joints. Generally, the follow- cusses this problem with respect to his observa- ing kinds of joints are present in the body: tions on the structure of the postabdomen and of postabdominal muscles in Eurycercus and 1. cylindrical joints, in which freedom of D. magna. These studies provide a starting point movement is controlled by their mechanical for further useful investigations. Little, if any, potential (e.g. joints of the antennal work has yet been done on this subject. branches); Chydorids crawl on substrata using thoracic 2. concertina-like joints (such as those at the limb I, use their postabdomen for pushing, base of the antennae); and occasionally swim by means of their 3. telescopic joints (as in segments of the antennae. In some chydorids (e.g. Alona affinis), abdominal part of the body); and the antennae are used in crawling on substrata 4. a lever joint (between the body and the and so may therefore be considered to form a postabdomen), which is more developed in pair of levers. bottom-living chydorids. By immobilizing Daphnia with D- tubocurarine chloride and measuring their 12.4.2 Levers oxygen consumption by means of a Cartesian diver, O’Conner (1950) found that muscle It is obvious that the general body form of activity consumes 32% of their total metabo- cladocerans is related to their locomotion. It is lism and during intensive activity may take up notable that the posterior part of the body is most of their oxygen consumption.

PHYSIOLOGY OF THE CLADOCERA 12.6 FATIGUE AND STRESS 157

12.5 IMMOBILIZATION this was confirmed by a lack of movement in response to stimulation with strong light. Now, we will discuss more about immobili- A mixture of chloroform and 96% ethanol at a zation. Both littoral and pelagic Cladocera are 1:10 (v/v) ratio efficiently immobilizes cladocera very mobile. Their investigation often requires (Daphnia and Bosmina), whereas rotifers are unaf- either retarding their movements or complete fected (Straskraba, 1964). Thus, planktonic crusta- immobilization, as practiced by various ceans can be separated from rotifers, allowing authors. There are two ways to immobilize cla- quantitative measurements to be recorded docera: physical (e.g. attaching them to some- separately. Gliwicz (1968) immobilized the pelagic thing, as mentioned in Chapter 2, “Methods”) cladocera Bosmina, Chydorus, Daphnia,and and chemical immobilization. Diaphanosoma using physostigmine salicylate (eserinum). This chemical caused complete paralysis of their appendages at a concentration of 12.5.1 Chemical Immobilization 50 mg/L within 3 5 min; it acts by depolarizing (Anesthesia or Narcotization) and thus inhibiting neuromuscular synapse activity by inhibition of cholinesterases (responsible for Immobilization may be caused by low tem- acetylcholine breakdown). The author noted that peratures or by exposure to carbon dioxide. In this substance is rapidly degraded in water. a solution saturated with carbon dioxide, Alona, Anaesthetization of Simocephalus and Bosmina,andChydorus sphaericus Scapholeberis, Daphnia with 1% urethane was used by and Simocephalus lost mobility within c. Philippova and Postnov (1988) to measure the 0.11min, and Daphnia in 14 sec (Nikitinsky energy required for movement; D. obtusa (10% and Mudrezowa-Wyss, 1930; Mudrezowa- solution) and Ceriodaphnia dubia have been Wyss, 1933). Sollman and Webb (1941) anesthetized with tricaine methanesulfonate observed that carbon dioxide produced general solution (Carmona and Snell, 1995). depression of D. magna, including decreased Cyclophosphane also causes paralysis in movement, and the Daphnia sank to the bottom. D. magna, which develops as a result of acute Of practical importance is narcotization with energy deficiency (Ivnitskii et al., 1998). These carbon dioxide (using a saturated liquid) prior authors found a high protective activity by to preservation (e.g. with formalin) (Infante, transporting forms of succinic acid and a sensi- 1978). Under these conditions, the animal, e.g. tizing activity by amino acids. Diaphanosoma, is preserved in a dilated state. In experimental practice, Daphnia have been immobilized with a chloretone solution 12.6 FATIGUE AND STRESS (Anderson, 1933), propylene phenoxetol (1% aqueous solution) (Young and Downing, 1976), Fatigue was observed by Clarke (1930) in and urethane (Postnov and Philippova, 1988); experiments with reactions of Daphnia to dim- and Moina has been immobilized with urethane ming of light. Having clearly reacted to the (Hubareva and Svetlichnyi, 1998; Svetlichny and first three stimuli the experimental animals Hubareva, 2002a, 2002b, 2004). D. magna was then demonstrated a response of about 1/3 efficiently anesthetized by bubbling halothane, magnitude and needed a 3-hour period of rest isoflurane, and enflurane through the culture (complete darkness in these experiments), medium (McKenzie, Calow, and Nimmo, 1992); before a normal response was produced.

PHYSIOLOGY OF THE CLADOCERA 158 12. LOCOMOTION

1 In response to environmental stress, heat 3.7 beats/sec at a Cd2 concentration of 50 nM. shock proteins (Hsps) are formed. Hsps were A decreased average swimming velocity has investigated in D. magna by Bond et al. (1993). also been demonstrated in D. magna exposed to In D. magna exposed to 34C, both Hsps gluta- above 5 ppb copper (Cu) using image analysis thione S-transferase were detected (Bond and (Untersteiner et al., 2003). Bradley, 1995). Compared with the effect of single com- Stress induced in D. magna by exposure to pounds, there was an approximately additive predators was demonstrated by increased Hsp effect of exposure to a mixture of polychlori- levels (Pauwels et al., 2005). After exposure to nated biphenyls and tributyltinchloride on predators, the level of Hsp60 increased; this D. magna swimming behavior, whereas there returned to normal levels within 24 h. was a synergistic effect on their reproduction (Schmidt et al., 2005). Immobilization by Cu was measured as the 12.7 IMPACT OF XENOBIOTICS number of immobilized organisms collected in ON LOCOMOTION the field after 48 h of exposure (Bossuyt and Janssen, 2005). It was lowest in Scapholeberis With increasing salinity, there is an immedi- mucronata (5.3 μg/L Cu), D. longispina,and ate decrease in the swimming velocity of B. longirostris,andhighestinDisparalona rostrata D. magna, followed by acclimation and a return (c. 71 μg/L Cu), Pleuroxus truncatus,and to the normal swimming velocity (Baillieul D. magna. Intraspecies differences were observed et al., 1998). The swimming activity of D. magna only in Ctenodaphnia. In the presence of is also decreased under conditions of acute, organic exudates of Anabaena,theCutoxicity(esti- sublethal cadmium (Cd) stress (3.55.0 g/L) mated by immobilization) significantly decreased (Wolf et al., 1998), and exposure to 10100 nM in Ceriodaphnia cornuta (Chouderi et al., 2009). 1 Cd2 (Baillieul and Blust, 1999) and tributyltin The time to immobilization of daphnid spe- chloride or polychlorinated biphenyls (Schmidt cies in response to various pesticides (Munn et al., 2006). Baillieul and Blust (1999) also et al., 2006) or the levels of immobilization in demonstrated in D. magna that the frequency of 48 h by metals (Deleebeeck et al., 2008) have antennal beats decreases from c. 4.6 to been used as a measure of toxicity.

PHYSIOLOGY OF THE CLADOCERA CHAPTER 13

Nervous System and Sense Organs

13.1 ANATOMICAL system of Cladocera was also discussed by BACKGROUND: SENSE ORGANS Leder (1913). More recently, Weiss et al. (2012) used mod- On the subject of Cladocera, Leydig (1860, ern methods for a comparative investigation of p. 3, translated from German) remarked that the nervous systems of three species of “[t]he nervous system, as the center of the ani- Daphnia. These authors concluded that the mal organism, deserves, similar to in other ani- function of the frontal filament (ventral frontal mals, a complete and precise investigation.” organ) is unclear and that the function of the Early data mainly dealt with the most dis- dorsal frontal organ is probably secretory. cernible cephalic region. The first detailed gen- They note that “however, it remains to be eral description of the nervous systems of investigated which substances are secreted.” Daphnia and Moina was made by Spangenberg By Nissl staining the head of Daphnia spp., (1877), who preferred to use young, recently they also revealed, “bulged cells” in the dorsal molted, specimens in his studies. He also dis- area and near the antennule, whose function is sected specimens that, according to his thought to be secretory. These authors do not description, were placed in a weak wood vine- cite the study by Angel (1967), who indicated gar solution for several days. The material was the presence of similar cells in Daphnia magna, then macerated for 2 days in a mixture of alco- but at least the cell near to the antennule corre- hol, water, and glycerol, and then stained with sponds to that described by Weiss et al. (2012). osmic acid. This study was followed by an Angel called them storage cells in the figure, investigation of the nervous systems of but no comments were made in the text. Bythotrephes, Leptodora, Sida, and Simocephalus The nervous system of Leptodora was studied by Samassa (1891) (Fig. 13.1). This author pre- in excellent detail by Kirsch and Richter (2007), served cladocerans in osmic acetic acid and who applied confocal scanning microscopy. The used histological methods for their analysis. ventral nerve cord consists, as shown e.g. with The principal traits of the nervous system in reference to Sida and Leptodora, of a double chain Daphnia were revealed by Fischel (1908) by of ganglia joined by longitudinal connectives means of intravital staining with alizarin and transverse junctions, thus producing a (Fig. 13.1). The general structure of the nervous ladder-like appearance. Each ganglion sends out

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00013-6 159 © 2014 Elsevier Inc. All rights reserved. 160 13. NERVOUS SYSTEM AND SENSE ORGANS nerves to the appendages. The largest anterior Fischel also demonstrated ganglion at the base ganglion is the brain (the suprapharyngeal gan- of setae natatoriae (dorsal setae at the bound- glion), which supplies the eye and the ocellus ary between the abdomen and postabdomen) with nerves. and nerves supplying the heart and thoracic Central parts of the nervous system send limbs. Some attempts at staining following nerves to various specific organs: the gut, Fischel’s methods have been unsuccessful. heart, and sense organs. According to Fischel The cephalic region of the nervous system is (1908), the brain supplies the ganglion opticum shown in Fig. 1.6. The brain and the adjacent and the occipital organ with nerves. The fron- optic ganglion were described in detail by tal organs have been examined by several Lidvanov and Lvova (2003). These authors authors but their functional role is unclear. arrived at the conclusion that the nervous

FIGURE 13.1 Nervous system. Left, Sida crystallina. Right, Daphnia. Source: left, redrawn from Samassa (1891); right, Fischel (1908).

PHYSIOLOGY OF THE CLADOCERA 13.2 NEUROSECRETION 161 system of Daphnia is homologous to that of 13.2.1 Neurosecretory Cells Anostraca. In Daphnia, most neurons of the cen- of the Nervous System tral nervous system are multifunctional, thus indicating that the system is primitive. They Sterba (1957) distinguished special cell also discovered that there are no structures for groups in the cephalic part of the Daphnia ner- the liberation of neurohormones into the hemo- vous system that differ in their structures. He lymph and that neurosecretions (the synthe- could clearly distinguish them from neighbor- sized neuropeptides) are liberated via axons. ing cells by their Gomori-positive staining and Angel (1967) observed that many cells in the also distinguished different cell groups by their central nervous system exhibit a glycogen varying secretions: he identified cells in the cycle. protocerebrum, the deuterocerebrum, a segment Following on from the ideas of Hutchinson of antenna II, and a segment of the mandible (1953), it may be said that all environmental (Fig. 13.2). Sterba found that secretions are liber- factors act together on all sense organs. ated from these cells without accumulation in a Hutchinson (1953, p. 156) states that “there are special reservoir, and that the amount of secre- an enormous number of interconnecting, inter- tion is greater at the beginning of egg matura- nal things.” This immensely complicates any tion and very low immediately before the eggs estimation of the resultant effect by scientists; are transferred to the brood chamber. During however, cladocerans themselves can do it, these periods molting takes place. thus performing every function and movement in their own way.

13.2 NEUROSECRETION

The major functions of the nervous system comprise general neurosecretory regulation and general control of the activities of sense organs. Further, as shown in daphnids, the cladoceran nervous system is cholinergic. Nervous impulses in Crustacea are transferred by acetylcholine (Ach) from nerve endings to particular organs. It is known (Prosser and Brown, 1967 [1962]) that Ach is synthesized in the presence of ATP and cholinesterase. Daphnid cholinesterase shows the characteris- FIGURE 13.2 Neurosecretory areas in the anterior tics of a pseudocholinesterase, since it prefers region of the Daphnia nervous system. AI, antenna I; FG, propionylthiocholine to Ach (Vesel et al., 2006). neurosecretory cells of “the frontal group;” KA, compound The characteristics of pseudocholinesterase eye; mFO, median frontal organ; NA, eyespot; NAII, (cholinesterase II) include inhibition by diiso- nerves of antenna II; NZ, neurosecretory cells on the circu- propylfluorophosphate, prostigmine, physo- menteric connective; NZm, neurosecretory cells on the mandibular ganglion; Oe, esophagus; OG1, optical gan- stigmine (eserine), tetraethyl pyrophosphate, glion 1; OG2, optical ganglion 2; T, tegmentarius, the nerve and hexcaethyl tetraphosphate (Prosser and to lateral frontal organ; VG, neurosecretory cells of “the Brown, 1967 [1962]). ventral group.” Source: Sterba (1957).

PHYSIOLOGY OF THE CLADOCERA 162 13. NERVOUS SYSTEM AND SENSE ORGANS

Four distinct cell groups were identified by the following regions of the nervous system: Angel (1967) in the supraesophageal ganglion scattered throughout the optic ganglion; on the of Daphnia (Fig. 13.3), and Halcrow (1969) anterior and ventral surfaces of the brain; on revealed areas of presumed neurosecretory circumenteric connectives; and at the junctions activity in the nervous system of D. magna of the second ventral commissure with ventral (Fig. 13.3) after staining with paraldehyde nerve cords. The presence of these groups was fuchsin. Fuchsinophilic granules are found in confirmed and reinvestigated by Bosch de

to MEDIAN FRONTAL ORGAN

to LATERAL FRONTAL ORGAN

to OPTIC GANGLION

VENTRAL GROUP 1

VENTRAL GROUP 2

VENTRAL GROUP 3 VENTRAL GROUP 4

to ANTENNULE

CIRCUMOESOPHAGEAL CONNECTIVE 4 5 3 1 2 a b

FIGURE 13.3 Neurosecretory areas in the anterior region of the Daphnia nervous system. 1, nerves to compound eye; 2, optic ganglion; 3, brain; 4, lumen of gut; 5, circumenteric connective. Sources: upper, Angel (1967); lower, Halcrow (1969).

PHYSIOLOGY OF THE CLADOCERA 13.3 SENSE ORGANS 163

Aguilar (1969, 1972), who found the following Neurosecretory cells have also been found in groups of neurosecretory cells in the cephalic the brain of Simocephalus (Zahid et al., 1980). ganglion of Daphnia: frontal; ventral; a group Supposed neurotransmitters or neuromodu- of esophageal connectives; groups of posteso- lators (and their metabolites or precursors) phageal commissures; cells of the mandibular were discovered in D. magna by Ehrenstro¨m ganglion; and neurosecretory cells at the site of and Berglind (1988). D. magna synthesizes and connection of the ventral chain and of nerves transforms these substances which include of the second pair of thoracic limbs (Bosch de 3,4-dihydroxyphenylalanine (l-DOPA), dopa- Aguilar, 1969); and the parapharyngeal group mine, noradrenaline, adrenaline, tyramine, (Bosch de Aguilar, 1972). In Podon intermedius, epinine, 3-methoxyturamine, 3,4-dihydroxy- neurosecretory cells were found in the proto- phenylacetic acid (DOPAC), l-tryptophan, cerebrum, at the boundary between the proto- 5-hydroxytryptophan, 5-hydroxytryptamine, cerebrum and the deuterocerebrum, at the and 5-hydoxyindolacetic acid. The range of base of the antennal nerve, and in the tritocer- test concentrations for these substances was ebrum (Bosch de Aguilar, 1971). 0.01À32.8 ng/mg protein. The quantity of In the nervous system of D. magna, a hyper- catechols l-DOPA, dopamine, and DOPAC in glycemic hormone and immunoreactive neu- Daphnia showed diurnal variations, reaching a rons are found (Zhang et al., 1997). Their wide diurnal peak at 8 AM, although other biogenic distribution is shown in Fig. 13.4. amines did not exhibit diurnal variations. A well-developed histaminergic system was Tonkopiy et al. (1994a) demonstrated that identified in D. magna and D. pulex, including M-cholinolytics decrease the toxicity of armine, in the visual system, by McCoole et al. (2011) aminostigmine, and arecoline for D. magna.On (Fig. 13.5); histamine takes part in the negative the basis of experiments with cholinomimetics, response of Daphnia to ultraviolet radiation H-cholinolytics, and M-cholinolytics, it was (UVR), but exposure to cimetidine (a blocking concluded that Daphnia possess a well- agent) inhibits this negative response. Further developed cholinergic system, containing cho- study led to the discovery of signaling dopa- linoreceptors that is analogous to that of mine, octopamine, and serotonin, as well as mammals. In D. magna, Podosinovikova et al. receptors and transporters for each amine (2001) inhibited the dopamine system by (McCoole et al., 2012). These studies demon- blocking the D2-receptors with haloperidol. strate that Daphnia possess a nervous system After this treatment, Daphnia were exposed to that controls the functioning of various, distant solutions of cholinoblockers (amedine, amizil, structures. atropine, cyclodol, norakin, pentifin) and their antihaloperidol activity was estimated. The swimming behavior of Daphnia has been shown 13.2.2 Neurosecretory Substances to become erratic at a dopamine concentration c. 100 mM (Pen˜alva-Arana et al., 2007). By observing the state of neurosecretory cells and the corresponding stages of molting and reproduction, Bosch de Aguilar (1969, 13.3 SENSE ORGANS 1972) concluded that the esophageal group produces a factor that inhibits molting, the 13.3.1 Anatomical Background mandibular group controls development of eggs, and the ventral group produces a factor For a long time, only two kinds of sense that inhibits formation of the ephippium. organs were recognized in cladocera: the eyes

PHYSIOLOGY OF THE CLADOCERA 164 13. NERVOUS SYSTEM AND SENSE ORGANS

FIGURE 13.4 Distribution of hyperglycemic hormone-reactive neurons originating in the thoracic ganglia of Daphnia magna. Br, brain; CEC, circumesophageal connective; NE, nauplius eye. Scale bar 50 μm. Source: Zhang et al. (1997).

PHYSIOLOGY OF THE CLADOCERA 13.3 SENSE ORGANS 165

sense organs, such as eyes, are anthropo- morphically understandable, most of the sense organs of Cladocera have nothing in common with those of mammals, although they may provisionally be designated as organs of chemical sense (chemoreceptors) and tactile organs To second antenna (mechanoreceptors). The latter may be involved in the perception of gravity and of equilibrium. The presence of sense organs that perceive vibration, e.g. stridulation of aquatic organisms, is also suspected and Dumont and Van de Velde (1976) suggested that one of the functions of head pores may be vibration detection. The olfactory setae (esthetascs) were examined and compared by Scourfield (1896), who also noted that there are nine in female anomopods, Sididae, and Leptodora, six in female Holopedium, and five in female Polyphemidae. Much later, Rieder (1987) described the esthetascs in detail (see Fig. 13.6). Sensilla were identified on the thoracic limbs of chydorids and macrothricids by Fryer (1963, 1974), Smirnov (1967) (Fig. 13.7), and

Thoracic nervous systemThoracic Brain Compound eye Dumont and Silva-Briano (1997). No such sensilla are present on the limbs of daphnids. Generally, on the body (limbs included) of a cladoceran there are numerous papilliform or setiform structures that are suspected to have a sensory function. In the head, there is a lateral frontal (parie- tal) sense organ (Gicklhorn, 1931b) (Fig. 13.1) and each of its cell clusters is supplied by a nerve arising from the cerebral ganglion To anus (Gicklhorn, 1931b; Fryer, 2004). Its function may be light perception or it may be a pres- FIGURE 13.5 Schematic of Daphnia nervous system sure gauge (Fryer, 2004). The median (ventral) showing histamine-like immunoreactivity sites (filled frontal organ is similarly innervated (Fryer, circles). Source: McCoole et al. (2011). 2004). Cladocera can perceive a wide range of (Figs. 1.1, 1.2, 13.1, and 14.1) and the esthetascs external stimuli such as various kinds of irra- on the antennules. Now various other sense diation, including daylight, colored light, and organs are also known to be present, or are sus- polarized light, plus various chemical, olfac- pected of being so, in cladocerans. While some tory, and mechanical stimuli.

PHYSIOLOGY OF THE CLADOCERA 166 13. NERVOUS SYSTEM AND SENSE ORGANS

+90 o

Track

Fix

Flick

–90 o

FIGURE 13.6 Behavioral regions on the compound eye of Daphnia. AÀK, lenses; fix, fixation; PA, photorecep- tor axons. Source: Consi et al. (1990).

13.4 VISION

13.4.1 Anatomical Background of Vision Most cladocera possess an eye and an ocellus (a pigment spot), both of which are black. The eye consists of integumental cells, an intercellular substance, lenses, and receptor cells FIGURE 13.7 Structure of an esthetasc of Daphnia containing pigment (Figs. 1.1, 1.2, and 1.6) magna and of its terminal part. The inner space (Is) and (Gueldner and Wolff, 1970). The structure of the basal bead (bb) of each of nine olfactory setae is sur- À the eye is compound, or ommatidium; a rounded at its base by five sheath cells (I V). The receptor cilia (c) extend toward the terminal pellet, tp. aÀg, cross Daphnia ommatidium is shown in Fig. 13.8. sections at different levels. Source: Rieder (1987). The ocellus is a pigment spot. Both the eye and the ocellus may be developed to various The relative size of the eye is different in extents or may be absent. The general trend is different species. It is very large in Dadaya and toward an increased ocellus in bottom-living in polyphemids it constitutes a major part of forms, in contrast to pelagic species. the total body volume. In some bottom-living

PHYSIOLOGY OF THE CLADOCERA 13.4 VISION 167

BM shows, it is either immovable (as in females of CoC Alona affinis, Alonella nana, Camptocercus fennicus, Disparalona rostrata, Graptoleberis testudinaria, Picripleuroxus laevis, and Pleuroxus MC CC trigonellus) or flutters within a very limited amplitude (as in Acroperus, Chydorus sphaericus, RC and Pleuroxus truncatus). The muscles of the eye seem to be short. In Eurycercus, the eye is situated at some PG distance from the brain and is moved by thin PC muscles, as shown clearly by Leydig (1860) MF R (Fig. 13.1). It flutters, and makes limited rota- tions, and is sometimes turned up by c. 180. As reported for daphnids, the eye is situated NF at some distance from the brain and is constantly rotated by means of thin muscles. Three MC pairs of muscles move the eye: the levator, the lateralis, and the depressor (Binder, 1931). FIGURE 13.8 Structure of a Daphnia ommatidium. These muscles are inserted ventrally, dorsally, BM, basilar membrane; C, cone cell; CC, crystal cone; CoC, and laterally onto the eye (Consi et al., 1987). It covering cell; MC, mitochondria; MF, microvilli; NF, neu- should also be mentioned, for comparison, that rofilaments; PC, process of cone cell; R, rhabdom; RC, retinula cell. Source: Ringelberg (1987). the eyes of vertebrates, including fish, are also supplied by six muscles: four rectus oculomotorius muscles and two oblique oculomotorius muscles. species, which obviously live in the dark (in Downing (1974) discovered that the Daphnia interstitial spaces), the eye may be small or eye is suspended in position by a membrane absent, whereas the ocellus may be increased that forms a water-tight seal between the eye in size, or both may be absent. In contrast, in and the hemocoel. planktonic species the ocellus is usually small The eye in preserved specimens may be or absent. The ocellus may be normally very discolored by alkaline solution and the num- small (as in Daphnia species) or absent (as in ber of lenses (ommatidia) may then be easily most Moina species). Abnormal, eyeless speci- counted. This number may be up to 10 in mens of Moina and Simocephalus have been Penilia and chydorids, 44À81 in other cteno- reported (Banta, 1921; Banta and Brown, 1922). pods (Korovchinsky, 2004), 28 in Eurycercus Fryer (1953) identified a specimen of D. pulex lamellatus,22inMoina micrura (Smirnov, that had no complex eye, although eyes were 1975), and 22 in Daphnia (Heberdey and present in its embryos. Mutant Daphnia have Kupka, 1942; Downing, 1974; Frost, 1975; been reported that lack black pigment in the Fryer, 2004). eye (Young and Downing, 1976). Both the eye Comparative investigations into the topog- and ocellus and their function have been stud- raphy of the eye and its muscles, and of eye ied using various experimental methods, movements, although they are possible and including surgical removal (extirpation). would be very informative, have never been In various chydorids, the eye is situated just done in bottom-living Cladocera (Chydoridae, on the cephalic ganglion. As direct observation Macrothricidae, and Ilyocryptidae).

PHYSIOLOGY OF THE CLADOCERA 168 13. NERVOUS SYSTEM AND SENSE ORGANS

Bottom-Living Cladocera formed (over a period of 44 days); however, if Few, if any, studies have been made into the returned to white light, the normal level of this vision of bottom-living and littoral Cladocera. pigment was reached after 90 days (McNaught, Occasional reports have been made on color 1971). vision (see below) in the littoral species Pleuroxus truncatus (syn. Peracantha truncata) 13.4.2 Environmental Background and Scapholeberis mucronata, which are attracted by blue illumination (Peters, 1927), and on Solar light rapidly becomes extinct in water, Ceriodaphnia, Kurzia, Moina, Pseudochydorus, especially long-wavelength (red) light (see, e.g. Sida and Simocephalus (Smith and Baylor, 1953). Hutchinson, 1957, Ch. 6). Red light (720 nm) extinction is complete at c. 4 m depth in pure water (Dodson, 2005). In water, polarized light Pelagic Cladocera: Experimental has great ecological importance; it is reflected Approaches and Direct Observation from various particles, including food algal cells. Most experimental evidence concerns planktonic species. The Daphnia eye rolls almost ceaselessly (i.e. is in a state of rotatory tremor) 13.4.3 Surgical Methods of Investigation as it is pulled by special muscles, and is thought to scan visual information in this way. Eyeless daphnids were used for investiga- It was assumed that such eye movements are tions into the effects of light on cladocera, related to the mechanism of vision, that illumi- despite the fact that such specimens (Daphnia nation is assessed by Daphnia in this way, and and Simocephalus) very rarely occur in nature. that the necessary angle in relation to the source of light is retained (Waterman, 1961). Frost (1975) determined four kinds of eye Extirpation of the Eye and Ocellus movement in D. pulex: For investigating the reactions of eyeless Daphnia, Schulz (1928) removed the eye 1. tremors at 16 Hz; through a puncture hole made by a fine nee- 2. a scanning movement at 4 Hz; dle. For this procedure, the daphnid was 3. large fast eye movements; and placed with its frontal side upon a slide and 4. optokinetic nystagmus, produced by excess water was removed so that the daph- moving striped patterns around a daphnid. nid’s head was outside the water. When the The first two kinds of movement were integument above the eye was punctured, the observed in diffuse light and the third kind in eye emerged above the integument and could the presence of spots of light crossing the be separated out directly or was shed with the visual field. Three other types of activity by the carapace at the next molting; this was D. magna compound eye were discovered by confirmed by Downing (1974), who repeated Consi et al. (1990): a “flick” caused by a flash of this method. The percentage of successful light, “fixation” in response to a stationary light operations was satisfactory and, after two stimulus, and “tracking,” i.e. following a mov- molts, all damage caused by the operation was ing stimulus (Fig. 13.6). When a light stimulus healed and the animals could be used for is situated at about 80 dorsal to the eye’s axis, experimentation. there is no response (this is the null area). Extirpation of the complex eye for experi- When D. schoedleri were cultivated in blue mental purposes was also described by Harris light, no pigment sensitive to red light was and Mason (1956), who copied the methods of

PHYSIOLOGY OF THE CLADOCERA 13.4 VISION 169

Schulz (1928). In their experiments, a Daphnia Mason, 1956). In summary, Daphnia generally was placed into a moistened groove made in do well without eyes. paraffin, on its back and slightly tilted to the In addition, C. sphaericus successfully propa- side. The carapace was then punctured above gated in the dark over a period of about the eye with a fine pin. If the hole was success- 2 months, and they had only slightly different fully made, the eye emerged above the punc- characteristics (e.g. number of progeny and ture. It was then removed and the animal was longevity) from controls kept in the light returned to the culture medium. After some (Smirnov, 1971). time, it became evident whether the animal had survived the operation; if so, such animals were used for experimentation. Generally, 13.4.4 Taxes these animals survived for several weeks and reproduced even more intensively than normal Reaction to Light: Phototaxis animals. Daphnia have been found to be primarily In a similar fashion, Schulz (1928) separated negatively phototropic and positively geotro- and removed the ocellus. Several animals sur- pic, but a reduction in light intensity reversed vived the operation, molted, and were used in the signs of tropism (Clarke, 1930). This reac- experiments into the effect of light on various tion persisted for a few minutes, showing that processes. Later, Consi and Macagno (1985) Daphnia exhibit labile behavior in relation to also removed the ocellus from D. magna immo- illumination. If illuminated from the side, bilized with carbonated water using a micro- daphnid turn toward the more strongly illumi- beam from a dye laser (Candela SLl-500; nated side. 450 nm) coupled to a Zeiss microscope. Several Wavelength peaks of light sensitivity are laser pulses at 26À27 kV were used to delete different in different species: in D. magna, they the ocellus. The animals recovered after 2À4 occur at 440, 470, 520, and 640 nm (Wechsler days and were used in experiments. Such ani- and St. John, 1960); and in D. retrocurva, they mals had the same spectral sensitivity as occur at 370, 435, and 570 nm, and less so at controls. 685 nm (McNaught, 1966; McNaught and In the operated animals (Schulz, 1928), the Hasler, 1966). reaction to light was identical to that of intact The threshold of visual sensitivity in Daphnia 2 2 Daphnia, and Schulz (1928, p. 543) concluded is 10 4À10 5 lx (McNaught and Hasler, 1964). that “the ocellus of daphnias as a rudimentary The response of Daphnia to illumination is organ does not play a noticeable part any dynamic. These reactions are not rigidly fixed more in the life of these animals.” In Consi’s and vary depending on changes in the envi- (1985) experiments, animals that had the ocel- ronment and on the condition of the animal lus removed had the same spectral sensitivity (Loeb, 1924). A reduction in light intensity as normal animals. reverses these reactions for a short time After extirpation of the eye and the ocellus, (Clarke, 1930), and periodic changes of photo- the reactions of daphnias to light remain tactic signs have also been observed under essentially the same due to their general light constant low illumination (Clarke, 1932). This sensitivity (Schulz, 1928; Harris and Mason, may be understood as exploratory behavior. 1956; Fryer, 2004). However, completely eye- Experimental reversal of the reaction of less Daphnia do not exhibit a dorsal light reflex D. magna to illumination was also observed by (Schulz, 1928). These blinded Daphnia are also Chernykh and Panasyuk (1964). The basic more sensitive to vibration (Harris and observations by Ringelberg (1964, 1987) were

PHYSIOLOGY OF THE CLADOCERA 170 13. NERVOUS SYSTEM AND SENSE ORGANS that under conditions of decreasing intensity It is thought that the muscles that move the of illumination, D. magna move toward the eye also transfer the stimulus to muscles of the light source; at increasing intensity, they move antennae: if daphnid eye muscles are strained away from it. A continuous decrease in light differently at each side of the eye, then the intensity led to upward swimming. antennal muscles act to change the position of De Meester and Dumont (1988) identified the body (Harris, 1953; Jander, 1959). Movement three phenotypes of D. magna with respect to of the D. magna complex eye may also be stimu- light: positive phototaxis, negative phototaxis, lated by its intermittent illumination (but not by and random wandering between areas of low the illumination of any other part of the body) and high light intensity. They consider these (Young, 1974). If the complex eye is illuminated phenotypes to be genetically determined. from above, then maximum movement is Thus, reactions to light may be individual: caused by the short-wavelength range of the some specimens may be strongly negatively spectrum; if illuminated from the side, then phototactic and others strongly positively pho- maximum movement is caused by the yellow- totactic, even within a single natural D. magna green range. population (Dumont et al., 1985). Daphnia spe- Genostrychnine intensifies the photokinetic cimens may deviate by up to 30 from the activity of D. pulex, and chlorpromazine ren- direction perpendicular to the polarization ders its occasional movements less frequent plane (Waterman, 1960, 1961). However, shoal- (Rimet, 1965). ing Daphnia congregate at the water surface, Extraocular light sensitivity also exists i.e. contrary to the aforementioned tendency (Ringelberg, 1987). Light is sensed through (Fryer, 2004). Scapholeberis also attach to the integuments, as well as the eyes, as shown for surface film of water under bright illumination D. pulex by Scheffer et al. (1958). However, and Holopedium ascends to the water surface light perception and phototactic movements by day and moves to deeper water by night occur in Daphnia when both the compound eye (Fryer, 2004). and the eye of the nauplius are extirpated Starved Daphnia tend to move, whereas sati- (Ringelberg, 1987). Fryer (2004, p. 46) also ated ones stay at the same level. Starved noted that “eyeless individuals manage their D. pulex swim upwards, but starved D. magna affairs reasonably well!” sink (Szlauer, 1962b). The orientation of a daphnid depends on Reversal of Phototaxis the direction of light. When illuminated from The reaction of Daphnia to light may be above, daphnias maintain a position with their reversed if some of the large constellation of dorsal surface turned toward the light (Schulz, factors that affect their behavior change (Loeb, 1928; Harris and Mason, 1956), an orientation 1924). For example, reversal of phototaxis in known as the dorsal light reflex. When illumi- C. sphaericus and C. ovalis by acids was shown nated from below, the daphnias swim upside by Bryukhatova (1928). Clarke (1932) also dem- down, i.e. with their dorsal side turned toward onstrated that phototaxis in Daphnia may be the source of light (Schulz, 1928; Harris and reversed by changes in external or internal Mason, 1956). These results deserve further factors. investigation. Skadovskiy (1939, 1955) noted that the reac- As a result of being attracted by light, some tion of D. pulex to light may be different Cladocera can also be collected in light traps, depending on the environmental circum- especially Bosmina coregoni, Bythotrephes, stances. In daphnias settling in summer to Eurycercus, and Leptodora (Szlauer, 1971). lower water levels in a eutrophic water body

PHYSIOLOGY OF THE CLADOCERA 13.4 VISION 171 the positive reaction to light becomes more light is the light reflected by algal cells and other intensive and they move to warmer and better particles, which are potential food items. Thus, aerated layers, whereas, with insufficient ali- sensitivity to polarized light plays an important mentation their negative reaction to light role in obtaining food. D. magna and D. pulex increases and they settle into colder water collected much more food in a zone of polarized layers with more abundant food. In the pres- light than in a zone of nonpolarized light of the ence of a sufficient quantity of undissociated same intensity (Verkhovskaya, 1940). carbonic acid molecules phototaxis becomes It has been shown that several species positive. Positive phototaxis also occurs in a (both littoral Ceriodaphnia, Kurzia, Moina, situation where there is insufficient oxygen. In Pseudochydorus, and Simocephalus spp. and negatively phototactic daphnias, the oxygen pelagic, Bosmina, Daphnia, and Leptodora spp.) consumption rate is considerably higher than that Cladocera illuminated by a vertical beam in positively phototactic ones. of polarized light swim perpendicular to the Various factors may be responsible for the polarization plane (Baylor and Smith, 1953; reversal of phototactic reactions in Daphnia. Waterman, 1960, 1961; Hazen and Baylor, The positive phototactic response in D. magna 1962). Gromov (1992) also observed that decreased as food became limited and this Daphnia can distinguish between vertical and response was also dependent on the particular horizontal beams of polarized light; in the ver- clone of this species being investigated (De tical beam, Daphnia movements are reduced by Meester and Dumont, 1989). The D. magna half. Following the addition of sodium bro- photoreaction also differs depending on their mide (NaBr), the response of D. pulicaria to lin- previous adaptation to temperature (Lobashev early polarized white light (90 e-vector and Ivanova, 1947). orientation) became more random (Goksen The phototactic reaction also changed in and McNaught, 1995). These authors suggest D. magna, Evadne, and Podon following the that this is due to a disturbance in the trans- 2À 5 2 addition of CdCl2 (1:10 1:10 w/v) mission of nerve impulses, as Br ion blocks 2 (Smirnova, 1960) to their environment; because chloride (Cl ) channels. it binds sulfhydryl groups, phototaxis could be restored by the addition of cysteine 2À 4 (1:10 1:10 w/v), a sulfhydryl group donor. 13.4.6 Perception of Colored Light The positive phototactic reaction was changed to negative in Evadne and Podon if CdCl2 was Ewald (1914) found D. pulex to be sensitive added to the water (Smirnova, 1960), and in to colored light, with two maxima: in the D. pulex if illumination was increased from green-yellow and blue-violet regions. Lumer 1300 to 5274 lx (Rimet, 1961). (1932) exposed D. pulex, D. magna, Moina, and Leptodora to a graded series of light levels of different wavelengths but equal intensity. All of the tested animals moved toward the orange 13.4.5 Perception and Effect À of Polarized Light light (620 640 nm). Moina was also attracted by green light (540 nm) with approximately Cladocera can detect polarized light, which the same efficiency; its secondary maximum influences their orientation and direction of was in the blue range (440 nm). Each species movement, i.e. they exhibit polarotaxis tested showed its own characteristic reaction (Waterman, 1961; Young and Taylor, 1990). In to light. The littoral species Pleuroxus truncatus the aquatic environment, a source of polarized (syn. Peracantha truncata) and Scapholeberis

PHYSIOLOGY OF THE CLADOCERA 172 13. NERVOUS SYSTEM AND SENSE ORGANS mucronata aggregated in the zone of blue light the light changed from blue to white and (Peters, 1927). downward when the light changed from yel- Sensitivity of the Daphnia complex eye was low to white. This response to color change shown to be highest to green light; it also per- was observed in both littoral Ceriodaphnia, ceives light through its integuments, but the Kurzia, Moina, Pseudochydorus, Sida, and maximum sensitivity in this case is to blue- Simocephalus and pelagic Daphnia, Bosmina, and violet wavelengths (Scheffer et al., 1958). Leptodora spp. (Smith and Baylor, 1953). Moina In most cladocerans, four visual pigments was almost paralyzed by a sudden exposure to have been found, with maximum sensitivity to blue illumination. Responses in Ceriodaphnia light of wavelengths 370, 430, 560, and 670 nm were preceded by a considerable time lag. (red) (McNaught, 1971). In oligotrophic lakes, When illuminated from above with red light the environment is predominantly blue but (about 600 nm) at a uniform intensity over the during eutrophication a redder environment is aquarium surface, Bosmina, Ceriodaphnia, formed. In the ommatidia of D. magna, Smith Daphnia, and Moina and were observed to gen- and Macagno (1990) identified four spectral erally dance upright, with a small horizontal classes of photoreceptors with peak sensitivi- vector in their movements, and thus stayed in ties at 348, 434, 525 and 608 nm for the dorsal the same area (Smith and Baylor, 1953). Under ommatidia; in contrast, there was only a small blue light (about 500 nm), these cladocerans difference in peak sensitivities for the ventral were distinctly agitated, leaning forward with ommatidia. a large horizontal vector, and thus swimming Both the normal and the eyeless Daphnia from one place to another. According to were particularly attracted by yellow and Stearns (1975), Daphnia tend to move vertically green light in Schulz’s experiment (1928). between 440 nm (blue) and 735 nm (red), and Color preference was also studied in Daphnia horizontally at 440 nm (violet) and under carinata. For this, animals were placed in a ves- white light. sel surrounded by black paper, with four If light falls on the D. magna eye through openings illuminated by lights of different the top of the head, the action spectrum peaks color (Maity and Saxena, 1979). After illumina- at low wavelengths, but if it falls on the eye tion for 15 min, the distribution of daphnias through the side of the head, the action spec- was recorded: D. carinata preferred light of the trum peaks in the yellow-green region following colors, in decreasing order: yellow, (Young, 1974). orange, red, violet, blue, and green. A negative effect of blue light has also been D. magna showed peaks of light sensitivity observed. Sida show a negative reaction to at wavelengths 440, 470, 520 (blue), and 640 blue light (Peters, 1927) and Moina is extremely (red) nM (Wechsler and St. John, 1960). sensitive to blue light, being almost immobi- Gromov (1992) also observed the influence of lized by it (Smith and Baylor, 1953). Prolonged light wavelength (or color) on the locomotory illumination with blue light (of about 500 nm) activity of Daphnia, as seen by their attraction killed cladocerans (Smith and Baylor, 1953). to light. The locomotory activity of mature Daphnia decreased within the range À 400 525 nm (violet-green). 13.4.7 Photoperiod When Cladocera were illuminated with a vertical beam of light, for which the wave- The photoperiod, i.e. length of the day (i.e. length was controlled using colored filters, light) in relation to the duration of darkness, they responded by swimming upward when plays an important role in controlling the life

PHYSIOLOGY OF THE CLADOCERA 13.4 VISION 173 cycle and onset of gamogenesis (bisexual The lethal effect was increased when Daphnia reproduction) in Cladocera (Fig. 11.4). In cul- longispina were exposed to UV-B than to ture, Pleuroxus denticulatus switched to gamo- UV-A; exposure to UV-A caused significant genesis under the influence of photoperiod: it oxidative stress, as detected by increased malo- was more prominent with a long day length in naldehyde, whereas exposure to UV-B was populations originating from the southern followed by increases in both malonaldehyde USA and at a short day length in northern and catalase (Vega and Pizarro, 2000). The populations (Shan, 1974). P. procurvus and addition of ascorbic acid reduced mortality P. truncatus started gamogenesis only under caused by UV-A, but not by UV-B. Therefore, the short day conditions. It has also been a protective mechanism against photooxidative shown in Daphnia that the formation of males stress is presumed to exist. UVR is also delete- may be induced by a decrease in day length to rious to newborn D. galeata at the water 12 h (Stross and Hill, 1965). surface, and this effect decreases with increasing depth (Winder and Spaak, 2002). Under UVR illumination, D. pulex is nega- 13.4.8 Effect and Perception of tively phototactic (Peters, 1927). D. magna illu- Ultraviolet Radiation minated by UV light also exhibit negative phototaxis (at a maximum spectral sensitivity The effect of UVR has been studied by of 349 nm), whereas phototaxis to visible light many researchers, and it has been shown that (120À600 nm) is positive (Storz and Paul, UVR seriously endangers Cladocera. At natu- 1998). Many species living at the water surface rally occurring intensities, it has lethal effects and at high altitudes develop melanistic color- e.g. on Daphnia middendorffiana (Luecke and ation, probably as a protection against UVR. O’Brien, 1983) and D. pulex (Wu¨ bben and It has been shown experimentally that Vareschi, 2001). Hurtubise et al. (1998) deter- downward migration provides effective pro- mined the median lethal dose (LD50) of UVR tection for D. pulex (Vareschi and Wu¨ bben, for Ceriodaphnia reticulata, D. magna, and 2001) and D. galeata (Winder and Spaak, 2002). Scapholeberis kingii using a solar simulator: the Siebeck (1978) compared the effect of UVR on μ 2 LD50 at 96 h, ranged from 4.2 to 84 W/cm . D. pulex (light brown) and D. galeata (unco- Scapholebris kingii turned out to be highly sen- lored) and found that the former was 1.5 times sitive, and D. magna and C. reticulata moder- better protected from UV. Melanic clones of ately so. Bosmina meridionalis showed no D. pulex and D. middendorffiana also survived change in mortality over the whole range of exposure to 20 W/m2 near UV light for twice UVR tested in comparison with the controls as long as unpigmented ones (Hebert and and in contrast to both Ceriodaphiunia dubia Emery, 1990). The melanic morph of D. longis- and D. carinata (Wu¨ bben et al., 2001). pina survived better than the hyaline morph The damage and repair processes induced under solar UVR (De Lange et al., 2000). by UVR in Cladocera may be interconnected. Melanin in the carapace reduces UV Following exposure to damaging UVR penetration. (280À320 nm), Daphnia, in the presence of both InthepresenceofUVR,alargeproportionof long wavelengths and visible radiation, exhib- D. pulicaria migrate downward, compared with ited a large increase in survival due to stimula- that are specimens shielded from UVR (Leech tion of photoenzymatic repair (the induction and Williamson, 2001; Leech et al., 2005). UV is of which is favored by UV-A radiation, therefore a factor that induces downward migra- 320À400 nm) (Williamson et al., 2001). tion of Daphnia spp. whereas species with a more

PHYSIOLOGY OF THE CLADOCERA 174 13. NERVOUS SYSTEM AND SENSE ORGANS highly pigmented carapace stay closer to the D. longispina, there was a positive correlation water surface when exposed to UVR (Rhode between the water absorbance and catalase activ- et al., 2001). ity, which could be related to photoinduced It has also been shown in D. pulex and hydrogen peroxide production. The activity of D. tenebrosa that melanin plays a major role in superoxide dismutase was high in D. longispina protection against UVR (Hessen, 1999). In the from a lowland humic pond. The activity of glu- absence of blue light and UV, Daphnia do not tathione dismutase was low in a melanic D. pulex reconstitute their carapace melanization after group. No difference in antioxidant activity was molting. The concentration of protective mela- found between the alpine melanic and nonpig- nin in Daphnia increased after the ice break-up mented D. longispina populations. in North Finland, i.e. in the period of maxi- Following UV exposure, catalase was found mum underwater UV intensity (Rautio and to be more active in Daphnia in the presence of Korhola, 2002). organic matter, whereas the opposite was The detrimental effects of UVR on D. magna shown for glutathione transferase (Hessen and D. tenebrosa increased in the presence of a et al., 2002). reduced calcium content in the solution A positive reaction to UVR (i.e. radiation with (Hessen and Rukke, 2000b). wavelengths of , 400 nm), was reported for lit- In D. catawba exposed to UVR, the respiration toral Pleuroxus truncatus (syn. Peracantha truncata) rates increased by 31.8% (at sublethal irradiation and Scapholeberis. However, Daphnia had the of 2.08 kJ/m2 UVB 1 visible photorepair radia- highest eicosapentaenoic acid content in ponds tion) and decreased by 70.3% (at 4.16 kJ/m2) with the highest UVR exposure, and sublethal (Fischer et al., 2006). The enhanced respiration damage of the gut preceded UVR-induced mor- rates were attributed to energetic costs associated tality (Zellmer, Arts, Abele et al., 2004). with the repair of damaged cell components. Photoinduced toxicity in D. magna (esti- With increasing temperature, UV-induced mated by immobilization) to various chemicals DNA damage is increased to D. pulicaria at the liberated by the pulp and paper industry water surface, as well as DNA repair rates; was ranked as pyrene . anthracene . retene thus net DNA damage is greater at lower tem- (Huovinen et al., 2001). The toxicity of these peratures, where survival may also be lower chemicals is thought to result from internal (Macfadyen et al., 2004). Photoprotection may photosensitization reactions caused by UVR. therefore be more effective than photoenzy- UV-damaged cells are thought to release hista- matic repair, and more effective under low mine. Indeed, small doses of antihistamines temperatures. (α-benz-hydrylether-β-piperidoniethane, 2-phe- D. magna exposure to UVR did not induce a nylbenzylaminoethyl-imidazolin, or α-phenyla- change in catalase activity and caused a slight mino-β-aminoethane) do suppress negative increase in glutathione transferase activity; phototaxis (Poupa, 1948). The minimum effec- however, no changes in enzyme activity were tive dose for the first substance is 2.5 μg/mL. recorded at different oxygen levels, although At higher doses, phototaxis became positive. survival increased at lower temperatures Histamine takes part in the negative response (Borgeraas and Hessen, 2000). of Daphnia to UVR (McCoole et al., 2011), and The activity of antioxidants (catalase, glutathi- exposure to cimetidine (a blocking agent) one transferase, and superoxide dismutase) was inhibited the negative response of Daphnia to studied in Daphnia spp. in order to assess their UV exposure. role in UV photoprotection (Borgeraas and Interestingly, completely eyeless Daphnia Hessen, 2002a). In alpine populations of are repelled by UV illumination (Schulz, 1928).

PHYSIOLOGY OF THE CLADOCERA 13.6 CHEMORECEPTION 175

UVR also affects food algae, thus influencing the bottom layers of water bodies the electric their consumers indirectly. It has been shown potential may change by 0.6-0.8 V over 1 cm. (Leu et al., 2006) that UVR exposure modifies Although they are normally negatively pho- the lipid content of green algae: the content of totactic, Daphnia swam to the anode in an elec- 18:1ω9 decreased, C18 polyunsaturated fatty tric field with an electric current density of acids (PUFAs) increased, and the ratios C:P 0.5À1 ma/cm2 (Clarke, 1932; Clarke and Wolf, and N:P decreased. In D. magna fed with these 1933). Exposure to electromagnetic radiation of algae, the content of 18:1ω9 decreased but the 50 Hz for 8 h over a period of 30 days delayed essential PUFA content did not. D. magna maturation and decreased fecundity, See also Chapter 3, section 3.2.1 on melanin. but was not lethal (Somov and Malinina, 2003). Exposure of D. magna containing embryos However, a low-frequency electromagnetic to low-frequency laser radiation of 633 nm for field (with a frequency of 50 Hz) accelerated 60 sec resulted in the production of larger juve- D. Magna maturation, whereas an electric field niles (Osipova et al., 2010). of 500 Hz negatively affected both survival and maturation. It also increased the proportion of nonviable progeny (Krylov, 2008) and 13.4.9 Perception and Effect of X-Rays reduced both the number and size of the offspring (Krylov, 2010). Irradiation with X-rays has a negative effect In a weak strychnine solution (i.e. a satu- on Cladocera. In Simocephalus, it decreases the rated strychnine solution diluted 1:2000), respiration rate and inhibits growth, possibly Daphnia became positively phototactic and also through changing cell permeability (Obreshkove swam to the cathode (Clarke and Wolf, 1933). and King, 1932a). In young Simocephalus,italso induces invagination of the brood pouch, which becomes worse at each molting (Obreshkove and King, 1932b). 13.6 CHEMORECEPTION Baylor and Smith (1958) showed that D. magna swim irregularly in a horizontal beam 13.6.1 Anatomical Background of red light and swim to the bottom in a vertical Chemoreception in cladocera is ascribed to X-ray beam; in contrast, outside this beam they the sensory papillae (olfactory setae and esthe- swim back up. These authors assumed that this tascs) on the antennules. The chemoreception action is a result of X-ray-induced free radicals function of these sensory papillae has been dis- having different actions on visual pigments in cussed by Gicklhorn and Keller (1926). A the compound eye and the nauplius eye. In detailed description of the structure of olfac- Moina macrocopa, X-ray irradiation is reported tory setae was provided by Rieder (1987) to be destructive to the facets of the compound (Fig. 13.7). The inner space (is) and the basal eye (Fuchikawa et al., 1995). bead (bb) of each of the nine olfactory setae of D. magna are surrounded at the base by five sheath cells (I-V), and the receptor cilia 13.5 EFFECTS OF (c) extend to the very end of the seta (to the ELECTROMAGNETIC FIELDS terminal pellet; tp). Sensilla were originally discovered on the Cladocera are sensitive to electric fields. thoracic limbs of E. lamellatus (Fryer, 1963) and Electric fields are a normal environmental con- later identified in many chydorid and macro- stituent and Skadovskii (1955) showed that in thicid species (Smirnov, 1971). Sensilla on the

PHYSIOLOGY OF THE CLADOCERA 176 13. NERVOUS SYSTEM AND SENSE ORGANS

FIGURE 13.9 Esthetascs on thoracic limb IV. Thoracic limb IV of Alona affinis (bottle-like, right), of Alona quadrangu- laris (globular, left). Source: Smirnov (1971). thoracic limbs may be finger-like, globular, or 13.7 MECHANORECEPTION bottle-like (Fig. 13.9). Dumont and Silva-Briano (1997) described the distribution of sensory The setae of various Cladocera seem to structures on the trunk limbs of chydorids in include a sense of touch in their function. In detail. chydorids crawling on substrata, antennules Chemoreception obviously combines taste with their esthetascs are in contact with the and olfaction. The notion of taste has been various clumps and filaments they encounter. applied to Cladocera only once, by Kerfoot and The outer distal lobes of their thoracic limbs Kirk (1991), who reported that small Bosmina I “are capable of independent movements” demonstrate some ability to discriminate (Fryer, 1963, p. 362) and one may observe how between food particles by means of taste, in con- a moving specimen touches its surroundings trast to Chydorus and larger cladocerans. with the setae of these lobes. According to Hardy and McDougal (1894, p. 3), The presence of mechanical wave receptors deglutition (swallowing) “starts at the mouth as in D. pulex was reported by Szlauer (1964). a result of the stimulation of the sensory sur- Later, Meyers (1985) studied in detail the setae faces there”; however, these sensory surfaces at the base of the swimming antennae of are not commented on further or named. D. magna and concluded that they are rheore- In crawling chydorids, esthetascs on the ceptive and “mediate gravity perception” dur- antennules face outward and contact the sub- ing the sinking phase of their movement. stratum (shown e.g. in Fig. 1.3); thus, they per- Goulden (1966, 1968) suggested that ceive the substrate quality. copulating males of Moina species recognize

PHYSIOLOGY OF THE CLADOCERA 13.7 MECHANORECEPTION 177 gamogenetic females of their own species using the movement phase lasts for c. 25 msec (Zaret the sensory papillae and setae on their grasping and Kerfoot, 1980). Strokes of its antennae pull antennules and by feeling the surface sculpture the body forward. of the female carapace (future ephippium), which Swimming is controlled by a complex set of is different in different species. One of the func- environmental factors, with illumination being tions of head pores, and of the attached tissues, is a prominent influence. D. magna and D. pulex also thought to involve mechanoreception swimming is oriented by the predominant (Dumont and Van de Velde, 1976). direction of light. If they are illuminated with In Cladocera, hearing was not completely light of the same intensity from all sides, they excluded by Leydig (1860, pp. 41 and 42, trans- rotate (Ringelberg, 1963, 1964); when the light lated from German): “Whether the organ of intensity is then increased from one direction, hearing belongs to sense organs of cladocerans the animal resumes its normal position. Under is doubtful ... it is quite possible that one day more normal conditions, if the illumination it will be shown that the antennules of daph- intensity decreases then the D. magna swim- nids form the organ of hearing.” ming rate increases (Ringelberg, 1964); however, below a certain light intensity, Daphnia swim normally, independent of the angular 13.7.1 Orientation in Space distribution of light. Littoral and bottom-living chydorids are oriented in relation to their substrata in vari- 13.7.2 Control of Body Posture ous ways. They may crawl on the substrate, mainly at a right angle to the substrate’s sur- The body of planktonic cladocerans face, although a cladoceran may also be (Bosmina and Daphnia) is generally oriented located on the top or the side of a clump of with its dorsal side upward and backward. debris. They may also swim over and between According to Szlauer (1962a), gravitation is the various littoral substrates. Of the macrothri- main factor controlling the orientation of cids, Lathonura rectirostris remains immobile D. magna movements. Due to gravity, the ani- for rather long periods and then makes sud- mals sink and their position is frequently cor- den jumps, whereas Drepanothrix dentata lies rected by strokes of their antennae. There are on the surface of bottom debris with its ven- alternating short periods of movement and tral side up and only its thoracic limbs mov- rest. According to Jacobs (1964), the elongated ing. The diverse methods of movement of head of some Daphnia counterbalances the part littoral cladocera, and their different orienta- of the body that is behind the antennae. tions to the substrata, have been insufficiently The position of the center of gravity, as noted studied and offer a wide scope for further by Scourfield (1900b), is one factor influencing observations. There are also specialists that the position of a cladoceran body in space. He mostly live attached to the substrate by their observed that the position of the body in space dorsal side, i.e. Sida and Simocephalus, the for- differs between Daphnia (head and the dorsal mer by their dorsal organ and the latter by side obliquely upwards) and Simocephalus means of hooked terminal setae on their (head and the ventral side obliquely upwards). antennae. Scourfield also changed the center of gravity Planktonic cladocerans are oriented with their by occasionally attaching a drop of glue to the longitudinal axis at some angle to the vertical, i.e. animal or introducing an air bubble under the with the head upward and forward. In Bosmina, shell. In such cases, he observed the animal to

PHYSIOLOGY OF THE CLADOCERA 178 13. NERVOUS SYSTEM AND SENSE ORGANS

Light γ Accumulation

β α Intensity Direction

Eye Eye muscles Antennae Gravity receptor

+ Ib– I–+ II

–+

Position formation

Central nervous system

FIGURE 13.10 Scheme of the Daphnia orientation system. I, phototaxis; II, geotaxis; δ, direction of light falling on the eye, depending on α, β, and γ. Source: Jander (1966). swim abnormally. Changes to the orientation Immobilized small Cladocera sink at a rate of of the body due to displacement of the center 2À3 mm/sec, or as a percentage of body length, of gravity may be observed e.g. in gravid 350À550% for Chydoridae and 250À72% for Eurycercus compared with juvenile specimens Sida (Daphniidae) (Smirnov, 1971). The likely of the same species. Female Eurycercus that perception of gravity by the antennal setae of contain numerous embryos in the brood pouch Daphnia was suggested by Bidder (1929) and swim with their head upwards and move with Grosser et al. (1953). The position of the body in their dorsal side forward (Smirnov, 1971, space may also be controlled by the tension p. 80), whereas younger specimens are ori- receptors of the oculomotor muscles transferring ented with their back upwards. No special stimuli to the antennal muscles (Harris and observations of this kind have been made in Mason, 1956). Harris (1953) also noted that a dif- representatives of other cladoceran genera. ferent type of muscle located on different sides of the eye causes strokes of the antennae that contribute to daphnid rotation. Jander (1966, Buoyancy 1975) suggested a more complex system of sup- Buoyancy also depends on the specific port for orientation in Daphnia, comprising com- weight of cladocerans; it is modified by oil mands from the eyes, the antennae, and a inclusions (hydrostatic effect) and the develop- gravity receptor (which is still unidentified) ment of slime envelopes. Planktonic cladocer- (Figs. 13.10 and 13.11). ans also possess thin exoskeletons. The In the dark, C. ovalis and C. sphaericus obvious hydrostatic role of oil drops in the become homogeneously distributed in water; body of cladocerans has not been investigated. illuminations is followed first by upward

PHYSIOLOGY OF THE CLADOCERA 13.9 EFFECT OF XENOBIOTICS 179

thin rod, whereas males approach it and may II even become attached to it (Szlauer, 1964). Gravity Eye receptor Movement related to predators principally involves the strategy of akinesis (mainly in I III littoral species; Chapter 14, section 14.3.1) or the strategy of escape (mainly in pelagic spe- Eye Antennae cies; Chapter 14, section 14.3.2). muscles

Command 13.8 ENDOGENOUS RHYTHMS generator OF ACTIVITY

LCT In continuous darkness, D. magna is least active at midnight and maximally active at noon (Harris, 1963), whereas under constant FIGURE 13.11 Later schematic of the Daphnia orientation, showing three negative feedback loops. C, chemi- illumination their activity period was revealed cal information; L, light intensity information; T, to be about 28 h. A circadian time of 27 h temperature information. Source: Jander (1975). 50 min in D. magna was also reported by Ringelberg and Servaas (1971). Under natural swimming and then they become concentrated conditions, a similar circadian activity rhythm at the bottom (Bryukhatova, 1928). In complete was found in D. pulex: its activity was low at darkness (as instantaneous photographs have night, maximum in the morning, and then demonstrated), daphnias maintain a position declined thereafter (Stearns, 1975). corresponding to that adopted in the presence of illumination from above (Jander, 1966). In addition, after extirpation of both the eye and 13.9 EFFECT OF XENOBIOTICS ocellus, the reactions of daphnias remain essentially the same owing to a general sensi- There are only a few examples of experi- tivity to light (Schulz, 1928; Harris and Mason, ments related to the effect of xenobiotics on 1956; Fryer, 2004). movement. In the presence of 0.02 mg/L 1 Thus, orientation of the daphnid body obvi- Cu2 , D. magna became less positively photo- ously depends on a combination of several tactic (Kien et al., 2001). Cr (as K2Cr2O7) varying stimuli. inhibits phototaxis in D. carinata,asshown It may also be noted that locomotory by a negative linear correlation between the 1 responses may differ in individuals of different phototactic index and Cr6 concentration sexes. Thus, D. pulex females run away from a (Wu et al., 2005).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 14

Behavior

14.1 DIFFERENCES IN BEHAVIOR into the bottom mud to running over the sur- AMONG SPECIES face of various bottom substrata. The action of pushing through narrow passages was termed Types of behavior characterize the lives of scrambling by Fryer (1968). different Cladocera species. It is likely that each Crawling over a flat substrate is characteristic of the numerous littoral and pelagic exhibits of Graptoleberis testudinaria, which also attach species-specific reactions. Representatives of the themselves to the substrata by sucking (Fryer, same genus may be strikingly different, e.g. 1968) and are thus able to glide along the sur- Chydorus gibbus is strictly bottom-living, face. The ventral side of this chydorid is flat, the whereas other species of this genus tend to outline is densely supplied with plumose setae swim in open water. The well-known urge of (Fig. 1.4), and the suction force is achieved by Sida crystallina to attach itself to any surface was pumping water from inside the valves. studied quantitatively by Szlauer (1973). Males Movements are notably slow in the bottom- show a strong drive for attaching to females, as living Ilyocryptus. After producing progeny reported in Chydorus sphaericus (Smirnov, 1965b, they remain in the same place surrounded by 1971; Van Damme and Dumont, 2006). their young (Chirkova, 1972a, b). There are also some species that live on the surface of mud with their ventral side up (Ilyocryptus and 14.1.1 Littoral and Bottom-Living Drepanothrix). Another species that lives on the Species mud surface, Lathonura, finds a convenient place, arranges itself ventral side up, and can The modes of locomotion related to sub- then be observed to be immovable for hours, strata include crawling, attachment to various with only its thoracic limbs moving. substrates, and attachment to the surface film Cladocerans that crawl on the bottom sub- of water (Scourfield, 1894, 1900a, 1923, 1929). strata can stay in any position while feeding Crawling is characteristic of the littoral forms, on the substrate. In bottom-living cladocerans, is combined with occasional swimming over the form of the shell is usually streamlined, substrata, and is diverse, ranging from burying frequently containing teeth at a ventroposterior

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00014-8 181 © 2014 Elsevier Inc. All rights reserved. 182 14. BEHAVIOR angle. This angle suggests that they serve to D. pulicaria males can detect a female at a max- direct away the water flow formed at the pos- imum distance of 6.4 mm (i.e. four body terior end of the animal, but the actual hydrau- lengths), principally via mechanoreception of lics have never been specifically studied. fluid disturbances created by the swimming females. The trails of swimming Daphnia were visualized and photographed by Brewer (1998) 14.1.2 Pelagic Cladocera by using a Schlieren optical path and a smooth density gradient. Pelagic cladocera, on the other hand, may live all of their life cycle without making contact Surface Film with the substrata. They support their body in Some cladocerans may attach themselves to the water by the strokes of their antennae the surface film of water with their ventral side (Fryer, 1991). There are two kinds of strokes: up (e.g. Scapholeberis and Dadaya) and thus feed slow, sufficient for keeping the sinking body in for long periods in an “upside-down” position place; and rapid, used to travel through space. on particulate matter and probably also on the Cladocera movement depends on various unimolecular film on organic matter. Both environmental factors; it is highly variable and behavior and the structure of the ventral side of dynamic but the physiological mechanisms the valves in Scapholeberis were examined by that transform the stimuli into the responses Scourfield (1894, 1900b) and later reported in are not always obvious. Hutchinson (1953) more detail by Dumont and Pensaert (1983) highlighted the random stimulation of all (Fig. 3.11). Scapholeberis sometimes swims within Daphnia sense organs by turbulence. the water column, in a similar way to other In rising temperatures, daphnias swim Cladocera, but once below the surface film it is upwards, whereas in falling temperatures they reluctant to leave it (Fryer, 1991). Fryer (2006) tend to sink (Gerritsen, 1982). The redox poten- also noted that despite this characteristic behav- tial also influences the direction of movement. ior there is no sign of reshaping of any of its In the presence of catechol (Eh, otherwise organs in response to such an inverted position. 51 termed E’o, 0.33 V) daphnids move upwards, and in the presence of cysteine Terrestrial Environment 52 (E’o 0.14 V) they move downwards (Baylor Bryospilus crawls in moist, moss-like and Smith, 1957). The transitional level is growths on the trunks of tropical trees (Frey, 51 about E’o 0.045 V. 1980). Despite having a morphology character- The behavior of females and males of the istic of other chydorids, it refuses to swim if same species is clearly different. For Daphnia placed in water. pulex, Szlauer (1964) observed that females fled from a thin rod while males swam toward it and sometimes attached to it. The mating 14.2 MIGRATION AND SWARMING behavior of Daphnia pulicaria was investigated by Brewer (1998) (Fig. 12.1), who showed that 14.2.1 Vertical Migration male swimming was faster than and orthogo- nal to that of females. Video recording of the Vertical migration is known to occur in both swimming trajectories of females and males littoral species (as indicated for Acroperus and revealed that males pursued and contacted Eurycercus by Szlauer, 1962a, 1963a) and many both females and males, the latter being con- pelagic daphnids, including Daphnia species tacted more often, although for a shorter time. (Bainbridge, 1961; McNaught and Hasler, 1964;

PHYSIOLOGY OF THE CLADOCERA 14.2 MIGRATION AND SWARMING 183

Wright et al., 1980). Cladocera can withstand centimeters to a few meters across (Young and the changes of pressure experienced during Taylor, 1990). Sometimes such shoals consist of vertical migration. mixtures of various species. Kotov (2000) Under conditions of decreased illumination, observed a shoal in Lake Glubokoe (Moscow littoral Daphnia magna and Ceriodaphnia reticulata region, Russia) that predominantly consisted demonstrate a rapid upward movement of Scapholeberis, but in which about 10% was (Meyers, 1980). Vertical, but delayed, ascent was made up of Bosmina, Ceriodaphnia, Pleuroxus also shown in C. sphaericus and Pseudochydorus truncatus, and other chydorids. In experiments, globosus (if they were not attached to a substra- Bosmina swarming behavior was observed tum), and their ascent was stimulated by only in the light and with abundant food decreased oxygen levels. (Jacobsen and Johnsen, 1988). Changes in the eye of Daphnia longispina, A central question here is: what stimuli such as an increased pigmented surface under induce individual cladocerans to gather into laboratory conditions of constant illumination, swarms? The mechanism may involve visual were found to precede its ascent during vertical landmarks or chemical stimuli (e.g. attractive migration; this therefore forms the expression of odors), or it may simply be behavioral. It was an endogenous rhythm (circadian rhythm) also observed that Bosmina and Polyphemus are (Cellier et al., 1998; Cellier-Michel and Berthon, reluctant to leave a swarm and change their 2003). Vertical migration may, however, occur swimming behavior in the marginal zone of a without participation of the eye; this has been swarm (Young and Taylor, 1990). D. magna shown both in the dark and in specimens with has a tendency to form and maintain aggre- an extirpated eye. Szlauer (1963a) demonstrated gates in response to chemical cues from fish that daily vertical migrations of D. magna occur and invertebrate predators (Pijanowska and in the absence of changes in illumination or in Kowalczewski, 1997). In addition, Polyphemus the dark. Daphniae that have had their eyes pediculus was found to excrete 1-heptadecene removed can still migrate vertically in response into the surrounding water; this substance to differences in light intensity (Harris and may be involved in the formation, mainte- Mason, 1956). Daily vertical migrations in nance, and recognition of swarms (Wendel D. magna may also be caused by fish kairomines and Ju¨ ttner, 1997). Thus, swarms may create a (Loose et al., 1993). special physiological environment that, in Gliwicz (1986) highlighted the dependence turn, influences the members of the swarm. A of Cladocera population density on lunar swarm may also create its own general water cycles, resulting from a combined effect on the movements, which would not otherwise take vertical migration of Cladocera and the feed- place. As they are uniformly orientated, the ing intensity of the fish. members of a swarm can create general water movements within the swarm. Individuals within a swarm are integrated into it and the 14.2.2 Swarming swarm may move as a single entity within a water body (Young and Taylor, 1990). Swarming has been observed in both littoral (Lastochkin, 1930) and pelagic (Birge, 1898; Ku¨ nne, 1926; Dumont, 1967; Johnson and 14.2.3 Hydrostatic Pressure Chua, 1973) cladocerans, including Bosmina, Ceriodaphnia, Daphnia, Moina, Polyphemus, and Cladocera have been found at depths of up Scapholeberis. Swarms may range from a few to 150 m (as summarized by Smirnov, 1971).

PHYSIOLOGY OF THE CLADOCERA 184 14. BEHAVIOR

For example, Eurycercus and Camptocercus have cladocerans, you will see that some of them been recorded at 150 m (Zschokke, 1911). In stop their usual movements and sink to the Lake Baikal, Alona labrosa was abundant at c. bottom. This behavior is called akinesis (sham 70 m and A. setosocaudata was present at death or “dead-man response”), and is an depths of up to 133 m (Vasilyeva and Smirnov, important reaction in arthropods, related to 1975). Thus, cladocerans can live at consider- the fact that predators will only attack moving able hydrostatic pressures. A water depth of prey (Kerfoot et al., 1980). It is manifested 10 m approximately corresponds to 1 bar principally in littoral cladocera (Smirnov, (1 atm 5 1.013 bar 5 14.696 psi). Another char- 1977), whereas planktonic species use a differ- acteristic is their ability to withstand changes ent method to avoid predators: they flee from in hydrostatic pressure during vertical migra- danger (they manifest “the escape reaction”). tion. It has been demonstrated experimentally When disturbed, Chydorids, Drepanothrix, that during vertical migration (in response to Lathonura (Smirnov, 1977), and Ilyocryptus light) D. magna is unaffected by high pressure (Mordukhai-Boltovskoi and Chirkova, 1973; (Lincoln, 1970). Fryer, 1974, p. 142; Smirnov, 1977) play dead for a few seconds. Eurycercus lamellatus and Lathonura remain immovable for more than 14.3 EMOTIONAL REACTIONS 3 min, while this may continue in Chydorus for several minutes (Kerfoot, 1978). In these motionless specimens, the thoracic limbs may Cladocera have immediate reactions to vari- either stop or continue beating, e.g. in ous stimuli, shown as a temporary increase in Ilyocryptus (Mordukhai-Boltovskoi and heart rate by about 20% (Smirnov, 1965b) fol- Chirkova, 1973). In Lathonura, either the tho- lowing e.g. a slight mechanical irritation. In racic limbs and heart both stop beating or only addition, upon seeing specimens of its own the thoracic limbs stop during akinesis. species, oxygen consumption in Polyphemus Akinesis may continue for rather a long time pediculus was shown to change: it decreases in until the animal resumes its usual movement parthenogenetic females containing eggs, and for no obvious reason. increases in females containing developed In planktonic Bosmina, it was impossible to embryos, newborn, gamogenetic females, and discern its brief akinesis by direct observation. males (Butorina, 1979, 1980). In newborns, the However, using cinematographic photography, increase can reach 69%. Kerfoot (1978) (Fig. 14.1)foundthatBosmina use The mating behavior of Daphnia pulicaria brief akinesis (sham death) when attacked by a was investigated by Brewer (1998) (Fig. 12.1). predator, and passively sink by 2À4timesbody Cladocera also exhibit an immediate reac- length at 0.6À0.8 mm/sec, just sufficient for the tion to disturbance: akinesis in most littoral attacking predatory cyclops to miss its prey species (i.e. they faint) or an escape reaction in (Kerfoot, 1978; Kerfoot et al., 1980). planktonic species (they “flee in fright”). I did not manage to find any references to investigations on the physiological background 14.3.1 Akinesis of akinesis in arthropods. It seems that attempts to provide a deeper explanation of When disturbed, chydorids and macrothri- this widely known fact have not been made. In cids stop moving, fall to the bottom, and addition, quantitative and qualitative ratings remain immovable, sometimes for a long time. of the stimuli that cause akinesis have never If you shake a glass vessel containing littoral been made.

PHYSIOLOGY OF THE CLADOCERA 14.3 EMOTIONAL REACTIONS 185

272

150 303

130 334

110 90 78 36 380 650 X 0 0 410 610 36 220 78 500 272 110 573 303 130 150 334

650 610 573

380

410 FIGURE 14.1 Trajectories and akinesis (dead-man response) in Bosmina attacked by Cyclops. An attacked Bosmina in the state of akinesis sinks by several its diameters. Source: Kerfoot (1978).

14.3.2 Escape Behavior approached the object and some even attached themselves to it. The highest escape ability was Most pelagic cladocerans (Ceriodaphnia, manifested by Diaphanosoma,andthelowestby Daphnia, Diaphanosoma, Leptodora, Polyphemus, Bosmina and Chydorus (Szlauer, 1965). By apply- Sida, and Simocephalus) escape predators by ing a similar method, Drenner et al. (1978) stud- fleeing and do not manifest akinesis, as shown ied capture frequencies (by a siphon tube) for e.g. by Szlauer (1964). The escape capacity of different cladocerans in relation to their distance sidids was discussed by Korovchinsky (2004). from the tube. Captures frequency of cladocerans Szlauer (1964) observed that female Daphnia was greater than that of copepods. avoid a tube or glass wand of a certain diameter Somersaulting has been observed in Daphnia when it is used to model a predator by moving it both in response to a predator and to chemical through a Daphnia culture at a certain speed. cues from crushed conspecifics (Pijanowska and Their escape ability was recorded as the inverse Kowalczewski, 1997). D. magna reacts to these of the number of specimens sucked into a tube. stimuli by escaping, sinking to the bottom, or Interestingly, males behaved differently: they making aggregates; specimens that experience

PHYSIOLOGY OF THE CLADOCERA 186 14. BEHAVIOR these cues were more successful in avoiding whereas upon exposure to carbaryl (1 ppb), predators (Pijanowska and Kowalczewski, D. pulex escape-like behavior increased and it 1997). exhibited extreme escape behavior at an acutely toxic level (40 ppb) (Dodson et al., 1995). Phototaxis was depressed in D. magna by 14.4 IMPACT OF XENOBIOTICS bacitracin and lincomycin, whereas aminosi- ON BEHAVIOR dine increased the reaction to light and eryth- romycin did not alter phototaxis (Dojmi di At sublethal concentrations, xenobiotics dis- Delupis et al., 1992). D. magna lost mobility turb the normal behavior of cladocera in various either completely or partly on exposure to ways. Upon exposure to deltamethrin, D. spinu- dimethoate or pirimicarb (Andersen et al., lata jerks as if prodded with a needle; its escape 2006). Their recovery following single-pulse reaction became less intense and is followed by exposures was also studied. erratic swimming (Alberdi et al., 1990). Upon Hyperactivity was observed in D. magna exposure to lindane, D. magna failed to migrate exposed to pesticides, followed by reduced in a directed manner (Goodrich and Lech, 1990), activity and death (Matthias and Puzicha, 1990).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 15

Ecophysiology

15.1 PHYSIOLOGICAL depend on physiological differences between BACKGROUND OF THE LIMITS the species. Therefore, physiological mechan- OF ENVIRONMENTAL FACTORS isms must be examined to provide explanations for these phenomena. Their morphological and physiological There exist acidophilic species—the macro- potential enables Cladocera to live in a variety thricid genera Acantholeberis, Streblocerus, and of situations. Most Cladocera are freshwater some Australian endemic chydorids—that are animals but some are marine species or live in confined to acid coastal dune lakes (Smirnov saline lakes. Their ability to live at different and Timms, 1983). On the other hand, alkaline salinities depends on their capacity for osmotic lakes are inhabited by a few other species of regulation (discussed in Chapter 8) Cladocera. Various different Cladocera species inhabit It should be remembered that water in a wide variety of environments: various sub- nature comprises a complex solution of diverse strates, open water, microtelmata (accumula- inorganic and organic substances, including tions of a few cubic centimeters of water in biologically active and toxic ones (the latter epiphytic plants) (Smirnov, 1988), interstitial may be partly of natural origin, e.g. phenols). capillary water (Dumont, 1987, 1995; Sabater, Dodson (2005, p. 176) summarize the situation 1987), surface films, moist, and moss-like thus: “Aquatic organisms live in an olfactory growths on tree trunks in tropical forests field of dozens, if not hundreds of biologically (Bryospilus) (Frey, 1980). significant chemical signals. ... For example, All aquatic invertebrates are adapted to the Daphnia can probably sense at least a dozen natural dynamics of their environment. different chemical signals. These signals pro- Investigation into the distribution of Cladocera vide information about whether to accept or species in relation to the ranges of environ- reject food and about the presence of macro- mental factors has revealed that at least some phytes, specific predators, competitors, conge- species are present within certain limits of neric individuals, and males.” The presence of those factors (Ma¨emets, 1961; Røen, 1962; complex and variable chemical factors should Fryer, 1993); their limited distribution may also be mentioned, as well as the fact that

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00015-X 187 © 2014 Elsevier Inc. All rights reserved. 188 15. ECOPHYSIOLOGY factors react and interact in solution, either macrothricids prefer warm water subtropical alleviating or enhancing the action of particu- and tropical situations. In Daphnia longispina, lar components. the growth rate and maturation time remain Primary and secondary producers are sensi- constant within 1420C (Sarviro, 1985). In tive to the presence of dissolved organic acids, Daphnia magna, the temperature range is vitamins, and so on, which are variously dis- 1130C, and the thermal sensitivity for avoid- tributed in space and time. These are “complex ance responses is about 1.5C (Lagerspetz, chemical landscapes,” according to Keller et al. 2000). In a temperature gradient, most (2001), and it should be added that such land- D. magna have been shown to collect together scapes, both experimental (Bowles et al., 2002) at 1820C (Skadovskiy, 1955); at 20C for and natural, are highly labile. adults and 25C for juveniles (Chernykh and Physical factors are also in a state of contin- Panasyuk, 1964); or at about 24C, depending uous change, and reactions may be different to on their previous acclimation (Verbitsky and constant and fluctuating factors. Thus, Rider Verbitskaya, 2011). et al. (2005, p. 15) formulated the dependence The upper limit of temperature for Chydorus of physiological processes on the environmen- sphaericus was determined to be 34C tal conditions as: “Environmental signals regu- (Mortimer, 1936) or about 32C(Bogatova, late a variety of key processes in animal 1962). For daphnids, the upper limit of tempera- physiology. In many genera, environmental ture [100% lethal dose (or LD100)] has been signals such as changes in day length or tem- determined as 32CforD. cucullata,35 for perature signal the onset or termination of D. magna,36CforD. pulex,and31Cfor reproduction. These environmental signals Scapholeberis mucronata (Mortimer, 1936); 30C typically stimulate the release of neuroendo- for Daphnia and Ceriodaphnia (Mallin and crine signaling molecules that initiate endo- Partin, 1989), and 36CforD. longispina. Moina crine cascades culminating in physiological macrocopa is inhibited by a temperature of 36C responses to the environmental cue.” (Maksimova, 1969) and perishes at 39.5C Cladocera species integrate various environ- (Brown and Crozier, 1927) or 41C(Maksimova, mental factors, which results in a certain quan- 1969). D. magna is eliminated from natural tity of progeny. Depending on the combination waters at this temperature, and D. pulex at of numerous and not precisely discernible fac- slightly over 30C (Brown and Crozier, 1927; tors, the result may be a peak or a decline in Goss and Bunting, 1976); so, there are clearly abundance. In a particular water body, certain differences between species (LaBerge and Hann, species produce peaks of abundance, although 1990). For Bosmina longirostris, the tolerant zone in different years such peaks are different in for life activities is up to 36C, but the zone of height and their timing may be significantly normal life activities is 1123C, according to different. Verbitsky and Verbitskaya (2002). It is likely that tropical macrothricids can withstand somewhat higher temperatures, 15.1.1 Temperature although this has not been investigated. Latonopsis and Alona cambouei, for example, Cladocera have been observed to develop in have been found in hot springs in India at temperate latitudes in warm seasons, i.e. from 3436 C (Padhye and Kotov, 2010). Macrothrix May till September in the northern hemi- spp. are abundant in Iraq in pools that are nor- sphere. It was therefore inferred that they mally hot under the normal weather condi- prefer warm waters. Most (but not all) tions of that country.

PHYSIOLOGY OF THE CLADOCERA 15.1 PHYSIOLOGICAL BACKGROUND OF THE LIMITS OF ENVIRONMENTAL FACTORS 189

A further increase in temperature eventually e.g. increased of the population growth after causes protein denaturation (Brown and previous cooling. Crozier, 1927). For Daphnia middendorffiana, the upper level of temperature was found to be 26 C, above which inactivation of respiratory 15.1.2 Some Physiological Events Occur enzymes started (Yurista, 1999); however, it at Low Temperatures was shown, with reference to D. magna and D. pulex, that Cladocera may survive sudden Farkas et al. (1984) assumed that D. magna temperature changes over the range of 530 C cannot overwinter in an active state due to a (Goss and Bunting, 1976). failure to adapt their phospholipid composi- The lower limit of temperature tolerance tion and the physical state of their membranes seems to be about 0 C, since C. sphaericus to low temperatures. Docosapolyenoic acid is remained active and survived at this tempera- not present in Daphnia phospholipids and the ture, and did not avoid melting snow (Smirnov, level of polyenoic acid does not increase when 1971). The life span of D. longispina at 5 Cis the temperature decrease from 20Cto10C. much longer than at 20 C(MunroandWhite, Schlechtriem et al. (2006) cultivated D. pulex 1975). Some cladocerans stay active and repro- at 11C and 22C for 1 month on a highly duce at a temperature of about 0 C(Rivier, unsaturated fatty acid (HUFA)-free diet. Long- 1986, 1992). They are known to exist on arctic term exposure to cold temperature caused a islands and in tundra, where they are exposed, significant increase in eicosapentaenoic acid at least for some of the time, to very low tem- (EPA; 20:5ω3) and conversion of C18 fatty peratures. Rivier (1986) described winter com- acid precursors to EPA and arachidonic acid munities from many ice-covered lakes and (20:4ω6) was observed. Schwerin et al. (2009) reservoirs in which Daphnia and Bosmina con- observed compensatory control of physiologi- tained embryos. She also reported the presence cal processes in the cold: in D. pulex,most in winter of Daphnia with numerous embryos at cold-repressed proteins comprise enzymes the bottom of ice-covered Lake Siverskoe, at a involved in protein digestion (trypsins, chymo- low level of oxygen (ca. 2 mg/L O2). trypsins, astacin, and carboxypeptidases); and Generally, rising temperature is followed by cold-induced proteins include several vitello- a rising intensity of activity. For example, the genin and actin isoforms and AAA 1 ATPase. heart rate of D. magna increased from 60 to 400 The increased actin concentration in cold- beats/min when the temperature rose from acclimated animals may contribute to the pres- 5 Cto28C (Mac Arthur and Baillie, 1929) ervation of their muscular performance. (Fig. 6.6). It has also been noted that daphnias Following ultraviolet (UV)-B irradiation, swim upward in rising temperatures, whereas Daphnia spp. showed increased survival and they tend to sink at falling temperatures removal rates of light-dependent DNA damage (Gerritsen, 1982). at 10C than at 20C (Connelly et al., 2009). Heating water from 15 Cto25C, with a According to Gainutdinov et al. (2000), the subsequent reduction to 20 C, resulted in thermostability of D. magna is modified by larger body sizes of Ceriodaphnia quadrangula the addition of glutamate, glycine, or adeno- young, shorter longevity and earlier matura- sine 30,50-cyclic monophosphate (cAMP), with tion of exposed juveniles (Romanovskiy and the changes being slow, strong, or fluctuating, Loganova, 2010). In Simocephalus, Verbitsky depending on the concentration of substance and Verbitskaya (2009) also identified the added. The authors interpreted this process as after-effects of previous temperature changes, “coding of the chemosensory information by

PHYSIOLOGY OF THE CLADOCERA 190 15. ECOPHYSIOLOGY the nervous system,” although it is unknown pH. These authors also suggested a model of which organs of Cladocera are involved in whole-animal CO2 transport. temperature perception. Preference for an acid medium is especially clear in the macrothricids Streblocerus serricaudatus and A. curvirostris. The latter retains 15.1.3 Oxygen Concentration sodium (Na) better than does D. magna, which is improved in acidic medium: the rate of Some Cladocera, e.g. Ceriodaphnia laticaudata, sodium loss at pH 3 is lower than at pH 7 can withstand low levels of oxygen (Fox, 1945; (Potts and Fryer, 1979). Burgis, 1967). Accordingly, this species developed in great abundance during the first year of existence of the Cherepovets Reservoir (in 15.1.5 Illumination the Upper Volga basin) in response to a general oxygen deficit caused by decomposition of Photoperiod (see Section 13.4.7) and sea- flooded vegetation and forest litter (Smirnov, sonal changes of illumination obviously influ- 1966). In subsequent years, not a single speci- ence the population dynamics of Cladocera. men of C. laticaudata was found, although However, some Cladocera can live in the dark. C. pulchella and C. quadrangula became domi- Species of the same genus may differ greatly nant instead. in their reactions to environmental conditions and in their behavior. For example, different species of Ceriodaphnia are known to tolerate 15.1.4 Acidity-Alkalinity Ranges different oxygen limits (Burgis, 1967), different Moina species either inhabit freshwater or live Most cladocera prefer a neutral pH. The in saline waters, Daphnia species are ecolog- upper limit of pH was determined for chydor- ically different, and Chydorus species either ids as 10.6 (Bogatova, 1962), while a lower limit swim or are confined to the bottom (e.g. of pH 3.7 has been reported for Acantholeberis C. gibbus). It is remarkable that the reactions of curvirostris, Alonella excisa, C. sphaericus, Cladocera to external factors are so labile and Ceriodaphnia setosa and Ophryoxus gracilis that the values that characterize cladocerans (Kitaev, 2007). Havas and Likens (1985) reactions to various factors are so variable. The showed that Daphnia catawba die below pH 5.0, numerical values of the physiological para- but that Holopedium can survive even at pH 4.0. meters discussed throughout this text are D. pulex usually lives in circum-neutral con- therefore not absolute. Such values depend on: ditions, and Weber and Pirow (2009) investi- 1. age and sex; gated its response to acid stress. At pH 7.8 and 2. previous adaptation; normocapnia (i.e. a normal partial pressure of 3. interaction with other factors (synergies and CO2) its extracellular pH is 8.33 and PCO2 is antagonistic interactions); and 0.56 kPa, and bicarbonate concentration in its 4. the presence within a morphologically hemolymph is 20.9 mM. Acidic conditions defined species of genotypes (clones) that caused a slight acidosis (ΔpH was 0.160.23), have different tolerances to environmental a3065% bicarbonate loss, tachycardia, hyper- factors. ventilation, and hypermetabolism. At pH 5.5, defense mechanisms were activated, extracel- This is why the characteristics of environ- lular PCO2 decreased to 0.33 kPa and the ani- mental factors are relevant to a discussion of the mals tolerated only short-term exposure to this geographic distribution of a particular species.

PHYSIOLOGY OF THE CLADOCERA 15.2 SYNERGISM AND ANTAGONISM AMONG ENVIRONMENTAL FACTORS 191

15.1.6 Adaptation exhibited a high mean thermoresistance, whereas in others it was low (Pashkova et al., It should be noted that the values given for 1998). D. longispina lineages have been shown ranges of stability to certain factors, and any to differ in their resistance to Cu (Martins such figures, are not constant but instead et al., 2005). Thus, it is likely that the elimina- depend on other variables and on previous tion of vulnerable lines may lead to a loss of acclimation. This has been shown e.g. for cop- genetic diversity. per (Cu) by Bossuyt et al. (2005), and for acclimation to different previous temperatures by Shkorbatov (1982): temperature shock in 15.2 SYNERGISM AND Daphnia acclimated to 28 C occurs at a higher ANTAGONISM AMONG temperature than that of Daphnia acclimated to ENVIRONMENTAL FACTORS 8C. However, beyond a certain limit physiological temperature adaptation may be Numerous dynamic factors can act “in con- replaced by a regular diapause (Mitchell, cert,” as Steinberg et al. (2010) noted, and the Halves, and Lampert, 2004). independent action of one factor may be modi- Survival may also depend on the previous fied by other factors. Antagonism (or syner- environment of the specimens being tested. gism) between ions in solution modifies their Shkorbatov (1953) highlighted the effects of individual actions in certain species. Tauson prolonged physiological adaptation of clado- (1924a, 1924b) was probably the first to dem- cera, e.g. Simocephalus vetulus taken from a onstrate this in cladocerans. She observed a pond survived in deoxygenated water much decrease in the death of various cladocerans in longer than did specimens taken from a river. the presence of certain combinations of cations In addition, Stroganov (1983) emphasized the in comparison to treatment with single ions. fact that individuals also differ in their stabil- Winberg (1933) demonstrated experimentally ity, as observed in D. magna in solutions of that the destructive action of the potassium ion 1 sodium pentochlorophenolate. (K )onC. sphaericus is decreased in the pres- 1 ence of the sodium ion (Na ), but is unaffected 1 by the presence of calcium ions (Ca2 ). In con- 15.1.7 Genotypes 1 trast, in the case of Chydorus ovalis Ca2 also 1 Green (1956) found “racial and clonal differ- moderates the negative action of K . ences” within the same Daphnia species in the Pairs of metal ions may be either antagonis- ability of individuals to synthesize hemoglobin tic (as estimated by toxicity in D. magna), e.g. (Hb) and thus of their adaptation to low oxy- Al 1 Mo, Cr 1 Co, and Cr 1 Mg, or synergistic, gen levels in the habitat. Quantitative estima- e.g. Cr 1 Se, Cr 1 Zn, and Fe 1 Se, as demon- tions of the response of D. magna to the impact strated by Tomasik et al. (1995). The relation- of environmental factors may also vary ships between ions also depend on their depending on genetic factors (Barata et al., concentration. Yakovlev et al. (2000) found 2000). In D. pulex, 92 genotypes have been that the combination of Zn 1 Cd is antagonistic found (among 227 specimens); in S. vetulus,18 toward D. magna survival, whereas Zn 1 Cu genotypes were identified (among 112 speci- are synergistic. According to these authors, mens), which showed physiological differences there are also still more complex interactions. (LaBerge and Hann, 1990). In 25 tested clones Combinations of metal ions are more dan- of D. magna, thermoresistance of the heart, gerous for D. magna than are isolated ions at when tested at 38C, was variable: some clones the same concentration. It has also been shown

PHYSIOLOGY OF THE CLADOCERA 192 15. ECOPHYSIOLOGY by Dediu et al. (1995) that combinations of change has profound alimentary significance, metals are more dangerous than separate as algal lipids are assimilated with little metals at the same concentration, as shown change. Following extensive analytical mea- for Cu 1 Zn, Cd 1 Zn, Hg 1 Zn, Cu 1 Cd, surements, Sushchik (2006) found that the Cd 1 Hg, and Cu 1 Hg in D. magna. Clifford growth of planktonic Cladocera (Daphnia) is and McGeer (2009) have shown that the zinc controlled by EPA and nitrogen (N) availabil- (Zn) toxicity toward D. pulex is alleviated by ity; a general conclusion was also made that 1 Ca2 whereas, in D. magna, in the presence of EPA and N levels indicate the quality of their Ca, the Cd, Ni, Pb and Zn uptake is suppressed food within a water body. The seasonal distri- and elevated Na concentrations increased the bution of EPA in different links of the trophic uptake rates of Cd and Pb (Komjarova and chain was determined from May till October Blust, 2009). Selenium uptake by D. magna by Sushchik et al. (2008) in phytoplankton (in increased significantly as sulfate concentrations May the diatom Stephanodiscus was dominant), decreased (Ogle and Knight, 1996). In addition, zooplankton, and fish (Carassius) in a reservoir Ca and Hg each inhibit the accumulation of the near Krasnoyarsk (Russia). The quantitative other by D. magna (Teles et al., 2005). distribution of EPA in zooplankton generally Glyphosate reduces the acute toxicity of Ag, resembled that in phytoplankton (with a peak Cd, Cr, Cu, Ni, Pb, and Zn (but not of Hg and of c. 120 g/L in phytoplankton in May). In Se) toward Ceriodaphnia dubia (Tsui et al., general, the quantity of EPA in a cyprinid fish 2005). The effect on C. dubia of spinosad and (Carassius) followed that in zooplankton. The R-11 (an adjuvant used with pesticides) was predominant fatty acids in the fish were 16:0, also found to be synergistic (Deardorff and 18:1, and 22:6ω3. Sushchik (2001) had previ- Stark, 2009). ously observed that Daphnia development is There are two possible explanations for controlled by polyunsaturated fatty acids these observations: effects on the direct toxicity (PUFAs) derived from diatoms and blue-green of particular ions or the more complex effect of algae. Sometimes, the diatoms were lacking in deviation from the natural equilibrium of ions 20:5ω3 or other PUFAs, and the blue-green in fresh or brackish water, to which certain algae contained most of the essential 18:3ω3. species are adapted. Kainz et al. (2004) observed that the concentration of an essential fatty acid (docosahexaenoic acid) in the food chain decreased in the 15.3 LIPID PATHWAY FROM ALGAE cladoceran link in all of the lakes studied. It VIA CLADOCERA TO FISH should also be noted that in lakes in which diatoms are the dominant primary producers, Mu¨ ller-Navarra et al. (2004) advocate, with as indicated by a sediment consisting of diato- good reasons and reference to Daphnia data, mite (e.g. in Kamchatka, in Iceland), the tro- that the HUFAs EPA (C20:5ω3) and docosahex- phic chain is based principally on lipids. aenoic acid (C22:6ω3) levels provide a strong predictor of zooplankton growth. These fatty acids are conservatively transferred up the 15.3.1 Lipids in Freshwater Fish food web to fish and to man. In temperate latitudes, diatoms (with their Fats are known to be incompletely trans- stock of lipids) dominate in the spring, fol- formed by their consumers; the composition of lowed by blue-green and green algae, the latter their fat is similar to that of their food items. of which mostly contain a stock of starch. This For their normal development, Fish mainly

PHYSIOLOGY OF THE CLADOCERA 15.4 ENVIRONMENTAL CONDITIONING BY CLADOCERA 193 require three long-chain PUFAs (Sargent et al., was about 50% of the lake volume per day 1999): docosahexaenoic acid (22:6ω3), EPA during most of the growing season (Balayla (20:5ω3), and arachidonic acid (20:4ω6). and Moss, 2004). Cladocera thus clarify water Following consideration of the experimental and their feces fall to the bottom. data, Herodek and Farkas (1967) suggested that most of the fatty acids in fish originate in the crustaceans they consume. 15.4.2 Impact on the Gaseous As seen from the fat composition of fish, cla- Conditions of the Environment docerans, and algae, few modifications are made Cladocera consume oxygen and release car- to fatty acids in the pathway from algae via crus- bon dioxide. The extent of their influence may taceans to fish. The extent of fatty acid modifica- be assessed from their rate of oxygen con- tion at the algae-crustacea and crustacea-fish sumption and the quantity of Cladocera per linksisthefocusoffurtherinvestigations. unit volume of water. With good reason, Arts et al. (2001, p. 122) indicate that fatty acids “are important “dri- vers” of ecosystem’s health/stability,” and 15.4.3 Enrichment of the Environment may determine a humanity’s past and future with Organic Matter health. In fish, man consumes the algal (slightly modified) fat. The fish smell not actu- Cladocera release considerable quantities of ally from fish; rather, it is from algal fat. N, phosphorus (P), and carbon (as dissolved organic C). While N is released as a final product of protein metabolism, the source of liber- 15.4 ENVIRONMENTAL ated P seems to be digested compounds from CONDITIONING BY CLADOCERA lipids and phosphoric acid. As a result of their abundance in the aquatic environment, 15.4.1 Biofiltration (Clearance) Cladocera make a large contribution to the complex solution that is natural water. This As a mass group, the cladocera within a water contribution can be quantitatively estimated by body filter great volumes of water (see also consideration of the available data. For such Chapter 4, section 4.1, “Feeding”). Their filtering quantitative estimations, see also Chapter 7, rate has been determined to be 0.95.15 mL/ section 7.2. individual (ind.)/24 h in 0.71.8 mm long Cladocera also release a variety of organic D. pulex, increasing with size in a curvilinear and inorganic compounds into the water (as fashion (Richman, 1958); in 2-mm long D. pulex, noted e.g. by Fish and Morel, 1983). The mean it is about 5 mL/ind./24 h in the daytime, release of urea by well-fed Daphnia was esti- increasing at night up to about 20 mL/ind./24 h mated to be 0.36 μg/mg/h and that of ammo- (Haney, 1985). As summarized in ctenopods by nia to be 0.76 μg/mg/h (Wiltshire and Korovchinsky (2004), it is 1248 mL/ind./24 h Lampert, 1999); in contrast, starved Daphnia in Diaphanosoma brachyurum s.l., 1058 mL/ind./ liberated 0.060.1 μg/mg/h of urea and 24 h in Holopedium,2657 mL/ind./24 h in 0.45 μg/mg/h of ammonia. The released urea Sida, and 32252 mL/ind./24 h in Penilia.In is presumed to influence the development and D. magna, it is 0.243.56 mL/ind./h depending morphology of green algae. on the food concentration (Porter and Orcutt, Fish and Morel (1983) determined that 1980). In one example, the clearance rate (i.e. D. magna release about 40 pmol/organism/h filtering rate) of a Daphnia hyalina population of organic Cu-binding compounds, thought to

PHYSIOLOGY OF THE CLADOCERA 194 15. ECOPHYSIOLOGY consist of molecules larger than amino acids. 1999). This infochemical has been character- D. magna also release a chemical (an organic ized as an olefinic low-molecular-weight car- substance of small molecular mass) into the boxylic acid. environment that induces colony formation in The quantitative scale of these processes the green alga Scenedesmus acutus (Lampert may be estimated by considering the afore- et al., 1994). Yasumoto et al. (2005) also dem- mentioned data. onstrated that D. pulex releases aliphatic sulfates that increase the size of Scenedesmus cenobia. 15.5 THE IMPACT OF EXTREME Immediately after molting of a single D. puli- LIMITS OF ENVIRONMENTAL caria, the activity of β-N-acetylglucosaminidase FACTORS AND OF XENOBIOTICS in the culture medium increased by 40100 times (Vrba and Machacek,ˇ 1994); this molting 15.5.1 Impact of Extreme Limits enzyme sustained its activity for . 2 days. of Environmental Factors Within swarms of M. macrocopa, the concentrations of various forms of N and P increase, Temperature protein and carbohydrate fractions appear, All of the evidence indicates that the winter and various amino acids are present, as well minimum temperature is not disastrous for the as palmitic, tridecanoic, and stearic acids cladoceran fauna, judging e.g. by the rich (Kalacheva et al., 2000). fauna in Yakutia, which is within the zone of Polyphemus pediculus releases 1-heptadecene global minimum temperatures. Egg latency into the surrounding water; 7 ng/L was found protects the local species of Cladocera. within a swarm vs. 1.5 ng/L some distance away (Wendel and Ju¨ ttner, 1997). Ultraviolet Radiation B. longirostris tends to increase the N:P ratio UV radiation (UVR) is thought to affect cla- in the surrounding water, thus increasing docera that inhabit water bodies at high alti- phosphorus limitation (Balseiro et al., 1997). tudes and high latitudes. These Cladocera Alkaline phosphatase and a small amount of develop black pigmentation, which is thought acid phosphatase are released by D. magna to be protective. Indeed, exposure to sublethal into the environment (Boavida and Heath, UV irradiation resulted in damage to the intes- 1984; Zhao et al., 2006). The liberation of tine in a large proportion of subarctic Daphnia enzymes by animals to their aquatic medium (Zellmer and Arts, 2005). It was shown by may be a common occurrence, as it has also Zellmer et al. (2006) that following exposure to been shown for fish (Kuzmina et al., 2010). sublethal solar UVR, D. pulex suffers damage to the intestinal system similar to that induced by fasting, although the function of its diges- 15.4.4 Infochemicals tive enzymes (amylase and cellulase) is not much altered. The substances released by invertebrates, as far as they are involved in chemical communication in the aquatic environment, are known 15.5.2 Impact of Xenobiotics as infochemicals. Thus, formation of cenobia in the green alga Scenedesmus is influenced by a Inorganic Xenobiotics chemical released by D. magna during its non- It was shown in D. magna exposed to tritiated digestive metabolism (von Elert and Franck, water with an activity of 5 3 1025 3 108 Bq/L

PHYSIOLOGY OF THE CLADOCERA 15.5 THE IMPACT OF EXTREME LIMITS OF ENVIRONMENTAL FACTORS AND OF XENOBIOTICS 195 thatthenumberofbroods,thesizeofeach MERCURY brood, life span, and growth rate decreased The scale of mercury (Hg) contamination within five generations; embryos developed may be illustrated by the case of Lake disproportionately; and their eggs decom- Managua (Nicaragua), where about 40 tons of posed (Gudkov and Kipnis, 1995). elemental Hg was introduced from a chloralk- ali industry during the first 12 years of its SILVER activity (Lacayo et al., 1991). One result was Silver (Ag) is one of the most toxic elements. the complete disappearance of Bosmina from D. magna and C. dubia have been shown to be the plankton. In the aquatic situation, Hg is transformed into methylmercury, which is highly sensitive to AgNO3 but less so to AgCl (Rodgers et al., 1997). even more toxic. According to Tsui and Wang (2004b), the uptake of Hg and methylmercury by D. magna is proportional to their ambient ALUMINUM concentration, and efflux rates are comparable; Daphnia catawba and Holopedium were not release takes place via excretion, egestion, found to be particularly sensitive to aluminum molting, and neonate production. In bioaccu- (Al) (Havas and Likens, 1985). mulation, methylmercury is predominant. In lakes in Wisconsin (USA), the methylmercury ARSENIC concentrations were 1211 ng/g in D. pulex, D. galeata mendotae, and D. ambigua, and It has been shown that trivalent arsenic (As) 40419 ng/g in Holopedium (Back and Watras, is more toxic than pentavalent arsenic to 1995). A low concentration of mercuric chlo- Daphnia carinata (He W. et al., 2009). μ 21 ride (HgCl2; a daily rate 0.01 gHg ) given with food to C. affinis stimulated reproduction, CADMIUM but survival and longevity decreased It was determined experimentally that (Gremiachikh and Tomilina, 2010). C. dubia is more sensitive than D. magna to cad- POTASSIUM mium (Cd; as CdCl2) (Suedel et al., 1997). The uptake rate and killing rate of Cd for D. magna Juvenile D. magna exhibit a very varied sen- increased with an increase in temperature sitivity toward potassium dichromate from 10 Cto35C (Heugens et al., 2003). (K2Cr2O7) (Klein, 2000), as noted by their immobilization. COPPER URANIUM The presence of Cu in water is directly toxic to cladocerans. It also decreases the oxygen It was determined after 21-days exposure of consumption in Daphnia (Knyazeva, 1994). The D. magna to free uranyl that the chemical toxic- effect of Cu depends on the clone, as shown ity of uranium (U) predominated over its for D. longispina (Lopes et al., 2005). Cu toxicity radiotoxicity (as estimated by decreased decreases in the presence of Na (De somatic growth and reproduction) (Zeman Schamphelaere et al., 2007) or organic matter et al., 2008). in the medium, especially humic acids, as has Comparative tests of Cd and Zn on been shown for D. magna (De Schamphelaere D. magna, D. pulex, D. ambigua, and C. dubia et al., 2004) and C. dubia (Sang Don Kim et al., demonstrated that D. magna is significantly 1999). more tolerant to these metals, or their

PHYSIOLOGY OF THE CLADOCERA 196 15. ECOPHYSIOLOGY combinations, than other daphnids (Shaw The effects of different xenobiotics are also et al., 2006). Acclimation of D. magna to Zn discussed in sections describing their particu- was demonstrated, which influenced determi- lar functions. nations of Zn toxicity (Muyssen et al., 2002). Organic Xenobiotics 15.6 CLADOCERA IN Acetylsalicylic acid is more toxic to D. long- WATER QUALITY TESTING ispina than to D. magna in acute assays, but is more toxic to D. magna under chronic exposure There are three main roles of cladocera to (Marques et al., 2004a). Its metabolites were consider: their use in measuring pollution also tested; these turned out to be most toxic levels, drinking water quality, and in testing to D. longispina (Marques et al., 2004b). human body fluids. Nonviable neonates and aborted eggs were First, Cladocera spp. have found a role in produced in addition to the direct toxicity the system of indicators of the saprobic level observed. of water (i.e. of pollution with organic sub- Fipronil (a phenylpyrazole insecticide) is stances) (Kolkwitz and Marsson, 1909) and in more toxic for C. dubia than its optical isomers subsequent modifications of this system. (enantiomers), racemate, or photodegradation According to these authors, they appear to product (fipronil desulfinyl) (Konwick et al., decrease pollution due to self-purification in 2005). both β-mesosaprobic and oligosaprobic zones. Obviously, their distribution depends on PHENOL hydrochemistry. Of the cladocera tested, S. vetulus was the Hrba´cek and Hrba´ckova´ (1980) discussed least sensitive to the presence of phenol and the use of planktonic Cladocera for the estima- Sida crystallina was the most sensitive tion of water eutrophication. Andronnikova (Luferova and Flerov, 1971b). These authors (1996) described mixtures of zooplankton char- mainly attribute the latter effect to the higher acteristic of oligotrophic, eutrophic, and poly- (almost double) beating rate of the thoracic humic lakes. limbs in Sida. The motor activity of D. longispina Second, Daphnia was the first organism increased at increasing phenol concentrations, used to determine the quality of drinking but abruptly decreased at a concentration of water (Naumann, 1929). Cladocera have fre- 70 mg/L (Luferova and Flerov, 1971a). Na quently been used for this purpose (see e.g. transport in D. magna is inhibited by 24 dini- Anderson, 1944; Lesnikov, 1967). C. dubia has trophenol (Stobbart et al., 1977). also been used for the biotesting of water Enantiomers of the same compound (pres- (Flerov et al., 1988). ent in pesticides) were found to have drasti- The characteristics and conditions for using cally different toxicities when tested on daphnids in toxicity testing have been D. magna and C. dubia (Liu et al., 2005). described by Lesnikov (1967), Adema (1978), The survival of cladocerans exposed to toxi- Leeuwangh (1978), Ten Berge (1978), and cants also depends on the period between Pieters (2007). Lesnikov (1967) suggested esti- repeat exposures (Naddy et al., 2000). D. magna mation of the condition of daphnias by scoring can withstand the acutely lethal organophos- their oogenesis, embryogenesis, coloration, and phate insecticide chlorpyrifos (at c. 1 μg/L), oil drop status, as well as fullness of the gut. provided there is adequate time for recovery In the wide field of aquatic toxicology, the between exposures (Naddy and Klaine, 2001). cladocera (mostly daphnids) are frequently

PHYSIOLOGY OF THE CLADOCERA 15.6 CLADOCERA IN WATER QUALITY TESTING 197 used as test objects or sentinel species (Lesnikov, Daphnia immobilization by particular organo- 1967; Isakova and Kolosova, 1988) and Daphnia phosphates at different concentrations. has been described as an excellent model The use of D. magna as an ecotoxicological organism for studying multiple environmental indicator is allowable if its genotype and previ- stressors (Altshuler et al., 2011). Daphnia was ous adaptation status are taken into consider- especially recommended for phenol assess- ation, as discussed by Baird et al. (1989). A fast ment (Tumanov et al., 1988). D. magna can also toxicity test using parthenogenetic D. magna be used to evaluate the quality (toxicity) of eggs in vitro was also suggested by Sobral et al. bottom sediments (Terra et al., 2010; (2001) and Krylov (2011). The rate of Romanenko et al., 2011). formation of males in D. magna was used to The suitability of Cladocera as indicators of measure the presence of pollutants with various environmental conditions has been dis- hormone-disrupting effects (Tatarazako and cussed by Walseng and Schartau (2011). The Oda, 2007). sensitivity of D. magna, D. pulex,andD. cucullata Of the littoral species, the use of C. sphaeri- to various chemicals was found to be similar cus was suggested for assessing water quality (Canton and Adema, 1978), although Lesnikov (Pieters et al., 2008) or sediment toxicity (1967) found that D. pulex is somewhat less (Dekker et al., 2006). For C. sphaericus,LD50 at sensitive and different lines (or clones) of this 96 h was determined by Dekker et al. (2006) to species noticeably differ in their sensitivity. Of be 46 mg ammonia (NH3) /L. course, pollutants act as a mixture of interacting Third, an attempt has also been made to use ingredients, the effect of which is unpredictable, Daphnia for testing the toxicity of human phys- as particular substances can react with each iological liquids: urine, blood serum, hydroptic other, leading to partial neutralization or enter- liquid, and pleural fluid, as well as various ing into synergistic relationships. biologically active substances (Billiard, 1925). The reaction of D. magna to a wide range of This author determined the average survival pesticides has also been determined (Frear and time of Daphnia in urine to be 7 min (Billiard, Boyd, 1967). The pesticides tested ranged from 1926). If the daphnias lived longer, the urine extremely toxic [median lethal dose (LD50)of was considered hypotoxic; if shorter, it was carbophenothion 5 9 ppt (parts per trillion)] to considered to be hypertoxic. In patients with 5 noticeably less toxic (LD50 of prometone lung inflammation and a high temperature, the 35 ppm). Tumanov et al. (1988) suggested a urine contained albumin and the daphnias quick method for measuring pollution using lived for an abnormally long time (Billiard and previously prepared calibration curves of Perrot, 1926).

PHYSIOLOGY OF THE CLADOCERA CHAPTER 16

A Cytological Perspective Margaret J. Beaton and Carli M. Peters Biology Department, Mount Allison University, Sackville, NB, CANADA

Despite their miniature size, as a group, cla- examinations were completed long before the docerans have been the focus of intense eco- links between chromatin, genes, DNA, and logical and life history study for nearly two heredity were established and consisted of centuries. Literature on their feeding/growth gross organization of chromosomes. rates far outnumbers that focused on the phys- Today, researchers are applying a burgeon- iological constraints of the group, or their ing assortment of microscopy and molecular genetics. Cytological work has been even less tools to address questions of genome structure/ prominent, despite the earliest publications organization and the pivotal roles that genes occurring only a few decades after Latreille play in cellular growth and development. named the order. These first studies generally These studies will contribute to our under- examined embryological development in standing of the genetic and developmental Daphnia, Leptodora, and Moina (for example, bases for an array of ecological and physiologi- Weismann, 1874; Grobben, 1879; Lebedinsky, cal traits in cladocerans. The ability to pinpoint 1891) and were published in German. As a the location of factors in cells as well as mea- result, complete translations are not readily suring upregulation of proteins, for example, available, severely impeding accessibility for will strengthen our understanding of intra- and many. However, these papers include detailed intercellular processes, and by extension and valuable drawings of tissue and cellular the physiology and biology of the organisms. structures; for example, Grobben’s (1879) sur- The highly advertised release of the nuclear vey of development in Moina was thought to genome sequence for a specific clone of one of be the impetus for later studies “on cell lineage the recently diverged species of the Daphnia and on formative substances” (Witschi, 1934). pulex lineage, D. arenata (Colbourne et al., 2011, Later investigations concentrated on cell size plus supporting online material) has revealed and basic morphology, as well as on specific some remarkable findings. Like its body size, structures such as the labrum and nervous sys- the genome of this model organism is diminu- tem (e.g. see Sterba, 1957). The first cytogenetic tive, but it still manages to contain many more

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00016-1 199 © 2014 Elsevier Inc. All rights reserved. 200 16. A CYTOLOGICAL PERSPECTIVE genes than are found in the human genome of the structure of chromosomes (for a brief (. 30,000, which is 5000À10,000 more). The review see Gregory 2002, 2005). For example, inflated gene count appears to be a result of Swift (1950) found a constant and characteris- duplications as well as the presence of novel tic amount of DNA per haploid chromosome genes; about one-third of the identified genes set for each of two plant species. He also have no known homology with the proteome demonstrated that cells in somatic tissues of any other species. Furthermore, using regularly contain a discrete multiple of the cDNA libraries and microarrays, Colbourne haploid amount. From Swift’s work, the term et al. (2011) found that expression of much of C-value (meaning genome size) was defined the transcriptome changes with altered envi- as the quantity of DNA that is contained in a ronmental (biotic and abiotic) stresses. This species’ haploid chromosome complement. represents an enormous step forward in our Mirsky and Ris (1951) provided the first two quest to understand the links between genet- crustacean estimates (for a decapod and a ics, ecology, and life history strategies of barnacle), but the first cladoceran estimate Daphnia. In addition, the project led to chro- was not published until much later (Rasch, mosome analyses that have provided us with 1985). further insights into their structure and orga- Genome size is thought to vary by more nization (see below). than 200,000-fold across eukaryotic species! Many cladoceran species make ideal study This range shrinks at lower taxonomic levels organisms due to their ease of maintenance in (excluding incidents of polyploidy), so compar- culture and their parthenogenetic life cycle. isons across members of the same order tend to However, in most species, the body carapace, show contracted differences. Gregory’s (2013) which restricts growth except at ecdysis, com- animal genome size database includes records bined with a diminutive body and similarly from more than 1500 invertebrates, 318 of small genome size, has generally hindered which are crustaceans (representing 278 spe- cytological research on the group. Members of cies). Such meager coverage for a group com- the genus Daphnia (especially D. magna and prising nearly 70,000 extant species D. pulex) have been at the center of much of demonstrates our limited knowledge in this this research, so comprehensive comparisons area. As estimates accumulate, patterns in the are often lacking and coverage of some topics data that seem obvious today may become is quite limited. To that end, the available liter- murky tomorrow and patterns may appear ature on cladoceran genome size variation, next week that were not obvious yesterday. cytogenetics, endopolyploidy, and cytological Among crustaceans, C-values range from studies of various tissues will be examined. 0.14 to 64.62 pg, with a mean of 4.45 6 0.43 pg One goal of this chapter is to identify gaps in (Gregory, 2013). This represents a greater than our cytological knowledge of the order. 400-fold range, the widest range of published estimates among invertebrate groups; members of the Platyhelminthes exhibit the second 16.1 GENOME SIZE largest invertebrate range (slightly less than AND POLYPLOIDY 350-fold) (Gregory, 2013). To place the crustacean range in perspective, 64.62 pg and 0.02 pg 16.1.1 Background represent the largest and smallest published invertebrate estimate (Gregory, 2013). So, Studies estimating the amount of chroma- where do cladoceran genomes fit within the tin present in a nucleus predate the discovery crustacean range of DNA contents?

PHYSIOLOGY OF THE CLADOCERA 16.1 GENOME SIZE AND POLYPLOIDY 201 5 16.1.2 Overview of the Techniques transformation, OD log10 (1/T) (Hardie et al., 2002). Arbitrary OD levels can then be con- The genome size, or amount of DNA in the verted into to a DNA amount (typically in haploid cell, has been determined via several picograms, pg) with the inclusion of “stan- methods over the years, including biochemical dards” (cells for which the DNA content is separation, photometric estimates (typically known) in each staining procedure. using the DNA-specific Feulgen reaction or Flying spot and scanning stage densitome- flow cytometry), and even genome sequencing. try both systematically measure OD across a The methods differ in their ease of use and nucleus, with the amount of light passing cost, as well as the expertise, time, and equip- through a small aperture recorded at each ment required. location. Multiple measurements collected Early eukaryotic measurements were pro- across a single nucleus can be integrated to duced via biochemical analysis. Genome size produce an OD measure for that nucleus. The was predicted from the molecular weight for a flying spot and scanning stage methods differ large tissue sample divided by a cell number in their mechanics. The former method uses a estimate; this provided imprecise but strong beam of light that moves across the nucleus, evidence of the constancy of DNA (Hardie whereas a stationary beam and a motorized et al., 2002). More recent methods have utilized stage gliding the slide (and thus the nucleus) the properties of the Feulgen reaction, which across the beam’s path is employed in the lat- selectively stains nucleic acids, in concert with ter (Hardie et al., 2002). Image densitometry densitometry. During the staining protocol, calculates the OD in the same manner but mild acid hydrolysis (typically using fixed tis- employs captured images. The benefit of this sue) cleaves purine bases, exposing free alde- method is that each image may include dozens hyde groups (Chieco and Derenzini, 1999). of nuclei, all of which can be processed simul- Subsequent staining with Schiff reagent allows taneously, thus tremendously accelerating the aldehyde groups to bind the pararosaniline rate of acquisition of nuclear measurements. present in the reagent, turning it from colorless Finally, flow cytometry has occasionally to magenta. The intensity of the stain is pro- been employed to estimate the DNA content of portional to the number of aldehyde sites cells. This technique involves creating a sus- available and, therefore, to the amount of pension of cells that have their nuclei marked DNA present. A key step in the reaction is with one of a number of DNA-specific fluores- acid hydrolysis; extending the duration of this cent dyes [e.g. 4’,6-diamidino-2-phenylindole step leads to increased breakage and loss of (DAPI) and propidium iodide). The fluores- sections of the DNA strands, thus decreasing cently tagged cells are passed through a laser the available staining sites and likewise the beam of the appropriate wavelength for the intensity of the stain. chosen dye, and a detector records the bright- Developments in densitometry techniques ness as the cells pass through the beam to measure staining intensity of Feulgen- (Hardie et al., 2002). This method can process treated nuclei have also progressed from flying thousands of cells at a time, but a drawback of spot to scanning stage, and most recently to flow cytometry is that cells from a tissue must image densitometry. The difference between be isolated from each other. As with Feulgen the level of light able to pass through a densitometry, the inclusion of a suspension of nucleus compared to the background is a mea- cells of known DNA content allows the inten- sure of its transmittance (T), which can be con- sity of fluorescence to be converted to a mea- verted into optical density (OD) by the simple sure of the DNA amount.

PHYSIOLOGY OF THE CLADOCERA 202 16. A CYTOLOGICAL PERSPECTIVE

16.1.3 Evaluation of Techniques species of Daphnia, and an array of other cladocerans, were the subject of DNA investigations A close inspection of Gregory’s (2013) data- (Rasch, 1985; Beaton, 1988; Beaton and Hebert, base reveals both perplexing and intriguing 1989; Beaton, 1995; Dufresne and Hebert, 1995; results for cladoceran measurements. In total, Korpelainen et al., 1997; Vergilino et al., 2009). 62 C-value estimates have been published, Of the surveyed species, D. pulex was by far representing 49 species and including multiple the most intensively studied, with determina- estimates for five species of Daphnia (D. arenata, tions based on multiple methods (Table 16.1). D. magna, D. pulex, D.pulicaria, and D. tene- Differences across estimates appear to be brosa). First, substantial intraspecific variation attributable to the method used for determin- is evident, which is probably due to the ing the C-value. Scanning Feulgen densitome- method of estimating genome size. Second, cla- try (by far the most commonly used method in docerans rank among the smallest of the crus- this group) seems to have consistently resulted tacean (and indeed, the invertebrate) genomes. in an overestimation, if the other estimates are Of the 318 crustacean estimates listed in correct. Flying spot densitometry and flow Gregory’s database, 101 are less than 1.00 pg cytometry both produced estimates slightly and include all of the cladoceran as well as a larger (0.23À0.33 pg) than predictions based on number of amphipod, barnacle, copepod, and the genome sequencing initiative (0.204 pg). ostracod estimates. However, Colbourne et al. (2011) remarked While interspecific differences are routine, that the 200-Mb draft genome assembly proba- the genome size is generally predicted to be bly represented 80% of the entire genome, constant and stable for members of a species. with the remainder comprising some dupli- Some differences reported within a species are cated genes and heterochromatic regions. probably due to the acquisition methods and Furthermore, a clone of D. arenata (not of experimental error. We can consider the magni- D. pulex) was used. This would shift the esti- tude of this error in an examination of cladoc- mate originating from genomic sequencing eran estimates. From 1985 to 2009, many from 0.204 to 0.256 pg. Based on this predicted

TABLE 16.1 Selected Daphnia Genome Estimates for Comparison of Methods

Species Method of Estimation C-Value (pg) Reference(s)

D. pulex Flow cytometry 0.28À0.33 Korpelainen et al. (1997) D. pulex Flow cytometry 0.23 Vergilino et al. (2009) D. pulex Flying spot Feulgen densitometry 0.23 Rasch (1985) D. pulex Scanning Feulgen densitometry 0.37À0.38 Beaton (1989, 1995)

D. arenata Flow cytometry 0.24 Vergilino et al. (2009) D. arenata Scanning Feulgen densitometry 0.42 Beaton (1995) D. pulex/D. arenata Genome sequencing 0.204 Colbourne et al. (2011) D. tenebrosa Flow cytometry 0.29 Vergilino et al. (2009) D. tenebrosa Scanning Feulgen densitometry 0.53 Dufresne and Hebert (1995) D. tenebrosa Scanning Feulgen densitometry 0.58 Beaton (1995)

PHYSIOLOGY OF THE CLADOCERA 16.1 GENOME SIZE AND POLYPLOIDY 203 genome size, measurements produced using or two estimates are drawn from three of the flow cytometry and flying spot densitometry other suborders. With such poor representa- are both slight underestimates. tion across the group, few conclusions can be Flow cytometry for D. arenata, D. tenebrosa, drawn and there are no obvious patterns that and D. pulex, consistently produce values that might indicate a genome size increase or are 54-62% of those obtained using scanning decrease from basal to derived groups densitometry. Since the genome size predic- (Table 16.2). In addition, C-values show similar tions for other cladoceran members were ranges across the three Daphnia subgenera. determined by scanning microdensitometry, In an examination of DNA content variation their DNA contents probably also represent among species of Daphnia, Beaton (1995) could overestimates and are even smaller than those find no association between genome size reported by Beaton (1988, 1995). Consistent and habitat preference; the ranges for overestimation means that the reported species preferring pond environments were C-values, while suspect for determining precise DNA contents, should still allow comparisons within a technique (but certainly not between techniques). It is possible that the tissues and species used as the standards (often blood smears from a variety of fish species (Beaton, 1988)) were too dissimilar and biased the conversion from pixels to DNA content. Regardless, the general finding that cladoceran genomes are minute, based on the scanning densitometry estimates, is probably not affected by the bias. As such, current estimates for Bosmina longirostris and Scapholebris kingii (0.19 and 0.16 pg, respectively) are among the smallest for a crustacean, with only two cyclo- poid copepods possessing C-values of about FIGURE 16.1 Distribution of genome size estimates of 0.14 pg (Gregory, 2013). cladoceran members.

TABLE 16.2 Ranges of C-Value Estimates 16.1.4 Patterns in Interspecific Variation and Ecological Correlates Order Genus/ No. C-Value Range with Genome Size Subgenus Species (pg) Anomopoda 45 0.16À0.53 Gregory (2013) compiled 62 cladoceran À C-values from 13 genera and 49 species. The Ctenodaphnia 13 0.21 0.53 nearly fourfold range in estimates forms a Daphnia 16 0.24À0.58 bimodal frequency distribution (Fig. 16.1), Hyalodaphnia 7 0.22À0.36 with most C-values being about 0.25 or 0.4À0.45 pg. More than 90% of the available Ctenopoda 2 0.22À0.47 estimates are from representatives of the sub- Haplopoda 1 0.28 order Anomopoda (80% of which are from one Onychopoda 1 0.22 of the three Daphnia subgenera) and only one

PHYSIOLOGY OF THE CLADOCERA 204 16. A CYTOLOGICAL PERSPECTIVE approximately the same as those that prefer and Hebert, 1997). In D. pulex, for example, lake habitats. Since pond-dwelling cladocerans neckteeth are present only during an animal’s tend to be larger than those found in lakes, an first two or three instars, but exposure to the association between body size and DNA content pertinent predator kairomone is required from might not be expected. However, eukaryotic early embryogenesis onwards to attain the genome size is positively correlated with cell most pronounced structure (Miyakawa et al., size, nuclear volume, cell doubling rate, and so 2010). Therefore, there must be a critical period on (Cavalier-Smith, 1985). When a limited num- in development during which induction is ini- ber of representatives are chosen, a relationship tiated, but since the embryonic period is quite between genome size and body size appears brief, neckteeth formation must be rapid. The strong among cladocerans; the large-bodied other induced head shapes are similarly cre- Eurycercus glacialis (0.63 pg) and D. magna ated in just a few instars, and the fast response (0.4À0.53 pg) have among the highest C-values time is probably necessary to minimize the for the group, whereas the tiny B. longirostris, time that animals are vulnerable to predation. Scapholebris kingii,andCeriodaphnia reticulata Since cell cycle length is positively correlated (0.19, 0.16, and 0.23 pg, respectively) possess the to DNA content across many taxa (e.g. group’s smallest estimates (Beaton, 1988). Bennett, 1972; Shuter et al., 1983; Cavalier- However, using a larger dataset for members of Smith, 1985), the logical extension seems to be Daphnia, a much weaker relationship was that for species under variable levels of preda- found. Some of the largest members of the tion pressure, a smaller genome would be genus (for example, D. cephalata, D. longicephala, advantageous, by allowing higher cell prolifer- and D. magniceps, with respective estimates of ation rates during key developmental periods, 0.27, 0.27, and 0.30 pg) each possess a genome and thus allowing prompt formation of the size comparable to the small-bodied members defensive structures. (for example, D. ambigua and D. parvula,with The second strong ecological correlate estimates of 0.24 and 0.28 pg) (Beaton, 1995). observed with genome size was a positive one There can be two ways to achieve a large with egg size (Beaton, 1995). This relationship body: increasing cell size (and by extension has also been observed for selected taxa, for genome size) or increasing cell numbers. The example, among actinopterygian fish (Hardie weak observed relationship points to the and Hebert, 2004), as well as members of the employment of the latter option by at least rotifer, Brachionus (Stelzer et al., 2011). This some species. may be a surprising result, as the size of the Beaton (1995) identified two ecological cor- egg can also be a reflection of the amount of relates with genome size: the ability to alter yolk laid down. However, Hardie and Hebert head shape (inducible defenses) and egg vol- (2004) noted that in fish, egg size limits fecun- ume. Members of Daphnia that are capable of dity and such a limitation would also occur in forming an inducible head defense possess cladocerans. A trade-off between brood size small genomes, whereas those whose head and neonate size may occur. Among small- shape is canalized tend to have larger gen- bodied organisms, the size of the brood pouch omes. This correlation is not perfect, but may severely limit the number of eggs, but in Beaton (1995) found it to be statistically signifi- larger species, many small eggs or fewer larger cant. Head defenses (neckteeth, spines, hel- ones are viable options. Some of the largest mets, and crests) are all formed from the species of Daphnia (such as D. cephalata and epidermal sheet situated just under the cara- D. longicephala) have opted for large clutches of pace via an increase in cell numbers (Beaton small eggs, leading to small neonates.

PHYSIOLOGY OF THE CLADOCERA 16.2 CYTOGENETICS 205

Finally, it must be noted that polyploid of a suspension of cells for examination is not clones have been found for some members of an easy proposition with cladocerans. Creating Daphnia. Ploidy assignments have been made a cell suspension from adults is fraught with using a combination of isozyme patterns and difficulties, including the separation of cells, as DNA content measurements. The use of in situ well as the confounding issue of rampant hybridization, as performed by Colbourne somatic polyploidy in members of this group et al. (2011), may offer an alternative means of (Beaton and Hebert, 1989). Using newly depos- confirming ploidy assignments when infer- ited subitaneous eggs (sometimes referred to ences made by allozyme and DNA content as summer eggs), a technique that is frequently analysis are inconclusive. Adamowicz et al. employed (Zaffagnini and Sabelli, 1972; (2002) summarized the geographical range of Zaffagnini and Trentini, 1975; Trentini, 1980) polyploid representatives of the D. pulex com- will generate cells that can be squashed, but plex as being the dominant form at latitudes pinpointing the ideal interval for egg collection above 70N and 46À54S (in Argentina). is critical and the number of viable smears will Triploids are believed to be the dominant poly- be reduced at this developmental stage. ploid level in the Canadian arctic (with tetra- Alternatively, whole animals can be fixed, ploids observed less frequently), whereas only blocked, and sectioned with a microtome, but tetraploids were recovered in Argentina the resulting counts can be variable when (Adamowicz et al., 2002; Vergilino et al., 2009). chromosomes in a cell do not lie within a single plane (Zaffagnini and Trentini, 1975). Finally, preparations of chromosome spreads 16.2 CYTOGENETICS can be obtained from early embryos or from the epidermis underlying the carapace. In 16.2.1 Background Daphnia (and presumably in other cladocerans), the epidermis is a single sheet of diploid Historically, karyotypes have been gener- cells, with polyploid cells restricted to the tis- ated from cells that are arrested in metaphase, sue margins (Beaton and Hebert, 1989, 1994a). when the chromosomes are in a condensed A synchronized cycle is often observed in the state. To ensure that this brief period in the epidermis, with a brief window of opportunity cell cycle is observed, a population of cells to examine metaphase chromosomes occurring may be treated with a dilute colchicine solu- a few hours postecdysis (the first 12 h typi- tion to inhibit spindle formation. Once a popu- cally). Although this method allows examina- lation of cells is fixed onto a slide, the material tion of large numbers of mitotic figures, tissue can be treated with one of several DNA stains collection is time sensitive. (aceto-orcein is a simple and commonly used dye) prior to visualization. There are likely to be at least two reasons 16.2.2 Cladoceran Karyotypes why cytogenetic work on cladocerans has been hampered. First, as a whole, this group pos- A minute genome, combined with diminu- sesses miniscule chromosomes owing to their tive body size and a constrained growth pat- condensed genomes packaged into reasonable tern, has probably contributed to the paucity chromosome numbers. Second, the choice of of published cytogenetic work for this group. tissue and methods employed to obtain karyo- In addition, information regarding centromere types have been linked to variable counts location and chromosome arm length has been (Zaffagnini and Trentini, 1975). The production difficult to obtain (because the chromosomes

PHYSIOLOGY OF THE CLADOCERA 206 16. A CYTOLOGICAL PERSPECTIVE are so small!). With the exception of a handful There have been both lower and higher of studies published since 1995, the bulk of reported chromosome complements for information has been limited to chromosome D. magna and D. pulex, but these are generally numbers. Overall, diploid counts in the group considered to be unreliable. For example, those have been obtained for members of only obtained from tissue sections have generally two of the suborders: Anomopoda and been refuted and dismissed, sometimes by the Onychopoda (Table 16.3). Chromosome com- authors themselves (Chambers, 1913 and plements of many other members of the group Ku¨ hn, 1908, based on my translation of are certainly required. Zaffagnini and Trentini, 1975). A diploid count At least some of the published images have for D. magna by Rossetti (1952, in Trentini, been obtained from colchicine-treated tissues. 1980) represents the only aberrant set of counts Trentini (1980) and Zaffagnini and Trentini for which we have no evidence favoring their (1975) obtained chromosome spreads of par- rejection, aside from the overwhelming num- thenogenetic eggs using a squash method. A ber of studies contradicting his counts similar technique, but employing colchicine- (Mortimer 1936, in Trentini 1980; Zaffagnini treated embryos to create a suspension of and Trentini 1975; Trentini 1980; Beaton and metaphase-arrested cells, was used for several Hebert 1994b). All aberrant records have been Moina spreads (Wenqing et al., 1999; Zhang placed at the end of Table 16.3 simply for et al., 2009). Beaton and Hebert (1994b) reference. obtained spreads using thoracic epithelium of The sequencing initiative by the Daphnia mature females treated with a weak colchicine Genomics Consortium succeeded in characteriz- solution. ing the nuclear genome of the water flea, D. pulex (arenata), including a detailed descrip- 16.2.3 Suborder Anomopoda tion of chromosome structure in this species. Colbourne et al. (2011) categorized the chromo- Daphnia has been far and away the most somes (ranging in length from about 1 to 6 μm) intensely surveyed genus of the group, with into three broad classes based on size: the first counts for 21 species. Conservation of chromo- group included only one chromosome pair, some numbers in each of the Daphnia subge- with the second group comprising the next nera has been found, with diploid counts of three largest pairs. Only about 25% of the total 20, 20, and 24 for members of Ctenodaphnia, chromosome area was found to be heterochro- Hyalodaphnia, and Daphnia, respectively matic and G-bands were only found on the larg- (Table 16.3). It should be noted that Daphnia est four chromosome pairs. While no attempt curvirostris was historically classified within was made to identify the position of the centro- the subgenus Daphnia (previously referred to mere, the three largest pairs, at least, appear to as the pulex group) based on morphological be meta- or subtelocentric based on their image characters, so its diploid complement of 20 (Colbourne et al., 2011). The chromosome ends was considered a puzzle. This led Beaton and were characterized as telomeres made of long Hebert (1994b) to question its placement in the stretches of repeated TTAGG, the same subgenus. Subsequently, molecular analyses sequence used by Bombyx mori and similar to have confirmed that this species is most that found at the ends of vertebrate chromo- closely aligned with members of the subgenus somes. Finally, fluorescence in situ hybridiza- Hyalodaphnia, concordant with chromosome tion (FISH) was used to locate the ribosomal numbers (Lehman et al., 1995; Colbourne and RNA (rRNA) arrays (Colbourne et al., 2011). Hebert, 1996; Kotov et al., 2006). Using two probes, Pokey, a transposon that

PHYSIOLOGY OF THE CLADOCERA 16.2 CYTOGENETICS 207

Diploid Chromosome Counts for a TABLE 16.3 Suborder/Family Genus/Species 2n Cladocerans Simocephalus Suborder/Family Genus/Speciesa 2n S. vetulus18,19 20 ANOMOPODA S. exspinosus19 20 Daphniidae Ceriodaphnia Moinidae Moina C. laticaudata18,19 14 M. affinis18 20 C. reticulata19 14 M. affinis24 26 C. pulchella19 20 M. macrocopa9,21,24 22 Ctenodaphnia M. micrura24 24 C. angulata20 20 M. mongolica22,24 24 C. cephalata20 20 M. rectirostris24 24 C. exilis20 20 M. rectirostris13 30 C. longicephala20 20 Onychopoda Bythotrephes longimanus2 4? C. lumholtzi20 20 Polyphemidae Polyphemus pediculus3 8 C. magna11,18,19,20 20 ABERRANT COUNTS C. salina20 20 Anomopoda Ctenodaphnia magna14 28À32 C. similis20 20 C. magna12 8F Daphnia D. pulex4 8(7À10) F D. catawba20 24 D. pulex10 8F,M;9S melanized sp. nov.20 24 D. pulex15 20 D. middendorffiana17,20 24 D. pulex16 16 D. minnehaha20 24 6 À À D. pulex 8 10 M, S D. obtusa18 20 24 S. vetulus5 8À10 M D. pulex7,8,11,14,17,20 24 M. paradoxa1 8 D. pulicaria20 24 M. rectirostris1 8 D. schodleri20 24 aNumbers represent count source: 1, Weismann and Ishikawa (1889) in Hyalodaphnia Makino (1951); 2, Weismann and Ishikawa (1891) in Makino (1951); 3, Kuhn (1908) in Mackino (1951); 4, Kuhn (1908) in Trentini (1980); 5, H. curvirostris19,20 20 Chambers (1913); 6, Taylor (1914) in Trentini (1980); 7, Fanghaut H. galeata mendotae20 20 (1921) in Trentini (1980); 8, Schrader (1925) in Trentini (1980); 9, Allen and Banta (1928) and (1929) in Mackino (1951); 10, Rey (1934); 11, H. hyalina18 20 Mortimer (1936) in Trentini (1980) ; 12, Lumer (1937) in Trentini (1980); 13, von Dehn (1948); 14, Rossetti (1952) in Trentini (1980); 15, 19 H. longispina 20 Ojima (1954); 16, Bacci et al. (1961); 17, Zaffagnini and Sabelli (1972);

20 18, Zaffagnini and Trentini (1975); 19, Trentini (1980); 20, Beaton and H. rosea 20 Hebert (1994b); 21, Wenqing et al. (1995); 22, Wenqing et al. (1999); 23, Daphniopsis tibetana23 24 Zhao et al. (2004); 24, Zhang et al. (2009). ?, questionable count; F, during oogenesis; M, during spermatogenesis; S, during mitosis of somatic cells. (Continued)

PHYSIOLOGY OF THE CLADOCERA 208 16. A CYTOLOGICAL PERSPECTIVE inserts into a specific site of the large subunit With just six species of Moina surveyed, rDNA (Eagle and Crease, 2012) and a region of diploid chromosome numbers in the genus the intergenic spacer (IGS), Colbourne et al. appear to be restricted to 22À26, although (2011) confirmed that the rRNA genes were counts for M. rectirostris (syn. Moina brachiata) present as a single tandem array on one chro- have been reported as 8 and 30, in addition to mosome pair, with the transposon inserted 24, and a count of 8 was reported for M. para- along the entire length of the array. doxa (Table 16.3). The highest and lowest Chromosome numbers for Ceriodaphnia have counts must be regarded as suspect; von been published for only three members, with Dehn’s (1948) estimate (2n 5 30) was based on two diploid complements observed: 14 and 20 tissue sections, and a description of how (Table 16.3). It is interesting to note that C. reti- Weismann and Ishikawa (1889, in Makino, culata (2n 5 14) and C. pulchella (2n 5 20), 1951) collected their data (2n 5 8) could not be which can co-occur, exhibit differences in both obtained. Similarly, the diploid count of 2n 5 8 body and egg size (Burgis, 1967). What might for M. paradoxa should be considered suspect not be expected is that C. pulchella has the unless confirmation for the species is possible. larger chromosome complement, but a smaller Zhang et al. (2009) obtained a diploid count of egg and body size. With no phylogenetic 24 for M. micrura, M. mongolica,andM. rectiros- assessment of the genus found in the litera- tris with diploid karyotypes of 10M 1 14T, ture, we cannot speculate on the basis for the 6M 1 18T, and 10M 1 14T, respectively. Their chromosome variation. spreads of M. affinis revealed 2n 5 26 chromo- With only two species of Simocephalus studied, somes (in contrast to Zaffagnini and Trentini, diploid chromosome numbers appear to be con- 1975, who obtained a count of 20), with a served at 20 (Table 16.3). Chambers (1913) karyotype of 12M 1 14T. Finally, for M. macroco- reported an aberrant diploid count of 8À10, but pa, a diploid karyotype of 2n 5 22 5 10M 1 12T as it was based on tissue slices, the count is con- has been described (Zhang et al., 2009). sidered unreliable. Simocephalus and Ceriodaphnia The basis of the discrepancies observed have been linked as sister taxa based on a phylo- among species of Moina (especially for M. affi- genetic reconstruction using three genes nis) must be investigated further. Errors in the (deWaard et al., 2006). Chromosome counts for counts, misidentification of individuals, or the members of these two genera (2n 5 20 and 14, presence of cryptic species complexes are pos- respectively) show the largest separation sible explanations. Obtaining karyotypes from observed across the Anomopoda to date additional individuals, populations, and spe- (Trentini 1980). A single diploid chromosome cies should be undertaken to definitively count of 24 has been recorded for Daphniopsis,a establish the correct counts. sister genus to Daphnia (Table 16.3). Zhao et al. (2004) further established that the diploid karyotype for Daphniopsis tibetana has three pair of 16.2.4 Suborder Onychopoda metacentric (M) and nine pair of telocentric (T) chromosomes (2n 5 24 5 6M 1 18T). This Only two early counts have been reported arrangement is reminiscent of the findings of for this suborder and these should be viewed Colbourne et al. (2011) for D. pulex (arenata). with some skepticism. The diploid counts Overall, among the members of the family of four and eight need to be confirmed and sev- Daphniidae, only three reliable counts have been eral more representatives of this suborder sur- reported, 2n 5 14, 20, and 24. veyed before any generalizations are possible.

PHYSIOLOGY OF THE CLADOCERA 16.3 ENDOPOLYPLOIDY 209

16.3 ENDOPOLYPLOIDY a necessary and normal act in the developmental process of most multicellular organisms. Endoreplication, or somatic polyploidy, is Trophoblast cells of rodents, many plant observed when DNA replication occurs in a embryo suspensor cells, and the giant neurons cell without cytokinesis. Separating the process of the sea hare, Aplysia californica, are but a few of DNA replication from one or more steps of examples of this widespread phenomenon cell division can lead to a single reconstituted (Nagl, 1978). In general, polyploid cells range 6 cell (rather than two daughter cells) that pos- from 4 to 10 C (where C represent the DNA sesses twice as much chromatin than expected. content of a single chromosome set) and those Repetition of this abbreviated cycle produces cells with the highest levels are often associ- an enlarged cell and nucleus containing 2n ated with secretory roles, although the neurons 6 copies of the nuclear genome (where n is the of Aplysia (approximately 10 C) contradict this number of completed endocycles or endomi- pattern. The widespread nature of somatic totic cycles). Endocycles bypass mitosis polyploidy could be an evolutionary mecha- entirely, so chromosomes do not condense, nism for allowing abrupt shifts in traits or creating multistranded chromatin as seen in physiological responses (Beaton and Hebert, larval Drosophila salivary gland cells. 1997). Endomitotic cycles, on the other hand, typi- Some of the first reports of endopolyploidy cally include prophase, metaphase, and ana- in cladocerans came from examinations of phase, but during telophase a single nuclear labrum and fat cells (Cannon, 1922; Jaeger, membrane is formed around all of the chroma- 1935; Sterba, 1956a, 1957b). More recently, tin (see Lee et al., 2009 for a brief comparison). Beaton and Hebert (1989, 1997, 1999) estab- As a consequence, the latter process will result lished the extent of polyploidy for multiple in multiple sets of the haploid chromosome D. magna and D. pulex tissues, with several complement (endopolyploidy). substantive patterns revealed. First, most This sweeping description of endoreplica- examined tissues in mature females were tion hints at the occurrence of some accident found to contain multiple polyploid cells, each or abnormality, as is the case with some with DNA content ranging from 4 C to 2048 C cancerous tumors. However, endopolyploidy (Tables 16.4 and 16.5). The Feulgen staining is a ubiquitous phenomenon, and is routinely pattern of many of the moderately polyploid

TABLE 16.4 Typical Number of Fat and Epipodite Cells per Abdominal Appendage Pair of Daphnia pulex and D. magna

Appendage Number Fat Cells Epipodites

D. magna D. pulex D. magna D. pulex

141À42 16À17 107À108 45À46

246À47 26À27 122À123 58À59 3 125À126 49À50 123À124 82À83 4 109À110 40À41 137À138 85 545À46 26À27 130À131 86

Summarized from Beaton and Hebert, 1989.

PHYSIOLOGY OF THE CLADOCERA 210 16. A CYTOLOGICAL PERSPECTIVE

TABLE 16.5 Summary of Occurrence of Endopolyploidy in Adult Female Daphnia

Tissue Number of Polyploid Cells Levels Observed (C) Specific Comments

D. magna D. pulex

Epidermis ND 35 8À64 16À32 C is most common

Epipodites 621 357 8À32 16 C is most common Fat cells, limbs 371 161 64À256 128À256 C are more common Gut All All 4À16 4 C for almost all nuclei Labrum 8 8 64À2048 distinct patterns for species Rostrum 156 83 8À128 32À64 C is most common Shell gland 300 102 8À256 16 C is most common

Number of polyploid cells represents the counts for the entire tissue (for example, for all five pairs of limbs). ND, cell counts (and corresponding ploidy levels) were not determined. Summarized from Beaton and Hebert, 1989.

cells (32À64 C) appears to be far more hetero- Third, the number of polyploid nuclei in a geneous than those at 2À16 C (Beaton, tissue, once established (typically before instar unpubl.; Sterba, 1956a, 1957b). Two of the tis- three or four), remained constant with age sues, the gut and the epipodites (the leaf-like (Beaton and Hebert, 1999). When individual lobes located at the base of each of the five polyploid cells in the epidermis are destroyed pairs of thoracic appendages), contain only via laser ablation they are not replaced polyploid cells and the labrum contains cells (Beaton, pers. comm.), suggesting that a reaching the highest levels (1024À2048 C). genetic control to establish these cells is in Overall, Beaton and Hebert (1997) estimated place. The inference can also be made that that nearly half of each individual’s DNA was polyploid cells in most tissues cannot exit the packaged in a polyploid state; with no mitotic endocycle and return the typical cell cycle to figures recorded from polyploid cells other restore cell numbers. The exception to this than those in the gut, it is assumed that poly- rule, however, was observed in gut cells, teny does not occur in Daphnia. which appear to reach the tetraploid level by Second, both tissue- and species-specificity instar 2, and then return to a normal cell cycle, was found with regards to levels and number which allows tissue growth without further of cells involved (Tables 16.4 and 16.5). For ploidy increase (Beaton and Hebert, 1999). In example, in newly mature D. magna and general, polyploid cells do not return to the D. pulex females, the labrum possessed only cell cycle once they have opted for the endomi- eight polyploid cells, but levels differ between totic cycle, so this finding is particularly species (Beaton and Hebert, 1997). In addition, intriguing. The significance of digestive cells for a given species, epipodites of mature maintaining a low ploidy level has not been females contained cells that were between 8 C investigated. and 32 C, but the number of cells in each epi- Finally, Beaton and Hebert (1999) found tis- podite differed both among thoracic limbs and sue specificity in D. pulex with regards to between species (Table 16.4). increasing ploidy levels (Table 16.5). During

PHYSIOLOGY OF THE CLADOCERA 16.4 CYTOLOGICAL OBSERVATIONS FOR SPECIFIC TISSUES 211 the first instar, 4 C was the common level whereas at least some of the epidermis must found in most tissues, but specific labral cells form the “sucker” found on the dorsal surface had already reached 64 C. Over subsequent of Sida; in addition, the carapace seems to have instars, the levels in labral cells continued to “fused” with the thorax in Leptodora (Olesen increase (although a doubling of the DNA con- et al., 2003). The function of polyploidy in tent with each instar was not observed), and some tissues will be considered further in the two of the eight polyploid nuclei reached next section. 2048 C by instar 14 when the study ended. The tissues most involved in feeding and growth, i.e. the epidermis (which lays down the new 16.4 CYTOLOGICAL carapace each molt), the labrum, and the fat OBSERVATIONS FOR SPECIFIC cells of the limb central core, all showed sub- TISSUES stantial ploidy level increases across the instars. By comparison, the cells located in the 16.4.1 Nuchal Organ and Epipodites epipodites typically remained at 16 C or 32 C from the sixth to the thirteenth instar. As a rule, cladocerans can actively regulate The functional significance of possessing the osmotic pressure of their fluids to maintain polyploid cells in some tissues can be inferred. homeostasis of their water content (Aladin and For example, a band of polyploid cells along Potts, 1995). Within the order, 95% of the spe- the underside of the rostrum are probably cies are confined to freshwater, 3% are found important for chemoreception. The presence of in brackish water, and only 2% are restricted 20À25 polyploid cells distributed along the to marine habitats (Bowman and Abele, 1982, ventral margins and dorsal fold in the epider- in Weider and Hebert, 1987). Osmoregulatory mal sheet that lies adjacent to the carapace strategies of the group are as diverse as the may act as the primary developmental centers environments that they inhabit. Hyperosmotic to control the growth rate of the animal. regulation of hemolymph is found in freshwa- Similar types of cells in the cephalic epidermis ter animals and those living in slightly brack- have also been hypothesized to act in this ish, whereas hypoosmotic regulation occurs in manner; mitotic rates in diploid cells near marine individuals (Aladin and Potts, 1995). A polyploid cells were higher than in cells in combination of hyper- and hypoosmotic regu- more distant positions (Beaton and Hebert, lation is found in cladocerans from both brack- 1997). Further evidence supporting this comes ish and highly mineralized waters (Aladin and from studies in which ablation of selected Potts, 1995). polyploid epidermal cells in the head of hel- The two primary structures utilized by the meted D. lumholtzi led to a 10À40% decrease group for osmoregulation are the epipodites in the size of the helmet in the next instar and the nuchal, or neck organ (Aladin and (Beaton, pers. comm.). It would be interesting Potts, 1995). Although they had commonly to know how ploidy levels (and polyploid cell been thought of as a gill, Koch (1934) sug- number) compare for members of the other gested that the selective permeability to silver three suborders, as well as for those with dis- stain suggested that the epipodites have an tinct physical characteristics, such as Sida, ion-regulatory role. The nuchal or neck organ Holopedium, and Leptodora. Each of these spe- has been described in a variety of larval and cies presumably uses the epidermal tissue for adult crustaceans, including branchiopods, different functions. In Holopedium, this tissue copepods, and malacostracans (Aladin and presumably lays down its distinctive jelly coat, Potts, 1995). Among malacostracans, the

PHYSIOLOGY OF THE CLADOCERA 212 16. A CYTOLOGICAL PERSPECTIVE nuchal or neck organ has been suggested to which develops into a broad shield containing serve in chemoreception, mechanoreception, numerous mitochondria-rich cells. Similarly, and baroreception. Branchiopod neck organs members of Onychopoda possess only a well- may have evolved from these sensory func- developed nuchal gland (Aladin and Potts, tions; as with these other functions, salt uptake 1995). is also only effective where a thin cuticle is Sandwiched between the outside medium present (Aladin and Potts, 1995). It seems and the hemolymph, both structures are cov- probable that the neck organ first evolved in a ered by an extremely thin cuticle (Aladin and branchiopod nauplius to allow salt uptake in Potts, 1995). The ultrastructure of the neck freshwater environments. This organ is now organ and epipodite are very similar: cells of retained in adult cladocerans that have rapto- both structures contain dense cytoplasm with rial limbs and no longer possess epipodites numerous mitochondria (Aladin, 1991). (Aladin and Potts, 1995). Members of the five Furthermore, their cell structure is unaffected orders of Cladocera, Ctenopoda, Anomopoda, by the type (hyper- or hypoosmotic) of ion reg- Haplopoda, and Onychopoda may use one or ulation employed. Structurally, these tissues both of these ion-regulating systems share similarities with ion-transporting cells of (Table 16.6). Among the Ctenopoda, epipodites the branchiopods, cells of similar function in are well developed in some members (Sida), the gills of other crustaceans, those in insects but are small for others (Penila). Furthermore, and fishes, and the salt-secreting glands of rep- in Sida it has been modified, allowing it to also tiles and birds (Aladin and Potts, 1995). function as an attachment device (Olesen, The structure of the nuchal organ in 1996; Pen˜alva-Arana and Manca, 2007). In Daphnia has been well studied. First instar Anomopoda, such as Daphnia, the nuchal individuals have a nuchal organ that, upon gland persists from embryo to the first instar, external observation, appears as an expanded although it may become inactive just a few portion of a dorsal ridge of the head. In hours after release from the brood pouch D. himalaya, a groove appeared to mark the (Benzie, 2005). This group possesses epipodites margin of the structure, which has a medio- upon release from the brood pouch. In con- dorsal hole (Pen˜alva-Arana and Manca, 2007). trast, haplopods possess only the nuchal gland, Schwartz and Hebert (1984), using a simple silver staining technique, reported that the posi- TABLE 16.6 Variation in Osmorgulatory Structures tion of the organ could be used as a diagnostic Present in Suborders trait between the subgenera, Ctenodaphnia and Suborder Epipodites Nuchal Organ Daphnia. Benzie (2005), on the other hand, asserted that its location is not specific to a Anomopoda Present Limited to embryo and subgenus, but it is situated at the border first instar between cephalic and dorsal shields. Ctenopoda Present in Modified to function for Internally, lying just below the cuticle, the varying sizes attachment nuchal organ consists of two cell types across species (referred to as dark and light) that fill a large Haplopoda Absent Present in all stages, portion of the hemocoelic space (Halcrow, surrounding the head 1982). The higher proportion of microvilli on within the shield the apical surface of both cell types makes Onychopoda Absent Well developed and them noticeably different than the surrounding incorporated in head squamous cells. The darker of the two cell shield types has microvilli that sometimes branch

PHYSIOLOGY OF THE CLADOCERA 16.4 CYTOLOGICAL OBSERVATIONS FOR SPECIFIC TISSUES 213 and may be smaller in diameter compared to The presence of two cell types in the epipo- those of the light cells. Deep within both cells dites raises some interesting questions. Beaton and at the bases of microvilli, are numerous and Hebert (1999) found that D. pulex epipo- mitochondria with prominent cristae. It is pos- dites routinely maintained nuclei with two sible to define the border of the nuchal organ ploidy levels, 16 C and 32 C (with occasional with permanganate staining: the cuticle overly- 8 C and 64 C cells) from the sixth through to ing it is densely stained, whereas the thicker the thirteenth instar. Could the cells with dif- surrounding cuticle is not (Halcrow, 1982). In fering ploidy levels correspond to the light D. magna, increased membrane surface area and dark cell types? In addition, how is the (microvilli) and numerous mitochondria are polyploid nature of these cells important for common features of nuchal organ cells that their function? Furthermore, we should ask if have osmoregulatory or ion-transporting roles osmoregulatory capability is sustained beyond in other crustaceans. This structure remains the sixth or seventh instar, since ploidy levels functional as an ion-transporter for a brief (and number of cells, for that matter) do not period of time; only approximately 12 h in the appear to increase, despite the continued free-swimming first instar animals (Halcrow, increase in body size. It seems logical that, 1982). Due to its rapid loss of function, it has without ploidy increases, as the animal grows been suggested that the nuchal organ is most each epipodite cell must experience a greater beneficial in these animals during their devel- osmoregulatory load, and at some point the opment in the brood pouch, when limb move- control of ion concentration could be ment is severely limited or nonexistent. These compromised. cells remain large in older animals, but have The nuchal organ of Leptodora is a saddle- reduced numbers of microvilli and mitochon- shaped area of integument almost completely dria (Halcrow, 1982). Beyond the first instar, encircling the head (Halcrow, 1985). The struc- epipodite epithelial cells presumably serve the ture is similar to that of other branchiopods in primary ion-regulatory role in D. magna mitochondrial abundance, varied cell types, (Halcrow, 1982). Early research suggested a surface area, and distinct integumental bound- respiratory function for epithelial epipodite ary. However, the microvilli that cover almost cells; however, since the cuticle of this tissue in the entire apical surface of the epidermal cells D. magna is approximately 4-5 times thicker in the nuchal organs of Artemia and Daphnia than is found in the surrounding limb epithe- are absent in Leptodora. This structural differ- lia, they probably represent poor gas exchange ence may be due to the organ’s larger surface surfaces (Goldmann et al., 1999). Two cell area in Leptodora or to a difference in metabolic types, dark and light, are found arranged in a activity, but is not believed to result from func- jigsaw-type pattern in the epipodite epithelia tional differences. Unlike the nuchal organ of (Kikuchi, 1983). Dark cells have many infold- Artemia and Daphnia, it persists in Leptodora ings, large mitochondria, and a complex tubu- adults, probably because epipodites are lack- lar system, and show an accumulation of ing on their stenopodial limbs (Halcrow, 1985). chloride ions, suggesting an ion-transporting Marine members of the Onchopoda retain function. The light cells have strong infoldings an undiminished neck organ throughout their of both basal and lateral cell membranes and lives (Meurice and Goffinet, 1983). Light, scan- tubule-like protrusions toward the blood ning, and electron microscopy of four mature space, but an osmoregulatory function has not and immature representatives (Podon interme- been suggested (Kikuchi, 1983; Goldmann dius, Evadne nordmanni, E. spinifera, and E. ter- et al, 1999). gestina) revealed that the neck organ hangs as

PHYSIOLOGY OF THE CLADOCERA 214 16. A CYTOLOGICAL PERSPECTIVE a mass from the dorsal carapace in the middle fat cells, have been the subject of some study. of the interantennary cephalic shield (Meurice In Daphnia, the digestive tract is basically an and Goffinet, 1983). The cells composing the extended tube, consisting of a foregut, midgut, organ have an apical zone with a dense cyto- hindgut, and a pair of intestinal ceca. The plasm that surrounds the nucleus and a basal walls of both the foregut and hindgut are com- zone containing many mitochondria distrib- posed of cuboidal epithelial cells (about 5 μm uted throughout a lacunar system (Aladin, tall) lined with chitinous intima (Schultz and 1991). Dense bundles of microtubules are Kennedy, 1976a). The midgut can be distin- found in the apical cytoplasm and many free guished from foregut and hindgut based on polysomes fill the cytoplasmic interstices. A cell structure: midgut cells are taller (about small number of cisternae near the nucleus 30.5 μm), the nucleus is basally located, mito- make up the rough endoplasmic reticulum chondria are found anteriorly, and the apical (Aladin, 1991). Juxtaposed plasma membranes membrane possesses long microvilli. Cell den- are held together by septate desmosomes, sity along the gut remains relatively constant while a thin basement membrane defines the across age classes despite increases in the ventral cell mass of the organ (Aladin, 1991). length of the tract as animals grow. Since the The most interesting features of the ploidy level of all gut cells stabilizes at 4 C, it Podonidae neck organ are the small number is likely that these cells exit their endomitotic and the structure of the cells that comprise the cycle and return to a mitotic cell cycle (Beaton tissue. The organ is composed of at most and Hebert, 1999). This is quite unusual, but 12 cells, compared to 50 to 60 cells in Artemia, has not been studied and further investigation and the ultrastructure of all cells are similar, in is required. contrast to the two cell types found in Daphnia Early descriptions of the labrum depicted (Meurice and Goffinet, 1983). Comparisons the structure as glandular and organized into between environmental salinities and hemo- two groups of cells in Simocephalus sima and lymph concentrations indicate that a hypoos- Acanthocercus (Cunnington, 1903 and Scho¨dler, motic regulating mechanism is utilized by 1846; both in Cannon, 1922). In S. sima, the marine gymnomeran Cladocera. four large distal cells (whose shape were lik- A third osmoregulatory structure utilized ened to a hollow bowl) were thought to be by many cladocerans is the closed brood replaced when they “lose their secretory pouch. When the brood pouch contains devel- power” by the proximal group of cells, which oping eggs and embryos, the osmolarity of the were smaller and thought to be epidermal in marsupial fluid equals that of the hemolymph, origin (Cunnington, 1903; in Cannon, 1922). but once the embryos develop a neck organ, The distal cells were linked to the outside by a the fluid ion concentration rises to that of the duct leading to the inner surface of the labrum. surrounding sea (Aladin, 1991). The ability to Cannon (1922) similarly found two groups of regulate the embryonic environment and pro- cells in the labrum of S. vetulus: the proximal tect developing embryos allows Cladocera to group was separated into two lateral parts of inhabit high-osmolarity water (Aladin, 1991). 20 epidermal cells each, both situated near the nerve of the first antennae. It is likely that these cells correspond to the polyploid cells of 16.4.2 Labrum and Fat Cells Daphnia that are located along the underside of the rostrum (Table 16.5). The distal group of Three structures that are essential in diges- cells consisted of eight large cells (four per tion and energy storage, the labrum, gut, and side) arranged in pairs, with anterior cells

PHYSIOLOGY OF THE CLADOCERA 16.4 CYTOLOGICAL OBSERVATIONS FOR SPECIFIC TISSUES 215 connected to the posterior ones. The two poste- as developing parthenogenetic oocytes fill with rior cells “embraced” two “duct cells” that yolk, lipid, and glycogen levels in fat cells were slightly larger than muscle cells (Cannon, diminish (Jaeger, 1935). These cells are the 1922), so presumably were 2À4C. most likely sites of vitellogenin synthesis, a Sterba (1957b) recognized three gland cell principle component of yolk (Zaffagnini and types in an examination of Daphnia (presum- Zeni, 1986). In addition to lipid droplets, the ably D. magna), which he referred to as head cytoplasm of the fat cells contain numerous bottom, replacement, and main cells (the free ribosomes, a well-developed rough endo- names of which are based on a partial transla- plasmic reticulum (indicating active protein tion of his work). He found that the multiple synthesis), mitochondria, and small Golgi com- head bottom cells, which are proximally plexes with enlarged cisternae (Zaffagnini and located, reached 128 C and the medial paired Zeni, 1986). Sterba (1956a) noted that the mito- replacement and distal main cells reached chondria are uniformly distributed throughout 2048 C. Sterba (1957b) also noted that the cells the cytoplasm until lipid stores fill the cells, at within each cell type were linked via plasma which point they arrange in chains (my trans- bridges. Beaton and Hebert (1989, 1999) simi- lation) and are moved to the cell margins. The larly observed polyploid nuclei in D. magna structural organization of fat cells in partheno- and D. pulex arranged in three groups genetic D. obtusa females appeared to be simi- (Table 16.5). From proximal to distal, the first lar to those of adult female insects in the group comprised four cells (maximum of vitellogenic phase and to subepidermal fat 512 C), the second group had two extremely cells of reproducing females of the amphipod, large cells (maximum of 2048 C), and the most Orchestia gammarellus. These similarities sup- distal group of two cells reached 256 C in port the hypothesis that the fat cells of Daphnia D. pulex and 1024 C in D. magna.InD. pulex, synthesize vitellogenin (Zaffagnini and Zeni, the largest of these cells appeared to be 1986). Y-shaped, suggesting a similar structure to The nucleus in each fat cell contains an that found in the labrum of the conchostracan, irregularly shaped, large nucleolus, which Caenesteriella (Larink, 1992). None of the four occasionally appears as two or more pieces. In most distal cells in D. magna exhibited the Daphnia, these cells are known to be highly Y-shape or folded conformation found in polyploid (Jaeger, 1935; Sterba 1956a). Beaton D. pulex, despite possessing commensurate and Hebert (1989) found fat cells in the limb ploidy levels. Overall, there is congruence in central core of D. magna and D. pulex females the pattern of polyploidy observed in the (typically corresponding to instar 4 or 5 for labrum (Sterba, 1957b; Beaton and Hebert, well-fed animals) that contained up to 256 1989, 1999). This tissue appears to possess cells times more DNA than is found in haploid with the highest ploidy level in the animal. cells. This level agreed with a study of fat cells Lipid and glycogen can constitute a massive completed by Sterba (1956a). An examination portion of the volume of a daphnid’s fat cells, of ploidy shifts in D. pulex associated with which are situated in the body core along the growth and development revealed that the length of the gut and in the endopodites of number of core limb cells was unchanged with thoracic appendages to the base of the epipo- instar, but that ploidy levels changed with dites. The level of lipid reserves in these cells food availability (Beaton and Hebert, 1999). appears to be intimately related to an organ- Body size and nutritional status apparently ism’s reproductive status. Prior to peak ovary influence the ploidy level present in this tissue; growth, the fat cells hold maximal lipid stores; this is an expected finding, since fat cells store

PHYSIOLOGY OF THE CLADOCERA 216 16. A CYTOLOGICAL PERSPECTIVE lipid reserves for potential oocytes. When food do not develop immediately, but undergo a supplies were low, inadequate glycogen and period of diapause prior to completing lipid supplies were amassed, resulting in embryogenesis. We must remember that sex decreased egg production or the requirement determination in this order is under environ- of additional lipid synthesis by the ovaries mental control. In cyclically sexual cladocer- during the period of sexual reproduction. ans, males are produced from clutches of Fat cells have also been implicated with epi- parthenogenetic eggs before the sexual eggs podite cells in the synthesis of hemoglobin can be formed. (Hb), the primary oxygen-carrying molecule in Contrary to early research, a recent exami- cladocerans. In contrast to malacostracan crus- nation of the “parthenogenetic” process by taceans that use hemocyanin as their oxygen Hiruta et al. (2010) revealed that, in D. pulex, carrier, many branchiopods utilize Hb as their these eggs are not produced mitotically but respiratory protein. The site of Hb synthesis instead undergo an abortive meiosis. Using had been pinpointed for members of several histological and immunochemical analyses, invertebrate phyla, such as Annelida and they observed two divisions, with paired Nematoda and one insect (Bergtrom et al., homologous chromosomes aligning at the 1976), but not for a crustacean until the fat equatorial plate during the first division cells of D. magna were identified as a primary (as happens during meiosis). Bivalents were manufacturing site. Goldmann et al. (1999) split to form two half bivalents that began sep- hybridized a probe based on a previously aration, but reassembled as a diploid equato- determined Hb cDNA sequence (Tokishita rial plate after anaphase I. During the second et al., 1997) to sections of adult specimens. division, one of the separated sister chromatid Both fat and epipodite cells produced mRNA sets migrated and was elevated above the egg signals for Hb; stronger staining in epipodites surface to form a tiny polar body-like daughter compared to fat cells (Goldmann et al., 1999) cell, reminiscent of meiosis II (Hiruta et al., may reflect relative levels of production. 2010). Hiruta and Tochinai (2012), on examining spindle assembly in D. pulex, revealed a distorted spindle shape during abortive meio- 16.5 OOGENESIS sis. Contrary to the normal mitotic silhouette, spindles appeared barrel-shaped and lacked Cladocerans have evolved two forms of centromeres. Furthermore, γ-tubulin appeared reproduction that may alternate depending on to be distributed along the spindle microtu- environmental conditions. For much of the bules; in contrast, during mitosis this protein is growing season, a population will be com- concentrated around the poles (Hiruta and posed of nearly all females that produce Tochinai, 2012). parthenogenetic eggs, which develop immediately. Early accounts asserted that eggs were created ameiotically, so simple mitotic divi- 16.5.1 Formation of Parthenogenetic sions (apomixis) were invoked to retain their Eggs diploid condition. Under adverse conditions, a switch to the second mode of reproduction Paired ovaries lie along the length of the occurs, and in most cladocerans, germ cells intestine in the thorax of cladocerans. The loca- undergo meiosis to produce haploid gametes tion of the germarium in the tubular ovary that must be fertilized to reconstitute the dip- appears to vary across the suborders; it is loid state. The eggs resulting from this process located in the posterior region in the

PHYSIOLOGY OF THE CLADOCERA 16.5 OOGENESIS 217

Anomopoda and Haplopoda (Rossi, 1980; Kato certainly indicate that the structure is comet al., 2012) and anteriorly in the Ctenopoda posed of polyploid cells, and DNA contents (Rossi, 1980). Among the Onychopoda, should be examined. Jorgensen (1933) found the germarium of Initially, the oocyte of Daphnia is indistin- Evadne nordmanni to be near the anterior of the guishable from nurse cells. The cytoplasm ovary, but Rossi (1980) positioned it at the pos- appears homogeneous and the nucleus con- terior end in Bythotrephes longimanus. Within tains a large, central nucleolus (Zaffagnini, the ovaries are oocyte clusters, comprising one 1987). As the oocyte initiates growth, cyto- oocyte and three nurse cells. Zaffagnini and plasmic RNA concentration increases; as the Lucchi (1965, in Zaffagnini, 1987) reported oocyte begins to differentiate, the RNA concen- that, in D. magna, intercellular bridges arising tration decreases slightly. At the onset of vitel- from incomplete cytoplasmic divisions connect logenesis, the oocyte appearance is clearly the cells within each cluster, indicating one different from the nurse cells; the cell is much oogonium to be the origin of the four cells. larger, the cytoplasm appears more granular, The oocyte is the second or third cell to exit its RNA concentration drops further, and lipid the germarium, and the four-cell cluster forms droplets become visible (Zaffagnini, 1987). The a single row, although in Daphnia the nurse presence of oil droplets is one of the first out- cells are shifted dorsolaterally or laterally ward sign that distinguishes the oocyte from (Rossi, 1980; Zaffagnini, 1987). At this time, the nurse cells. oocyte’s cytoplasm appears homogeneous and During vitellogenesis, the ooplasm accumu- the nucleus contains a large nucleolus. Somatic lates yolk globules via pinocytosis, and as indi- cells in the area form an incomplete follicle sur- vidual globules enlarge, they concentrate in rounding just the oocyte (Zaffagnini, 1987). The the cell periphery. Lipid droplets also increase presence of follicular cells may be unique to in size and number, and mitochondria are Daphnia; their absence has been noted in mem- sometimes observed to cluster around them bers of all suborders (for example, Bythotrephes, (Zaffagnini and Lucchi, 1965, in Zaffagnini, Leptodora, Moina, and Sida) (Rossi, 1980). 1987). Microvilli develop on the surface of the In Leptodora, Moina, and the onychopods, oocyte near the follicular cells and pinocytotic embryos develop in a closed brood pouch and vesicles and endoplasmic reticulum form in are nourished by a maternal glandular struc- the immediate vicinity (Zaffagnini, 1987). ture, the Na¨hrboden, which was first described Kessel (1968) observed periodic accumulations by Weismann (1877, in Patt, 1947). Using of yolk bodies in the cisternae of the rough Polyphemus pediculus as a model, Patt (1947) endoplasmic reticulum. The nucleus also confirmed Weismann’s findings. The structure experiences change; as the nucleolus grows, its can be seen for the first time during embryo- interior becomes less dense. Furthermore, the genesis as a line of cells situated between the nucleolar RNA concentration increases gut and future brood pouch. Patt (1947) throughout differentiation until vitellogenesis, reported that as the animal grows the structure indicating active RNA synthesis. also grows via cell expansion. In these species, The function of the nurse cells, therefore, the Na¨hrboden appears not to be functional seems less clear. As vitellogenesis proceeds, when females produce amphigonic eggs. the nurse cells progressively shrink and degen- When parthenogenetic eggs are deposited into erate. Simultaneously, RNA-rich bodies appear the brood pouch, Na¨hrboden cells increase in around each nucleus before being transferred size to a maximum just before embryos pos- to the oocyte. However, since the oocyte sess limb buds. These observations almost actively manufactures RNA and the nurse cells

PHYSIOLOGY OF THE CLADOCERA 218 16. A CYTOLOGICAL PERSPECTIVE do not synthesize lipid or yolk, the nutritive 16.6 CONCLUDING REMARKS function of the nurse cells doesn’t appear to be essential (Zaffagnini, 1987). While a great deal of cytological information has been amassed for selected cladocerans, there is still a great deal of work left to be 16.5.2 Formation of Amphigonic Eggs done. For much of this chapter, we have attempted to highlight some cytological pat- Generally one or two sexually produced eggs terns that have emerged from the last hundred are formed at a time. The ovaries of examined or so years. However, some prominent gaps in members of Onychopoda and Anomopoda our knowledge are evident, even from a cur- (Evadne, Podon,andDaphnia) appear similar in sory examination. Much of our understanding parthenogenetic and sexual females (Zaffagnini, of the subject is based on studies of one or 1987; Egloff et al., 1997). The amphigonic oocyte two species of Daphnia and occasionally of one is formed with three nurse cells in the same additional cladoceran member. Only four manner as a parthenogenetic oocyte. The genome size estimates are known for repre- nucleus and nucleolus of both oocyte types also sentatives from outside the suborder appear to be similar. However, in marine ony- Anomopoda. Our understanding of chromo- chopods, the brood chambers of gamogenic some numbers is also limited to Anomopoda, (sexual) and parthenogenetic females differ. except for two ancient and unsubstantiated Cells of the former are cuboidal or hexagonal estimates. Many more surveys of DNA con- and may contribute to the egg’s chitinous layer, tent and chromosome number, which repre- whereas cells of the latter are transparent and sents some basic science, is required. The amorphous (Kuttner, 1911 and Rivier, 1968; ultrastructure of some tissues has been care- both in Egloff et al., 1997). fully completed for several tissues (but again Several characteristics have been reported for with heavy emphasis on Daphnia). However, Daphnia that distinguish sexual from partheno- questions of the function and development of genetic eggs: the amphigonic oocyte has a more some structures will benefit substantially from granular cytoplasm and contains no lipid dro- the burgeoning molecular tools that are avail- plets; and the yolk globules do not accumulate able to researchers today. A much clearer in the periphery, but fill the ooplasm uniformly understanding of the mechanism used for cre- with many tiny yolk balls that have phospho- ating parthenogenetic eggs has only recently lipid composition and concentration differences been described, due in no small part to the compared to a parthenogenetic oocyte developments in immunohistochemistry. The (Zaffagnini and Minelli, 1968, in Zaffagnini, function of nurse cells needs to be reevaluated 1987). Furthermore, although the nurse cells of via gene expression and in situ hybridization both types of oocytes seem to be similar in both studies. Similarly, molecular studies focused appearance and RNA content, those of the sexu- on structures such as the Na¨hrboden of ally produced oocytes cease growth by time that Polyphemus will clarify the types of material vitellogenesis is initiated (Zaffagnini, 1987). that cells of this tissue provide to parthenoge- Finally, upon completion of vitellogenesis, the netic embryos and how they function when amphigonic egg occupies nearly the entire ovary amphigonic eggs form. There is clearly much volume (one egg per ovary). left to be investigated.

PHYSIOLOGY OF THE CLADOCERA CHAPTER 17

Immunology and Immunity Stuart K. J. R. Auld School of Natural Sciences, University of Stirling, Stirling, Scotland, UK

17.1 INTRODUCTION work, Metchnikoff carefully observed Daphnia magna that were infected with a yeast parasite Infectious diseases are a challenge for clado- (which he referred to as Monospora bicuspidata; cerans, as they are for almost all living organ- now known as Metschnikowia bicuspidata). He isms. Cladocera are host to numerous parasites made detailed drawings of parasite spores and and pathogens from a wide variety of taxa, host immune cells throughout the duration of including amoeba, bacteria, fungi, nematodes the infection (until host death), and observed and microsporidians (Green, 1974; Stirnadel Daphnia cells enveloping yeast spores and and Ebert, 1997; Goren and Ben-Ami, 2013) yeast spores destroying Daphnia cells, i.e. the (see Table 17.1), and the fitness consequences battle between host and parasite. More recent of parasitic infection also vary greatly accord- research on cladocerans has extended ing to the combination of host and parasite Metchnikoff’s work by exploring variation in species. Cladoceran-parasite systems have host immune activity and linking measures of been pivotal in improving our understanding host immune activity with infection outcomes of the causes and consequences of host immu- and both host and parasite fitness (Mucklow nity to infection, both in the laboratory and in and Ebert, 2003; Mucklow et al., 2004; Auld the wild. This is especially true of species from et al., 2010; Decaestecker et al., 2011; Duneau the genus Daphnia (Ebert, 2005, 2008) and, as a et al., 2011; Graham et al., 2011; Pauwels et al., consequence, much of the research discussed 2011; Auld et al., 2012a, Auld et al., 2012b). in this chapter concerns various Daphnia Below, I take a step-wise look at cladoceran species. defenses against disease, not all of which The father of modern immunology, Elias involve the host systemic immune system. I Metchnikoff, noted that “[t]he common water start by looking at how shifts in host behavior flea or Daphnia seems quite suitable for studies and phenology allow the avoidance of para- on pathological processes and may be able to sites and reduce the likelihood of infection, throw some light on many general questions and then go on to describe how host barrier in medicine” (Metchnikoff, 1884). In this early and systemic defenses contribute to preventing

TABLE 17.1 Parasites of Cladocera, Infected Tissues, Host Species and Known Distribution

Parasite Group Infected Host Locations Reference(s) Tissue

Amoebidium Amoeba Epidermis Ceriodaphnia laticaudata Italy Green (1974) parasiticum Daphnia obtusa Italy Green (1974) Moina macrocopa Czech Green (1974) Republic

Moina micura Cameroon Green (1974) Simocephalus vetulus Greenland Green (1974) Pansporella Amoeba Hemolymph Daphnia longispina UK Stirnadel and Ebert (1997) perplexa Daphnia magna UK Stirnadel and Ebert (1997) Daphnia pulex UK Stirnadel and Ebert (1997) Pasteuria Bacteria Hemolymph Ceriodaphnia dubia USA Auld et al. (unpubl. ms) ramosa Ceriodaphnia dubia USA Auld et al. (unpubl. ms) Daphnia dentifera USA Duffy et al. (2010); Auld et al. (2012)

Daphnia longispina UK Stirnadel and Ebert (1997) Daphnia magna Finland, Green (1974); Stirnadel and Germany, UK Ebert (1997); Carius et al. (2001); Mitchell et al. (2004) Daphnia pulex UK Stirnadel and Ebert (1997)

Daphnia pulicaria USA Duffy et al. (2010) Simocephalus vetulus Czech Goren and Ben-Ami (2012); Republic, Green (1974) Israel, UK Spirobacillus Bacteria Hemolymph Daphnia atkinsoni Israel Goren and Ben-Ami (2012) cienkowskii Acroperus harpae Macedonia Green (1974) Bosmina longirostris UK Green (1974) Chydorus sphaericus Finland, UK Green (1974) Daphnia ambigua UK Green (1974) Daphnia curvirostris Uganda Green (1974) Daphnia dentifera USA Green (1974); Duffy et al. (2010) Daphnia hyalina Czech À Republic, UK Daphnia laevis Uganda À

(Continued)

PHYSIOLOGY OF THE CLADOCERA 17.1 INTRODUCTION 221

TABLE 17.1 (Continued)

Parasite Group Infected Host Locations Reference(s) Tissue

Daphnia longispina Uganda Green (1974)

Daphnia magna Finland, Goren and Ben-Ami (2012); Israel, UK, Green (1974) Ukraine Daphnia obtusa UK Green (1974) Daphnia pulex Finland Green (1974) Pleuroxus aduncus UK À

Pleuroxus trigonellus UK À Pleuroxus uncinatus UK À Sida crystallina UK À Simocephalus vetulus Israel, UK Goren and Ben-Ami (2012); Green (1974) Simocephalus expinosus Czech À Republic, UK Aphanomyces Fungi Body cavity Daphnia hyalina ÀÀ daphniae Daphnia pulex ÀÀ

Blastulidium Fungi Hemolymph, Bosmina obtusirostris UK Green (1974) paedophthorum embryos Camptocercus lilljeborgi Denmark Green (1974)

Ceriodaphnia megalops UK Green (1974) Chydorus sphaericus France Green (1974) Daphnia ambigua UK Green (1974) Daphnia longispina UK Green (1974) Daphnia magna UK Green (1974) Daphnia obtusa France Green (1974)

Eurycercus lamellatus UK Green (1974) Pleuroxus laevis Denmark Green (1974) Scapholeberis mucronata UK Green (1974) Sida crystallina UK Green (1974) Simocephalus vetulus France, UK, Green (1974) USA Metschnikowia Fungi Hemolymph Alona affinis UK Green (1974) bicuspidata

(Continued)

PHYSIOLOGY OF THE CLADOCERA 222 17. IMMUNOLOGY AND IMMUNITY

TABLE 17.1 (Continued)

Parasite Group Infected Host Locations Reference(s) Tissue

Bosmina longirostris UK Green (1974)

Ceriodaphnia reticulata UK Green (1974) Ceriodaphnia rotunda Czech Green (1974) Republic

Daphnia dentifera USA Duffy et al. (2010) Daphnia longispina UK Green (1974) Stirnadel and Ebert (1997)

Daphnia magna Israel, Russia, Goren and Ben-Ami (2012); UK Metchnikoff (1884); Stirnadel and Ebert (1997) Daphnia pulex UK Stirnadel and Ebert (1997) Ilyocryptus sordidus UK Green (1974)

Scapholeberis mucronata UK Green (1974) Glugoides Microsporidia Gut wall Bosmina longirostris UK Green (1974) intestinalis Daphnia dentifera USA Green (1974) Daphnia magna UK Green (1974) Daphnia obtusa UK Green (1974) Daphnia pulex Albania, Green (1974) Czech Republic, Macedonia Daphnia longispina UK Stirnadel and Ebert (1997) Eurycercus lamellatus Albania, Green (1974) Macedonia Simocephalus vetulus UK À Gurleya vavrai Microsporidia Epidermis Daphnia pulex UK Green (1974); Stirnadel and Ebert (1997) Daphnia longispina UK Green (1974) Stirnadel and Ebert (1997) Hamiltosporidium Microsporidia Fat body, Daphnia magna Finland, Goren and Ben-Ami (2012); sp. gonads Israel, Haag et al. (2011) Sweden Octosporea Microsporidia Fat body, Daphnia magna Czech Ebert et al. (2001); Green (1974) bayeri ovaries Republic, Finland, UK

(Continued)

PHYSIOLOGY OF THE CLADOCERA 17.2 PREVENTING INFECTION 223

TABLE 17.1 (Continued)

Parasite Group Infected Host Locations Reference(s) Tissue

Echinuria Nematode Hemolymph Daphnia magna Canada, UK Green (1974) uncinata Daphnia pulex Canada, UK Green (1974) Simocephalus vetulus Canada, UK Green (1974) Daphnia obtusa UK Green (1974) Caullerya Unknown Gut wall Daphnia galeata Austria, Bittner et al. (2002) mesnili Germany, Switzerland

Daphnia hyalina Austria, Wolinska et al. (2006) Germany, Switzerland Daphnia longispina UK Stirnadel and Ebert (1997) Daphnia magna UK Green (1974) Stirnadel and Ebert (1997) Daphnia obtusa UK Green (1974)

Daphnia pulex Denmark, UK Green (1974); Stirnadel and Ebert (1997) Polycaryum Fungus Connective Daphnia pulicaria USA Johnson et al. (2006) laeve tissue

infection (Fig. 17.1). In cases in which infection Pre-infection Post-infection occurs, I discuss how hosts can limit the negative fitness impacts of infection by shifting Avoid Eliminate allocation into different life history traits. parasites infection Finally, I examine some evolutionary and coevolutionary consequences of cladoceran- parasite interactions.

Barrier Minimize defenses virulence 17.2 PREVENTING INFECTION 17.2.1 Avoiding Parasites The likelihood of parasitic infection depends on (1) the probability of the host encountering FIGURE 17.1 Stages of cladoceran defense against infectious parasite transmission stages and (2) parasitism. the efficacy of host barrier and immunological

PHYSIOLOGY OF THE CLADOCERA 224 17. IMMUNOLOGY AND IMMUNITY defenses following parasite exposure (hence- Auld et al., 2012b); Metschnikowia bicuspidata forth, intrinsic resistance) (Combes, 2001) (see and S. cienkowskii epidemics occur in the fall in Fig. 17.1). Parasite avoidance is thus an effec- North America (in D. dentifera populations) tive mechanism for reducing the likelihood of (Ca´ceres et al., 2006; Duffy et al., 2010; Duffy parasitic infection and its associated disease, et al., 2012); and epidemics of Caullerya mesnili, especially since parasite transmission stages peak in the fall and winter (in various are often unevenly distributed over both space European Daphnia populations) (Wolinska and time. Nevertheless, avoidance is fre- et al., 2006). As host population density quently ignored as a mechanism of host increases, the likelihood of an individual host immunity. consuming an infectious parasite transmission The transmission stages for many cladoc- stage also increases, as does the probability of eran parasites, including Metschnikowia bicuspi- parasite transmission from diseased to healthy data, Pasteuria ramosa, and Spirobacillus hosts. Sickness thus begets more sickness. cienkowskii, lie in the sediment at the bottom of Some genotypes of D. magna switch from ponds and lakes (Decaestecker et al., 2004), asexual to sexual reproduction during P. ramo- where they await consumption by susceptible sa epidemics in the wild (Duncan et al., 2006) hosts. Potential hosts may minimize the risk of or when surrounded by high densities of infection by keeping close to the water’s sur- infected hosts in the laboratory (Duncan et al., face. Indeed, D. magna were found to be more 2009). Moreover, parasite-susceptible D. magna likely to suffer parasitic infection if they were genotypes are most likely to switch from an negatively phototactic (i.e. repelled by light) asexual to a sexual reproductive mode and spent most of the time at the bottom of (Mitchell et al., 2004). These sexually produced the water column near the mud; infection was eggs are encased in a tough casing, and they reduced when Daphnia were positively photo- hatch in the following year (i.e. after the cur- tactic (i.e. attracted to light) and spent most of rent epidemic has passed). Therefore, by their time at the top of the water column near reproducing sexually, P. ramosa-susceptible the surface (Decaestecker et al., 2002). D. magna mitigate the fitness consequences of However, being positively phototactic comes infection by ensuring that their offspring avoid at a cost: Daphnia that are attracted toward the the current epidemic and instead face a differ- water’s surface are more probably to fall vic- ent parasite population in the future (to which tim to visual predators, such as fish. This they may be more broadly resistant). trade-off between susceptibility to parasites and vulnerability to predation can thus maintain diversity in Daphnia and prevent the evo- 17.2.2 Barrier Defenses lution of universal host resistance to parasitism (Decaestecker et al., 2002). Upon encountering parasite transmission The risk of suffering parasitic infection var- stages, a cladoceran’s first line of defense con- ies over time as well as space. Epidemics of sists of both the carapace and the epithelium. To P. ramosa occur in the fall in North America (in infect, a parasite must attach to and (usually) Daphnia dentifera, D. pulicaria,andCeriodaphnia penetrate host tissues (Fig. 17.2,stages2and3); dubia (Grippi, Auld, and Duffy unpubl. data; the parasite can only establish an infection and Duffy et al., 2010) and in the summer in Europe proliferate after it has overcome at least one of (in D. magna, D. pulex,andD. longispina) these barrier defenses (Figs. 17.1 and 17.2,stage (Stirnadel and Ebert, 1997; Little and Ebert, 4). Since invertebrates have a single open body 1999; Mitchell et al., 2004; Duncan et al., 2006; chamber (the hemocoel), it is prudent for them

PHYSIOLOGY OF THE CLADOCERA 17.3 THE INNATE IMMUNE SYSTEM 225

backcrossed and selfed genetic lines), Luijckx 1 et al. (2011b) found that D. magna display either complete resistance or complete susceptibility to a purely clonal P. ramosa strain and 2 5 that resistance is inextricably linked to the par- 4 asite attachment mechanism. This shows that D. magna resistance to this particular P. ramosa 3 strain (which stems from the ability of the host Gut lumen Body cavity Environment to prevent spore attachment to the gut) depends on a single host gene. Further, it sug- FIGURE 17.2 A typical infection sequence for a cladoc- gests that the resistance gene has two alleles eran parasite. 1, Host exposure to the parasite transmission and that resistance is dominant over suscepti- stage. 2, Attachment to the gut epithelium. 3, Penetration bility (Luijckx et al., 2011b). However, both into the host’s body cavity. 4, Proliferation within the host’s body cavity. 5, Parasite transmission into the environment. D. magna and D. dentifera can exhibit strong genetic specificity with P. ramosa i.e. infection success depends on the specific combination of to invest heavily in keeping parasites out. host genotypes and parasite isolates (Carius Conversely, vertebrates can more easily isolate et al., 2001; Luijckx et al., 2011a; Auld et al., parasitic infections in certain tissues and are 2012c), so resistance to one P. ramosa strain thus better at fighting disease-causing organisms may come at a cost of susceptibility to another. within the body (Theopold et al., 2004). Since Daphnia frequently encounter multiple Daphnia research has been key in improving P. ramosa genotypes in the wild, resistance in our understanding of host barrier defenses, in general is likely to be a quantitative trait. particular, by revealing that barriers are not In any case, the ability of P. ramosa to attach necessarily unsophisticated walls against para- to and penetrate host barrier defenses is a sites and that they instead vary in efficacy major determinant of infection outcome. depending on the particular immune chal- Further supporting this, D. magna genotypes lenges. Studies using the parasite P. ramosa mount a strong cellular response (a systemic have shown that spore attachment to the immune response that occurs within the body esophageal gut epithelium is crucial to the cavity) only to P. ramosa isolates that can cause infection process: P. ramosa spores were found infection, and the magnitude of this cellular to successfully bind to the esophagus and then response increases with the dose of infectious infect certain genotypes of D. magna and a parasite spores (Auld et al., 2012a). These find- genotype of D. dolichocephala; and infections ings suggest that the cellular immune response only occurred where attachment was success- is an indicator of the capacity for parasite ful (Duneau et al., 2011). P. ramosa failed to spores to successfully overcome host barrier attach to and infect individual genotypes of defenses in the gut (Auld et al., 2012a). Daphnia arenata, Daphnia barbata, Daphnia lumholtzi, and Daphnia similis (Duneau et al., 2011); however, since just a single genotype was 17.3 THE INNATE IMMUNE studied for each of these species, we cannot SYSTEM draw broad conclusions regarding their ability to prevent P. ramosa attachment. Once the barrier defenses have been brea- By taking a classical genetics approach ched, parasites face the next round of host (using parental, F1, and F2 generations, defenses: the systemic immune system.

PHYSIOLOGY OF THE CLADOCERA 226 17. IMMUNOLOGY AND IMMUNITY

Vertebrates possess an immune system with D. pulex and its parasites (McTaggart et al., both innate and adaptive components, whereas 2009). It is important to realize that many of invertebrates have only an innate immune sys- these genes are putative immune genes; they tem (Schmid-Hempel, 2011). Adaptive immune are similar to genes that are known to be defenses consist of highly specific responses to involved with an immune response in other an ongoing immune challenge (Janeway et al., organisms, but their link with Daphnia immu- 2001; Schmid-Hempel, 2011) and are therefore nity has yet to be demonstrated. comparatively slow to respond to an immune challenge. Conversely, innate immune responses are much less specific—they are 17.3.1 Pathogen Recognition thought to distinguish between broad classes of parasite, e.g. Gram-positive bacteria, Gram- Pathogen recognition receptors (PRRs) negative bacteria, and fungi (Janeway and detect molecular patterns on the surface of Medzhitov, 2002; Ferrandon et al., 2003) (see both prokaryote and eukaryote pathogens and later Sections 17.5.1 and 17.6.2). Innate immune complete the first step of initiating an innate responses can, however, be mounted relatively immune response. Daphnia species possess soon after immune challenge: the D. magna cel- multiple types of PRRs, which are discussed lular immune response can occur within hours individually below. of an immune challenge (Metchnikoff, 1884; Gram-negative binding proteins (GNBPs) Auld et al., 2010). are a major class of PRR. GNBPs recognize Like other invertebrates, Daphnia innate and bind to β-1,3-glucans and activate immune immunity genes fall into three functional defenses (including the prophenoloxidase groups: (1) pathogen recognition, (2) immune cascade, which is known to be present in regulation, and (3) attack. Although much of D. magna). Although they bind principally to what we know about invertebrate immunology Gram-negative bacteria and fungi, it is impor- stems from studies of Dipteran insects (in par- tant to note that GNBPs are also known to ticular Drosophila melanogaster and Anopheles bind to Gram-positive bacteria in Drosophila gambiae), recent investigations of the D. arenata, (Gobert et al., 2003; Filipe et al., 2005). D. pulex D. magna, D. pulex, and D. parvula immune sys- has 11 GNBP copies—more than A. gambiae tem have expanded our knowledge greatly; (7), A. aegypti (7), D. melanogaster (3), T. casta- indeed, D. pulex was the first crustacean naeum (3), or A. mellifera (2) (McTaggart et al., genome to be sequenced (Colbourne et al., 2009), and five of these GNBPs have also been 2011). This work has revealed many similari- sequenced in D. parvula (McTaggart et al., ties between the innate immune systems of 2012a). Ten of the 11 D. pulex GNBPs are these cladocerans and other invertebrates, as grouped together, along with a GNBP gene well as some interesting differences. from Eisenia foetida (an annelid), suggesting McTaggart et al. (2009) found 82 putative they are relatively ancient; the remaining gene immune genes from 21 families in D. pulex. is more derived and groups with GNBP genes This is fewer than in D. melanogaster, more from A. gambiae (McTaggart et al., 2009). than in Apis mellifera, and similar to numbers Another invertebrate PRR group includes in Tribolium castanaeum. Furthermore, D. pulex peptidoglycan recognition proteins (PGRPs). immune genes undergo more adaptive evolu- Peptidoglycan is a key constituent of the cell tion than genes that are not thought to be asso- wall of Gram-positive bacteria and PGRPs are ciated with the immune system, pointing known to bind to this peptidoglycan and initi- toward a coevolutionary history between ate immune cascades involved in

PHYSIOLOGY OF THE CLADOCERA 17.3 THE INNATE IMMUNE SYSTEM 227 phagocytosis, generation of immune cytotox- SR-As: two of these proteins are orthologous ins, and generation of antimicrobial peptides to Tequila/graal/Scrasp1 (Dappu-SCV3 and in Drosophila (Hultmark, 2003). Remarkably, Dappu-SCV4), and one is orthologous to Corin D. pulex does not possess any PGRPs (unlike (Dappu-SV2). Therefore, Dappu-SCV2, Dappu- A. gambiae, A. aegyptii, D. melanogaster, T. casta- SV3, and Dappu-SV2 may all have an immune naeum, and A. mellifera) (McTaggart et al., role (McTaggart et al., 2009), although this 2009). We can only speculate that D. pulex’s remains to be tested. numerous GNBPs compensate for this and Down syndrome cell adhesion molecules provide defense against Gram-positive bacteria (Dscams) are important for arthropod neural (McTaggart et al., 2009). development, as well as being PRRs (where Thioester proteins (TEPs) are also important they are principally involved in phagocytosis PRRs in many species. Seven TEP genes were (Watson et al., 2005). Arthropod Dscam genes found on the D. pulex genome, including the have an immunoglobulin (IgSF) domain, like well-studied alpha-2-macroglobulin (α2m) gene, their vertebrate counterparts, and are espe- which inhibits protease production in Gram- cially interesting because they contain clusters positive bacterial cells, while promoting phago- of variable exons that can be alternatively cytosis in vertebrates (deBoer et al., 1993). spliced during transcription, thus facilitating Anopheles gambiae TEPs bind to pathogenic cells the generation of tens of thousands of unique and await phagocytosis (Levashina et al., 2001), transcripts (Du Pasquier, 2005; Watson et al., although TEPs may have other nonimmunologi- 2005; Brites et al., 2008; Brites et al., 2011). This cal functions in other organisms. A study of ability to generate a multitude of transcripts is D. magna, D. pulex,andD. rosea found strong consistent with Dscam genes being PRRs; it has evidence of positive selection in the bait region been hypothesized that these alternative tran- of the α2m gene, which is consistent with the scripts may be functionally equivalent to verte- hypothesis that coevolutionary interactions brate antibodies (Watson et al., 2005). between Daphnia and Gram-positive bacterial pathogens have fostered an arms-race evolution α α in 2m (Little et al., 2004). However, 2m tran- 17.3.2 Immune Regulation scription in D. magna does not increase after exposure to P. ramosa (Decaestecker et al., 2011) Signal transducers play a pivotal role in (see Section 17.6.1). developing immune responses. For example, the Scavenger receptor-A proteins (SR-As) are a Toll pathway and associated Toll receptors inter- conserved and structurally similar to a group act with GNBPs and PGRPs elicit the appropri- of PRRs found on both cell surfaces and in sol- ate attack responses to particular pathogens uble form; they have two roles. SR-As provide (including phagocytosis). Seven Toll receptors a housekeeping role by “mopping-up” cellular have been found in D. pulex (McTaggart et al., debris (Munier et al., 2004). They can also bind 2009) and three have been sequenced in D. par- to and promote the encapsulation of bacteria: vula (McTaggart et al., 2012a). Many of the the D. melanogaster protein Tequila/graal and D. pulex Toll receptors show similarities to those the orthologous A. gambiae protein Scrasp1 are of D. melanogaster.Inparticular,D. pulex Dappu- upregulated following an immune challenge TOLL1 and Dappu-TOLL3 are within the same (Danielli et al., 2000; Munier et al., 2004). The clade as D. melanogaster Dm-Toll-1, which is D. melanogaster protein Corin is also upregu- upregulated following exposure to fungi and lated following a bacterial or fungal immune bacteria (Lemaitre et al., 1996; Michel et al., challenge (Irving et al., 2001). D. pulex has six 2001), and Dappu-TOLL2 and Dappu-TOLL4

PHYSIOLOGY OF THE CLADOCERA 228 17. IMMUNOLOGY AND IMMUNITY cluster with Dm-Toll-9 and are similar to mam- cavity, numerous ameboid hemocytes bound malian Toll like receptors. firmly to the spores to isolate the parasite and that the parasite often swells and becomes yellow-brown. Metchnikoff concluded that 17.3.3 Pathogen Attack these hemocytes caused this change in the parasite spores and neutralized the infection; he Pathogen attack responses are the best- supported this claim with observations of M. known components of cladoceran systemic bicuspidata spores that had partially penetrated immune responses. They consist of both cellu- the gut epithelium. Of spores in the process of lar and humoral defenses, which are discussed penetration, only the sections that were within below. the host’s body cavity became covered with Circulating hemocytes are a key component hemocytes and underwent digestion; sections of antiparasite defenses in numerous inverte- of the spore that remained in the gut lumen brates (Ataev and Coustau, 1999; Elrod- were free of hemocytes and appeared undi- Erickson et al., 2000; Kraaijeveld et al., 2001; gested (Metchnikoff, 1884). However, this host Canesi et al., 2002). Invertebrate hemocytes cellular immune response was not always (Fig. 17.3) can either phagocytose (or encapsu- effective: sometimes M. bicuspidata spores did late) parasitic organisms or generate immune not fall victim to the host’s hemocytes, leading cytotoxins such as reactive oxygen species and to the establishment of infection (see reactive nitrogen species (Strand, 2008). At the Section 17.6.1). very beginning of this chapter, I discussed Nitric oxide (NO) is a highly oxidative free- Metchnikoff’s (1884) study of Daphnia cellular radical gas that can severely damage enzymes responses to the yeast M. bicuspidata. He found and DNA, making it generally toxic to a vari- that once the needle-like transmission spores of ety of parasitic organisms (Nappi and the parasite had penetrated the host’s body Ottaviani, 2000; Rivero, 2006). NO is generated in vertebrates, invertebrates, and plants by nitric oxide synthase (NOS) genes. NOS genes are either constitutively expressed [constitutive NOS (cNOS)] or their expression is induced upon encountering an immune challenge [inducible NOS (iNOS)]. However, its highly oxidizing nature means that NO can be toxic to the host that produces it, i.e. it can cause immunopathology (in which host disease is caused by host immune activity). NO is thus a highly potent weapon, but the host risks potentially severe collateral damage (Rivero, 2006). Unlike all other investigated invertebrates, D. pulex has two NOS genes (other invertebrates possess only one), and these two genes are very different in structure: the resulting proteins are only 44% identical (Labbe´ FIGURE 17.3 Differential interference contrast image et al., 2009). McTaggart et al. (2009) hypothe- of a Daphnia magna ameboid hemocyte. Scale bar, 5 μm. sized that this could be due either to a relaxa- From Auld et al. (2010). tion from a selective constraint or to positive

PHYSIOLOGY OF THE CLADOCERA 17.5 EVOLUTION AND COEVOLUTION IN CLADOCERAN-PARASITE SYSTEMS 229 selection that has promoted evolutionary evolved elaborate defense systems to prevent divergence between the two NOS genes. In infection (e.g. immune systems). However, either case, duplication of the NOS gene did preventing infection is just one component of not occur recently. these defenses; the other component involves The prophenoloxidase (PO) cascade, which reducing virulence, which often involves hosts involves the generation of PO, is another cyto- shifting resource investment into different life toxic immune defense known to be important history traits. in numerous invertebrates (Soderhall and A prime example of a virulence-limiting life Cerenius, 1998). D. magna produce more PO history shift occurs in D. magna.WhenD. magna when they experience wounding (Mucklow are at the early stages of infection with the steril- and Ebert, 2003) and PO has been shown to izing parasite P. ramosa, they accelerate to pro- limit parasite success in multiple parasite spe- duce a clutch of offspring before the parasite cies (Mucklow et al., 2004; Pauwels et al., 2011; fully sterilizes the host (termed fecundity com- see Section 17.6.1). pensation) (Ebert et al., 2004; Vale and Little, Chitinases are another potentially impor- 2012). This is also true of D. magna exposed to tant immune effector. Chitinases hydrolyze the horizontally transmitted microsporidian, chitin, a polysaccharide found in arthropod Glugoides intestinalis: although it does not fully exoskeletons, as well as the cell walls of fungal sterilize its hosts, G. intestinalis-infected D. magna spores and amoeba cysts. Proteins similar to alter their life history to produce more of their chitinase have been found to be upregulated offspring early in life (Chadwick and Little, in A. gambiae when the host is exposed to 2005). By inducing fecundity compensation, Gram-positive bacteria (Shi and Paskewitz, D. magna can reduce the virulence suffered from 2004), and there is evidence of rapid adaptive parasitic infection without necessarily limiting evolution in a plant chitinase, probably caused parasite proliferation. by coevolution with a fungal parasite (Bishop et al., 2000). Chitinases are thus a useful weapon for both animals and plants against 17.5 EVOLUTION AND many pathogenic organisms. Seventeen chiti- COEVOLUTION IN CLADOCERAN- nase genes were found in the D. pulex genome, PARASITE SYSTEMS which is a similar number to that found in A. gambiae (13), A. aegyptii (19), D. melanogaster Cladoceran hosts exhibit abundant genetic (16), and T. castanaeum (16), but considerably variation in both their susceptibility to infec- more than in A. mellifera (5) (McTaggart et al., tion and the virulence they experience as a 2009). However, we have yet to determine result of parasitism. Indeed, when genetic vari- which, if any, of these chitinase genes have an ation for parasite resistance has been tested in immunological function in D. pulex. wild Daphnia and C. dubia populations, it has been found in every case (Auld et al., in prep.; Ebert, 2005), suggesting that parasites are an 17.4 MECHANISMS TO LIMIT ever-present selective force in these host popu- THE SEVERITY OF INFECTION lations. Furthermore, there is abundant evidence that parasite epidemics select for host Parasites, by their very nature, reduce the resistance, thus driving rapid evolution in nat- fitness of the hosts they infect. This parasite- ural host populations (Duncan and Little, 2007; induced decline in host fitness is termed viru- Duffy et al., 2012; Auld et al., in press). Yet, lence. As discussed above, cladocerans have despite the ubiquity of parasites, susceptible

PHYSIOLOGY OF THE CLADOCERA 230 17. IMMUNOLOGY AND IMMUNITY host genotypes persist and are sometimes are exposed to (Luijckx et al., 2011a). favored by selection. There are two main Moreover, genetic specificity can occur at the hypotheses for the maintenance of host varia- species level: in both D. magna and D. dentifera, tion for susceptibility: genetic specificity and infection depends on the combination of the the costs of resistance. Daphnia genotype and the parasite species (Decaestecker et al., 2003; Auld et al., 2012c). Genetic specificity is important because it 17.5.1 Genetic Specificity can maintain both host and parasite genetic Genetic specificity occurs when the likeli- diversity and drive host-parasite coevolution hood of successful infection depends on the (providing the parasites cause a high degree of precise combination of host and parasite geno- virulence). This is because each parasite genotypes (Schmid-Hempel and Ebert, 2003; type (or species) can only select against a sub- Lambrechts, 2010); no one host genotype is set of host genotypes (and vice-versa). It also resistant to all local parasite genotypes and no means that individual parasite genotypes parasite genotype is infectious to all local host select against the most common host genotype genotypes (Fig. 17.4). Daphnia-parasite systems (and vice-versa), leading to negative provide some excellent examples of genetic frequency-dependent selection and a cycling of specificity: for both D. magna and D. dentifera, both host and parasite genotypes (Hamilton, the likelihood of suffering infection with 1980). This form of genotype cycling is referred P. ramosa depends on which host genotypes to as Red Queen dynamics after the Red are exposed to which parasite isolates (Carius Queen in Lewis Carroll’s Through the Looking et al., 2001; Auld et al., 2012c), with particular Glass, who had to constantly run in order to P. ramosa strains either achieving 100% or 0% stay still (Carroll et al., 1971). A recent study infection success depending on the hosts they looked for evidence of Red Queen dynamics in hatching dormant stages (resting eggs) of D. magna and P. ramosa from sediment cores that represented decades of time. Consistent Host genotype with the Red Queen hypothesis, they found 1 2 3 4 P. ramosa isolates were best adapted to contem- porary D. magna genotypes (i.e. had the highest 1 infection successes) (Decaestecker et al., 2007). 2 However, genetic specificity is not univer- 3 sally present in cladoceran systems. For example, there is no evidence for genetic specificity 4 in the D. dentifera-M. bicuspidata system (Duffy Parasite genotype Parasite genotype and Sivars-Becker, 2007). Remarkably, there is Infection no evidence for genetic variation in M. bicuspidata for infection success: M. bicuspidata sam- No infection ples taken from different populations at different times do not vary in their ability to infect D. dentifera (Duffy and Sivars-Becker, FIGURE 17.4 Genetic specificity. Host genotypes have 2007). Despite this, there is considerable that same mean resistance to infection (50%) and parasite genotypes have the same mean infectivity (50%), but the genetic variation for D. dentifera resistance to likelihood of infection depends on the combination of host M. bicuspidata (Ca´ceres et al., 2006; Duffy and and parasite genotypes. Sivars-Becker, 2007; Auld et al., 2012c, Duffy

PHYSIOLOGY OF THE CLADOCERA 17.5 EVOLUTION AND COEVOLUTION IN CLADOCERAN-PARASITE SYSTEMS 231 et al., 2012). What could be responsible for i.e. intrinsic resistance (Hall et al., 2010). That maintaining this variation? The answer stems is not to say that there are not evolutionary from the fact that resistance to parasitism often costs associated with intrinsic resistance in the comes at a cost. D. dentifera-M. bicuspidata system—there may well be—but such costs have not yet been 17.5.2 Costs of Resistance found. Since overall resistance is a complex trait that depends on both host exposure and Initially, one would expect organisms with intrinsic resistance, one might expect the mag- the strongest resistance to parasites to have the nitude of any evolutionary costs of resistance highest fitness and be favored by natural selec- to vary over time or space or both. tion, i.e. for parasite-resistant genotypes to It is important to note that the evolutionary dominate host populations. However, immu- costs of resistance are not a general phenome- nity is not free; it can come at the expense of non in cladoceran-parasite systems. D. magna one or more other fitness-related traits. Indeed, that are susceptible to the microsporidian these are termed the evolutionary costs of resis- Octosporea bayeri are not more fecund in the tance,orresistance trade-offs (Sheldon and absence of infection (Altermatt and Ebert, Verhulst, 1996; Kraaijeveld and Godfray, 1997). 2007), nor are D. magna that are susceptible to A recent study found that while D. dentifera P. ramosa (Little et al., 2002). populations that suffered large M. bicuspidata Evolutionary costs of resistance are not the epidemics evolved increased resistance to only type of cost: there may also be costs associ- M. bicuspidata, populations that experienced ated with activating defense mechanisms (e.g. relatively small epidemics evolved increased immune responses). Kraaijeveld and Wertheim susceptibility (Duffy et al., 2012). Further, (2009) liken evolutionary costs to the costs asso- parasite-mediated disruptive selection has also ciated with building an army and activation been observed in this system: an M. bicuspidata costs to the costs associated with sending the epidemic was found to simultaneously favor army to war. Studies have tested for an activa- both the most resistant and the most suscepti- tion cost of resistance in both the D. magna- ble host genotypes in the population, and P. ramosa (Little and Killick, 2007; Labbe´ et al., select against those of intermediate resistance 2010) and the D. dentifera-M. bicuspidata (Auld (Duffy et al., 2008). Both of these studies point toward an evolutionary cost of resisting M. bicuspidata infection; indeed how else could susceptible hosts be favored? Hall et al. (2010) determined a simple underlying mechanism for this cost, which hinges on the filtration rate of the host: D. dentifera genotypes with a high filtration rate obtain more algal food and can therefore pro- Fitness trait duce more offspring, but they are also more likely to consume M. bicuspidata spores and Resistance to suffer infection (Fig. 17.5). This trade-off influ- parasitism ences overall host resistance by mediating exposure to parasite transmission stages; it FIGURE 17.5 Evolutionary cost of resistance. There is does not concern the costs associated with the a trade-off between resistance and another fitness-related evolution of particular host immune defenses, trait (i.e. fecundity).

PHYSIOLOGY OF THE CLADOCERA 232 17. IMMUNOLOGY AND IMMUNITY et al., 2012c) systems by comparing the fitness (see Chapter 11) means that genotypes can be of hosts that were exposed to, but resisted, infec- propagated clonally and thus that one can tion from a particular parasite with hosts that examine the relative contributions of genetic were not exposed to the parasite. While Little and environmental factors to immune strategies and Killick (2007) found evidence for a cost of and infection outcomes. Second, there is a rich resistance in terms of D. magna survival when body of research examining variation in suscep- exposed to P. ramosa, more extensive study in tibility to various parasites both within and this system failed to uncover activation costs across host species (Green, 1974; Stirnadel and (Labbe´ et al., 2010). No activation costs of resis- Ebert, 1997; Little and Ebert, 1999; Wolinska tance in terms of host fecundity or survival et al., 2006; Duffy and Sivars-Becker, 2007; were found in the D. dentifera-M. bicuspidata sys- Duffy et al., 2010; Goren and Ben-Ami, 2013). tem (Auld et al., 2012c). Third, there is an ever-growing body of genetic Why are activation costs of resistance so resources, in particular from the recently rare in these particular cladoceran-parasite sys- sequenced D. pulex genome (McTaggart et al., tems? The explanation probably lies in the 2009; Jansen et al., 2011). Finally, one can easily infection process. If attachment of spores to the pair controlled laboratory experiments with gut wall explains most of the variation in observational study of immune phenotypes in infection likelihood (as it does in the D. magna- the wild—indeed, Daphnia research is making P. ramosa system; see above), then host resis- key contributions to the emerging field of wild tance does not stem from the mobilization of immunology (Auld et al., 2012b). costly systemic immune responses. Evolutionary ecologists have often assumed that a larger immune response leads to 17.6 LINKING IMMUNOLOGY TO improved immunity and greater host fitness in IMMUNITY: STUDIES INTO the face of parasites. This is largely true in DAPHNIA MAGNA AND many host-parasite systems. However, PASTEURIA RAMOSA research with D. magna and P. ramosa has emphasized that immune responses can also 17.6.1 Ecological Immunology be a consequence of parasitic infection: upon exposure to P. ramosa, Daphnia that mount Much immunological work is conducted on strong cellular immune responses go on to suf- a limited number of host genotypes and para- fer infection with sterilizing bacterium and site strains in the laboratory, but in the wild, thus experience genetic death (i.e. an end to variation abounds. Students of evolutionary their fitness potential) (Auld et al., 2010; Auld ecology have long been interested in how and et al., 2012a). Further, both wild-caught and why some hosts resist parasitic organisms and laboratory reared D. magna have a greater others do not. This has given rise to the field of baseline number of circulating hemocytes than ecological immunology—the study of the varia- their healthy counterparts when suffering a tion in host immune strategies and their under- P. ramosa infection (Auld et al., 2012b). lying costs and benefits (Rolff and Siva-Jothy, Immune gene expression studies have also 2003; Schmid-Hempel, 2003; Sadd and Schmid- highlighted the danger of assuming that Hempel, 2009; Graham et al., 2011). Cladocerans greater immune activity causes host resistance (Daphnia in particular) and their parasites are to infection. When P. ramosa (a Gram-positive well poised for eco-immunological study for bacterial parasite) was exposed to both resis- numerous reasons. First, the fact that many tant and susceptible D. magna genotypes, there Daphnia species are cyclically parthenogenetic was no significant increase in expression of

PHYSIOLOGY OF THE CLADOCERA 17.7 SUMMARY 233

α2m relative to controls (Decaestecker et al., parasite genotypes and modulate their defenses 2011). As discussed earlier, α2m is a PRR accordingly. D. magna that were exposed to known to target Gram-positive bacteria in and successfully resisted P. ramosa were more Drosophila. While this brings into question the likely to resist infection when reexposed to role of α2m in defending D. magna against infectious spores 48 h later (McTaggart et al., P. ramosa, it is important to note that TEP pro- 2012b). Even more remarkably, upon exposure teins can be highly specific to particular to P. ramosa, D. magna are known to be able to immune challenges: D. melanogaster TEP6 pass on strain-specific resistance to their off- (referred to as the macroglobulin complement- spring (Little et al., 2003): when a mother is related gene) is known to be highly specific to exposed to a particular P. ramosa isolate, her the yeast Candida albicans (Stroschein- offspring have increased resistance to that par- Stevenson et al., 2006), so D. magna’s α2m gene ticular isolate (but not to other P. ramosa iso- may play a role in defending against a parasite lates). Cladoceran immunity therefore has the or parasites other than P. ramosa. potential to reach across generations. Another study demonstrated that P. ramosa- infected hosts had higher constitutive PO levels, but in this case, PO activity was nega- 17.7 SUMMARY tively associated with parasite burden in infected hosts (Pauwels et al., 2011). This study, Cladocerans, and Daphnia in particular, have along with those examining the D. magna cellu- taught us much about the nature of immunity. lar response, provides a compelling example of Early work by Metchnikoff gave us insight to how immune responses can indicate cata- the importance of hemocytes in invertebrate strophic failures in other more important com- immunity, and more recent analyses of the ponents of the host’s immune system. Immune newly sequenced D. pulex genotype have failure is particularly disastrous for D. magna uncovered many putative immune genes. Much exposed to P. ramosa because the parasite steri- of what we understand about the ecology and lizes its host and thus renders it a genetic evolution of infectious disease also stems from dead-end. Immune activity and immunity are studies conducted in cladoceran-parasite sys- not always one and the same: a strong immune tems. In particular, they have helped us to elu- response can herald the onset of parasitic infec- cidate the mechanisms that maintain host (and tion and a rapid decline in host fitness. sometimes parasite) genetic diversity and drive host evolution and host-parasite coevolution. 17.6.2 Priming The challenge now is to bridge the gap between mechanistic immunology and whole- It has long been thought that immune speci- organism measures of infection traits—to exam- ficity (i.e. the ability to distinguish between ine variation in immune activity within and very similar infectious agents) and immune across cladoceran species and to link it to mea- memory (i.e. the ability of an immune system sures of parasitism. Which immune responses to react more rapidly and effectively to chal- are effective against which parasites? What are lenges it has previously encountered) are prop- the costs associated with maintaining or mobi- erties of the adaptive immune system only, lizing different immune responses? What is the and thus only possible in vertebrates (Janeway mechanism underlying transgenerational et al., 2001; Schmid-Hempel, 2011). However, it immunity? And finally, how do particular has now been shown that D. magna can immune functions shape coevolution between “remember” immune challenges from specific cladocerans and their various parasites.

PHYSIOLOGY OF THE CLADOCERA CHAPTER 18

The Genomics of Cladoceran Physiology Do¨rthe Becker1, Kay Van Damme1, Elizabeth Turner2, Joseph R. Shaw2, John K. Colbourne1 and Michael E. Pfrender3 1Environmental Genomics Group, School of Biosciences, University of Birmingham, Birmingham, UK, 2School of Public and Environmental Affairs, Indiana University, Bloomington, IN, USA, 3Department of Biological Sciences, Environmental Change Initiative, University of Notre Dame, Notre Dame, IN, USA

18.1 INTRODUCTION acclimation to long-term genetic alterations of adaptation. Linking organismal change across Understanding the tolerance of organisms these disparate time frames requires delving to environmental change, and the underlying deeply into the genetic mechanisms underly- mechanisms that define the thresholds of ing phenotypic plasticity. A plastic (flexible) physiological capacity (e.g. Hofmann and phenotype is the outcome of modification to Todgham, 2010) are major objectives for mod- the functional genome, for example by altering ern biological investigation. Against the back- transcription or translation (e.g. Whitehead, ground of a rapidly changing world, there is 2012). As Whitehead (2012) noted, “environ- an increasing research emphasis on linking mental change that emerges across generations organism-environment interactions to physio- can also be accommodated by plasticity, or logical responses and phenotypic plasticity. To alternatively may drive structural change of obtain a thorough understanding of the origin genomes by adaptive and demographic evolu- and maintenance of phenotypic variation of tionary processes that sort allele frequencies populations in the natural world (i.e. how within populations across generations.” environmental stimuli affect individual fitness Making connections between the functional and the evolutionary potential of populations), genome and the interaction with the environ- we need to decipher the molecular machinery ment that give rise to the phenotype is a chal- that regulates phenotypic responses to envi- lenging task. One central difficulty is that ronmental conditions. These responses span environmentally sensitive physiological traits the short-term physiological changes of are rarely controlled by single, or a few, genes

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00018-5 235 © 2014 Elsevier Inc. All rights reserved. 236 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY of major effect. They are often highly poly- Current genomic research seeks to address genic traits (West-Eberhard, 2005; Aubin- the following key questions in cladoceran Horth and Renn, 2009; Ayroles et al., 2009; physiology: Flint and Mackay, 2009), which dictates that 1. Are physiological responses reflected by entire regulatory networks need to be consid- responses in the functional genome? ered. Studying the genetic basis of cladoceran 2. What are the genomic elements or physiology provides an ideal context for regulatory programs that allow plastic understanding the generality of genomic responses (i.e. by which mechanisms is a mechanisms underlying physiological change in the environment sensed, responses. Cladocerans, with their pronounced integrated, and transformed into distinct phenotypic plasticity and particular mode of physiological responses)? reproduction, are useful organisms to study 3. How are physiological systems integrated, genome evolution (Colbourne et al., 2011). and how do genes interact to describe the Only through a better understanding of the regulatory networks underlying the phenotype (e.g. morphology and physiology) biological processes and pathways and the genomic underpinnings of phenotypic involved? variation can linkages be forged between genes and physiological functions. In this chapter, we explore the current Functional genomics allow a new approach knowledge on the genetic basis of cladoceran to explore the mechanisms that drive physio- physiology, drawing largely from our growing logical change and help to unravel key pro- understanding of Daphnia, which has been the cesses that enable organisms to cope in their most extensively studied genus within the natural environment (Aubin-Horth and Renn, group. 2009; Bell and Aubin-Horth, 2010). This area of investigation is embodied by the emerging field of ecological and evolutionary genomics 18.2 A LONG HISTORY (EEG), which seeks to link gene functions to OF RESEARCH: phenotypes and ecological factors (e.g. Renn THE PRE-GENOMICS ERA and Siemens, 2010; Pavey et al., 2012). Gene- environment interactions can be best studied The genus Daphnia has been the focus of in animals with well-known ecologies, on a biological and ecological research for over a physiological timescale as well as in an evolu- century, making it one of the oldest study sys- tionary context (Carroll et al., 2007; Hoffmann tems in ecology, physiology, and evolutionary and Willi, 2008; Pfrender, 2012; for a review biology among freshwater invertebrates. When see Hodgins-Davis and Townsend, 2009). it comes to genetics, it is the most well-known Cladocerans lend themselves to such research, cladoceran. Today’s in-depth knowledge of as their ecology is well studied (at least for the these organisms results from their prominent planktonic groups) and the genomic resources ecological role as keystone species in many required are being rapidly developed; “by freshwater ecosystems. It is worth noting that examining genome structure and the func- while the genetic investigation of physiological tional responses of genes to environmental traits in cladocerans has only recently been a conditions within species with traceable ecolo- major focus of investigation, the framework for gies, we further our understanding of gene- detailed inquiry is well in place. Extensive environment interactions in an evolutionary genetic investigation at the population level context” (Colbourne et al., 2011). and the formation of explicit phylogenetic

PHYSIOLOGY OF THE CLADOCERA 18.2 A LONG HISTORY OF RESEARCH: THE PRE-GENOMICS ERA 237 hypotheses for several groups of cladocerans Somewhat surprisingly, research that exam- form the backdrop for understanding the evo- ines phylogenetic relationships in Cladocera lution of physiological traits. In the pre- as a whole, or in the higher taxonomic level of genomics era of the past century, the genetic Anomopoda, is currently limited. Molecular architecture of natural populations of Daphnia phylogenies at these phylogenetic levels often was investigated through population genetic, fail to resolve relationships among the deeper quantitative genetic, and phylogenetic cladoceran branches (de Waard et al., 2006; approaches. Among the early studies were Stenderup et al., 2006; Van Damme et al., examinations of the characteristic alterations in 2007). The phylogenetic relationships within the phenotype in response to predator chemical many cladoceran families have not been well cues (Woltereck, 1909) and regulation of sexu- investigated, and for some groups the first ality and diapause (Banta and Brown, 1939). molecular phylogenetic studies emerged only With the advent of readily available molecular recently, (e.g. Bosminidae; Kotov et al., 2009; genetic techniques (i.e. protein gel electropho- Chydoridae; Sacherova´ and Hebert, 2003; resis) in the 1970s, genetic studies revealed Belyaeva and Taylor, 2009; Leptodoridae Xu populations and species with high levels of L. et al., 2011; Polyphemidae; Xu S. et al., genetic variation indicative of local adaptation. 2009). As a result, no other cladoceran genus In the following decades, research on cladoc- has been genetically characterized as well as eran genetics shifted with the techniques avail- Daphnia. The rapid development of a genetic able, from allozyme studies (1970À1990) to the characterization of Daphnia prompted Hebert amplification and the sequencing of nuclear (1987) to predict that “the future may well and mitochondrial genes (Hebert and Taylor, also see the broader use of cladocerans as 1997). Whereas allozyme analysis provided a model systems to examine problems of more useful overview of population structure, and general theoretical importance.” The extensive is still often used as a diagnostic tool to iden- understanding of phylogenetic diversity and tify naturally occurring hybrids (Hebert et al., ecology in Daphnia has made this genus the 1989; Cerny and Hebert, 1999), DNA sequenc- model group of cladocerans. Expansion of ing became the most widespread technique to research into other relatively unexplored cla- understand speciation and phylogeny in the doceran lineages will probably reveal a vast group. Cladoceran phylogenies have been vari- amount of ecological, genetic, and physiologi- ously constructed with mitochondrial genes cal variation. (12S, 16S, and COI) (e.g. Crease and Little, Parallel to the development of population 1997; Colbourne et al., 1998, Crease, 1999), genetic and phylogenetic studies using molecu- nuclear genes (18S, 28S, and EF-1) (e.g. Crease lar markers, quantitative genetic studies leverag- and Taylor, 1998), or combinations of both ing the clonal reproductive mode of Daphnia, (e.g. Adamowicz et al., 2009). Within cladoc- investigated the extent to which phenotypic var- eran families, or at the level of genera, DNA iation in life-history traits and fitness has a markers have proven to be very useful, as genetic basis. These studies provided links repeatedly shown for Daphnia in a genus-wide between phenotypic variation, mutation, and phylogenetic context (e.g. Lehman et al., 1995; heritable genetic variation (e.g. Lynch, 1985; Adamowicz et al., 2009), or in the exploration Spitze, 1993; Pfrender and Lynch, 2000). of the genetic diversity and biogeography of Building on this base of population- and quanti- selected species groups (Taylor et al., 1996; tative genetic studies, novel approaches linking Hebert et al., 2003; Kotov and Taylor, 2010; quantitative genetic variation with environmen- Crease et al., 2012). tal context were advanced in Daphnia.These

PHYSIOLOGY OF THE CLADOCERA 238 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY studies make a statistical association between system for ecological, ecotoxicological, and the patterns of population subdivision in quanti- evolutionary genomics (e.g. Colbourne et al., tative traits and molecular genetic markers. 2011; Lampert, 2011; Miner et al., 2012). With Excess levels of population subdivision in quan- the first draft of the D. pulex genome made titative traits and molecular genetic markers pro- available in 2006, a recent publication of the vide evidence of natural selection and local first annotated crustacean genome (Colbourne adaptation (Spitze, 1993; Lynch et al., 1999; et al., 2011) marks the beginning of a new and Morgan et al., 2001). This approach has recently promising research era, which seeks to bridge been extended to examine patterns of adaptation “the gap between genotype and phenotype” in through time by leveraging the historical legacy an ecologically relevant framework (Dow, genotypes in diapausing embryos trapped in 2007; Gregersen, 2009). But why is Daphnia a lake-bottom sediments (see Section 18.7) useful model? (Cousyn et al., 2001; Brendonck and De Meester, One of the most intriguing features in 2003; Orsini et al., 2012). Daphnia biology is its extraordinary ability to While these studies have been essential in cope with changes in the environment. In establishing the framework for the detailed response to environmental challenges, clado- investigation of physiology and physiological cerans can produce extremely divergent phe- mechanisms, they are best suited to describing notypes, which include altered morphologies patterns of genetic variation within and among (Tollrian and Dodson, 1999; Laforsch et al., species, and provide limited insight into 2004), changes in their physiology (Kobayashi mechanisms. Linking the extensive phenotypic and Yamagata, 2000; Paul et al., 2004) and variation in physiology and the wide range of behavior (Wiggins and Frappell, 2002; Zeis habitats occupied by cladocerans requires new et al., 2004; Zeis et al., 2005), and switches in approaches that leverage modern high- the reproductive mode from parthenogenetic throughput DNA sequencing technologies and to sexual reproduction cycles (Zaffagnini, draw on the rapidly expanding resource of 1987; Olmstead and LeBlanc, 2003; genome sequences, functional genomic assays Decaestecker et al., 2009). Modern genomic of gene regulation, and proteomic and metabo- approaches have transformed cladoceran lomics data. This post-genomic investigation research. Investigators working with this into the tremendous phenotypic and physio- group of organisms are poised to tackle one logical variation in cladocerans will provide of the main objectives in modern biology to insights into physiological mechanisms that identify the mechanistic basis of plastic were unattainable in the past. responses to environmental change. At present, researchers explore how the developmental and physiological “program” of an 18.3 DAPHNIA AS A MODEL organism can be modified in response to dis- SYSTEM FOR ECOLOGICAL AND tinct environmental stimuli. However, the lat- EVOLUTIONARY PHYSIOLOGY ter requires “first and foremost the understanding of its genomic make-up” The field of cladoceran genomics is in the (Aubin-Horth and Renn, 2009). Even though early stages of development, yet advances rap- numerous investigations in diverse taxa have idly. A decade ago, in 2002, the first genome reported the existence of environmentally project for a cladoceran species (i.e. Daphnia induced phenotypic plasticity, there has been pulex) was launched (see Section 18.4). This relatively little success in identifying the initiative aimed at creating a leading model empirical patterns of cellular control

PHYSIOLOGY OF THE CLADOCERA 18.3 DAPHNIA AS A MODEL SYSTEM FOR ECOLOGICAL AND EVOLUTIONARY PHYSIOLOGY 239 mechanisms that govern the plastic traits in 4. Their maintenance in the laboratory is question. This lack is mainly due a paucity of comparably cheap and easy. study organisms with well-developed geno- 5. Their large brood sizes and fast mic resources that are also empirical models reproductive cycle are ideal for for investigating phenotypic plasticity. experimental genetics (with the generation Currently, knowledge on genes, genomes and time in the laboratory rivaling that of their regulation is predominantly based on nearly all model eukaryotic systems). traditional model systems such as 6. The clonal nature of this organism Caenorhabditis elegans, Drosophila melanogaster, provides an outstanding opportunity to zebrafish, and mice. As all these species are study the genetic responses to of limited relevance in their natural habitats environmental stimuli in a defined and and ecosystems, and consequently lack a sig- constant genetic background with nificant context outside the laboratory, there unlimited replication. is limited data available on how environmen- 7. Daphnia are transparent throughout life, tal factors may contribute to genetic and phe- allowing studies of tissue-specific gene notypic evolution. Moreover, many genes that expression at any life stage (e.g. to changes respond to ecological conditions have in oxygen). unknown functions, and information from 8. The extensive phenotypic diversity of this laboratory model species may be insufficient. species provides ample raw material to Consequently, ecological genomic approaches study gene function and genome by require empirical annotations of new genome environment interactions. sequences from a broader range of species 9. Daphnia-specific genes have been shown to tested under a variety of natural and ecolog- respond rapidly to environmental ically relevant conditions (see Section 18.6). disturbances. As a model for ecological and evolutionary 10. Research is supported by a large, and research (e.g. Lynch and Spitze, 1994; Lampert, expanding, community of scientists, i.e. the 2011; Miner et al., 2012), Daphnia now bridges Daphnia Genomics Consortium. this gap, as it allows us to increase our under- At present, our understanding of the cellu- standing of the linkages between phenotypic lar processes by which plastic responses to responses and underlying genotypic and envi- environmental stimuli are triggered is still ronmental effects. There are several compelling very limited (Aubin-Horth and Renn, 2009). reasons why Daphnia is particularly well suited Genome-wide association studies that aim at to this research agenda: linking genes to phenotypes strongly suggest 1. A complete genome sequence of one that complex traits are governed by many loci species is now available. (Flint and Mackay, 2009). In Daphnia,mostof 2. The data resources are publicly accessible the alternative phenotypes can be generated (including genomic and cDNA libraries, from the same genetic background through the microarrays, tiling arrays, genetic linkage perception of cues from the environment. maps, web-based bioinformatics portals, These cues lead to modifications of gene regu- and annotation and gene expression lation that alter the physiological state and databases). developmental trajectory of individuals (e.g. 3. The phylogenetic position of Daphnia (i.e. the formation of males). The clonal character branchiopods, which are basal crustaceans) of daphniids facilitates dissection of the genetic is excellent for comparative genomics. and environmental components of plastic

PHYSIOLOGY OF THE CLADOCERA 240 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY responses (Shaw et al., 2008, Simon et al., evolutionary adaptation processes in response 2012). The remarkable ability of these organ- to the environment. Whereas earlier investiga- isms to cope with environmental change is tions were often constrained to hunting for associated in part with its unique gene inven- single genes associated with particular pheno- tory and genomic structure (see Section 18.4). types, employing functional genomic method- To decipher the relationship between phe- ologies at genome-wide scales offers a more notypic plasticity, and adaptive evolution, it is comprehensive strategy. These tools will facili- important to understand the genetic basis of tate a genuine understanding of how the envi- interactions between genotype and environ- ronment interacts with (and shapes) the ment (Miner et al., 2005; Pigliucci, 2005; West- genome and how genome regulation and vari- Eberhard, 2005). The regulation, expression, ation are linked to phenotypic plasticity and and function of many genes are highly context phenotypic evolution. dependent and are only manifested under particular environmental conditions. It is pres- ently unclear to what extent such genetic 18.4 DAPHNIA’S ECORESPONSIVE mechanisms are species-specific. Ecological GENOME genomics aim to understand how natural populations adapt to their local environments. Modern physiological investigations will Daphniids are known to have colonized radi- increasingly draw on a genomic perspective to cally different environments on multiple occa- discover underlying mechanisms and to under- sions, with a characteristic pattern of stand the patterns of variation in natural popu- convergence of adaptive traits linked to spe- lations occupying diverse environments and cific habitats (e.g. Colbourne et al., 1997). confronted with varied physiological chal- Consequently, the genetic variation found lenges. Developing a genomic perspective within natural populations of Daphnia pro- requires a high level of genomic infrastructure, vides an exceptional opportunity to evaluate including genome sequences and functional the nature of genomic and phenotypic adapta- genomic tools. Of all Cladocera (in fact, of all tions (e.g. De Meester et al., 2011). Comparing crustaceans), the widespread D. pulex (e.g. different Daphnia lineages that show habitat- Crease et al., 2012) is the best-developed spe- specific reaction norms allows us to decipher cies in this area. It is the first cladoceran species whether similar environments can and do to have the complete genome sequenced and induce similar genetic outcomes, and whether annotated (Colbourne et al., 2011). A second different Daphnia lineages evolve the same species, D. magna, already a well-studied way to meet these environmental challenges ecotoxicological model, is currently undergoing (see e.g. Colbourne et al., 1997; Pfrender et al., similar development. What makes the first cla- 2000; Scoville and Pfrender, 2010). doceran genome so special? Even though the The recent publication of the Daphnia D. pulex genome is small compared to that of genome (see Section 18.4), coupled with a other organisms, measuring only circa 200 Mb, wide range of phenotypic diversity, make this it contains almost twice as many genes as any organism an outstanding model system to other arthropod yet sequenced, and has a explore the genetic basis of complex pheno- greater number of genes than in most other typic traits, including physiology (e.g. Miner eukaryotic genomes, including the human et al., 2012). It allows unprecedented insight genome. So far, 30,907 protein-coding genes are into the responses to environmental perturba- predicted on the 12 chromosomes. Sequencing tions by tackling physiological acclimation and and assembling the waterflea’s genome

PHYSIOLOGY OF THE CLADOCERA 18.4 DAPHNIA’S ECORESPONSIVE GENOME 241 revealed a significant number of non-protein- genome. As Colbourne et al. (2011) showed, coding genes including microRNAs, ribosomal the elevated number of genes in the D. pulex RNAs, transfer RNAs, and several families of genome results not only from acquiring but transposable elements. For a detailed discus- also from retaining a large number of genes. sion on the D. pulex genome and its potential as The retention of duplicate genes and the neo- a genetic model, we refer to Colbourne et al. functionalization of these paralogues may be (2011), on which the current section is based directly linked with the plasticity of cladocer- (unless stated otherwise). In addition to the lat- ans and their ability to respond to a large ter work, the authors selected 50 thematic pub- range of environmental challenges (Colbourne lications (the “companion papers,” by the et al., 2011; Simon et al., 2012). Daphnia Genomics Consortium, published As gene duplication events are considered between 2005 and 2011), which constitute the to play a key role in shaping genomes, they initial exploration of the genome (see http:// represent one of the most significant sources of www.biomedcentral.com/series/Daphnia). The evolutionary novelty. If, after duplication, genome itself can be explored through the pub- selection maintains both copies of a gene, then licly available, interactive genome database, one gene version commonly retains its original wFleaBase (Colbourne et al., 2005), which also function while the other may functionally includes data on the expression patterns of a diverge; the latter is called a paralogue (for large number of protein-coding genes under reviews see e.g. Innan and Kondrashov, 2010; different experimental conditions. Proulx, 2012). Most of Daphnia’s paralogues Comparisons of the D. pulex genome with have evolved context-dependent functional that of several insects revealed that intron sizes specifications and the expression patterns appear to be rather reduced in the Daphnia diverge with the age of the gene. Whereas genome, with an average intron length of only more recent duplicates, which have different 170 bp. Moreover, the intron density in the cla- gene sequences, showed rather indistinguish- doceran seems similar to that observed in Apis able gene expression patterns under several mellifera, but there are at least twice as many tested environmental conditions, more ancient introns per gene than in Drosophila, for exam- duplicates revealed divergent expression pat- ple. As outlined by Colbourne et al. (2011), the terns over time. These duplication and diver- majority (i.e. 78%) of the intron gains observed gence events in various gene families represent in the D. pulex genome appear to be unique to an impressive evolutionary mechanism that the Daphnia lineage (or perhaps to the bran- enables these animals to express a selective chiopods). That is to say, that the Daphnia suite of functionally distinct molecules genome is more compact with respect to the depending on the environmental condition. size of its introns, but not in the number of The genome can thus mediate diverse cellular introns acquired. processes to maintain or restore cellular integ- Exploring the Daphnia gene inventory rity in response to the environmental chal- revealed a higher propensity for gene duplica- lenges faced. However, the D. pulex genome tion and retention when compared to other was also shown to contain a number of recent organisms. Gene duplication events account duplicates that have nearly identical nucleo- for approximately half of the genes in the tide sequences but show rather differential genome. In fact, the rate of gene duplication in gene expression patterns in response to differ- D. pulex seems three times as high as that ent environmental parameters. These expres- observed for Drosophila and Caenorhabditis, and sion patterns may arise by integrating novel about 30% greater than that of the human genes into a new genomic location

PHYSIOLOGY OF THE CLADOCERA 242 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY

(chromosome) or dissociating them from their (2011) could show the coexpansion of genes in previous regulatory framework (e.g. distinct metabolic pathways (e.g. androgen- pathways). estrogen metabolism and sphingolipid biosyn- Due to their high degree of homology, thesis metabolism), which indicates that the duplicated genes (and especially tandemly duplicated genes may be interdependent. In clustered genes) are prone to undergo genetic fact, half of the expanded metabolic genes exchanges via unequal crossing over and uni- were found to belong to a subset of seven dis- directional gene conversion (GC) (Hoffmann tinct pathways, and the expression patterns of et al., 2008). In the Daphnia genome, 47% of all these genes codiverged according to their genes were revealed to contain tracts of GC; in pathway and not according to their evolution- comparison, in five different Drosophila species, ary history, which suggests the functional only 12À18% of genes were shown to be divergence of expanding genes within affected by such genetic homogenization pathways. events. However, GC events were shown to be Finally, a large number of genes (36%) in the less common among more recent duplicates D. pulex genome lack any detectable homology and within gene families containing only few with any other species yet sequenced. paralogues. An exceptional example of wide- Phylogenetic analyses that account for the spread GC events in the Daphnia genome com- expansion and contraction of all gene families prises the di-domain hemoglobin (Hb) gene within pancrustacean and several deutero- family (see Section 18.5). stome genomes suggest a net increase in the Even though the high number of genes in number of paralogues within the lineage lead- the Daphnia genome may indicate that these ing to Daphnia. That is to say, the vast number genes arose by whole-genome duplication, the of lineage-specific genes appears to result from number of synonymous substitutions among disproportionate expansions of particular gene all pairs of duplicated genes in D. pulex rather families distinctive to this crustacean lineage suggests high and steady rates of tandem and the fast divergence of most of these genes. duplication events in the lineage’s evolution- Using whole-genome tiling arrays or EST (i.e. ary history. Similar to other genomes in which expressed sequence tag) sequencing, it was new duplicates are found in clusters, the shown that Daphnia-specific genes in particular D. pulex genome has approximately 20% of its (as far as it is known) appear to play important genes in tightly arranged groupings. Each clus- roles in the organism’s ecology. These “orphan ter can contain up to 80 paralogues. Two gene genes” may particularly account for the plastic families that have gained and retained an responses of daphniids, as they were shown to exceptional number of duplicated genes in have distinct gene expression patterns that are D. pulex include the photoresponsive genes of context specific (and may thus only be the opsin family (Rivera et al., 2010) and di- expressed under particular ecological condi- domain Hbs (Colbourne et al., 2011), which tions). Similarly, another set of D. pulex tran- play critical roles in coping with complex light scripts showed condition-dependent gene regimes or allowing adequate responses to expression patterns in response to diverse envi- oxygen deprivation in aquatic environments, ronmental challenges. These transcriptionally respectively. active regions (TARs), which have The expansion of gene families in D. pulex predictable exon-intron intervals, are not yet was shown to be also associated with distinct assigned to any known gene annotation mod- metabolic processes. By analyzing the func- els, and thus need to be functionally and struc- tional role of paralogues, Colbourne et al. turally characterized.

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18.5 THE GENETIC BASIS OF and mechanistic bases of physiological accli- PHYSIOLOGICAL PLASTICITY: mation and evolutionary adaptation processes A CASE STUDY are gained by investigating the structural variation and regulation of this protein in 18.5.1 Daphnia’s Hemoglobin Genes Cladocera. Numerous investigations on the functional Hb is found in virtually all kingdoms of liv- processes involved in oxygen regulation by Hb ing organisms and is arguably among the best- have been carried out in Daphnia, with initial studied proteins tied to natural conditions studies dating back to the late 1940s and early (Weber and Vinogradov, 2001). First character- 1950s (e.g. Fox et al., 1949; Fox, 1950; Green, ized by Hu¨ nefeld in 1840, its central role in 1956b). These aquatic poikilotherms are maintaining cellular oxygen homeostasis has exposed to a wide range of environmental con- garnered markedly detailed molecular biologi- ditions (Lampert, 2004; Lampert and Sommer, cal investigations since the late 1990s, includ- 2007; Brede et al., 2009; Weider et al., 2010; ing the comparative study of DNA and protein Paul et al., 2012); both diurnal and seasonal sequence, and of the molecular assemblage fluctuations in oxygen and temperature are and functional properties of this respiratory among the major abiotic challenges that protein across diverse lineages of animals (for Daphnia face. Their diurnal vertical migration reviews see Weber and Vinogradov, 2001; (DVM) between the epilimnion and hypolim- Hardison, 2012; Storz et al., 2013). Although nion in deeper lakes demands a high plasticity Hbs are remarkably diverse in structure and in their molecular responses to cope with dif- function, their sequence similarity and con- ferent oxygen conditions in nature within short served gene structures indicate that all globins time intervals. Both environmental hypoxia described to date are descended from a shared and temperature-induced mismatches between ancestral gene (Goodman et al., 1987; oxygen supply and demand provoke cellular Goodman et al., 1988; Hardison, 1998; oxygen deficiency in aquatic organisms (e.g. Vinogradov et al., 2005). Expansion and diver- Ekau et al., 2010; Po¨rtner, 2010). Daphniids in sification of the Hb gene family, both within particular, but branchiopods in general, show and across species lineages, is frequently an extraordinary plasticity to cope with such shown to correlate with the physiological oxy- environmental conditions; they have evolved gen demands that organisms face in their natu- various regulatory mechanisms that perceive ral habitats (Weber et al., 2002; Storz et al., change and compensate for substantial varia- 2007; Storz and Moriyama, 2008; Storz et al., tion in ambient conditions (Guadagnoli et al., 2010; Tufts et al., 2013). Because an animal’s 2005). So far, the mechanisms by which daph- ability to cope with environmental change is niids sense changes in the amount of oxygen in predominantly determined by its capacity to the ambient environment are not entirely maintain performance and oxidative metabo- understood. However, some studies (e.g. Gorr lism (e.g. Po¨rtner and Knust, 2007), variations et al., 2004; Gorr et al., 2006b) have revealed in the protein structure and differential expres- that cellular responses to environmental chal- sion of distinct hb genes consequently repre- lenges in Daphnia are mediated via distinct reg- sent central mechanisms that enable proper ulatory elements that bind to intergenic regions system functioning and survival. Due to the and control the gene expression level of physi- extensive background knowledge on the phys- ologically important proteins (see below). iological role of Hb and its structure-function In response to short-term changes in the relationships, critical insights into the genetic ambient conditions, waterfleas can counteract

PHYSIOLOGY OF THE CLADOCERA 244 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY oxygen deprivation via alterations in their ven- may ultimately increase the animals’ overall tilation and perfusion rates, which restore ade- fitness (Fox et al., 1951; Sell, 1998; Wiggins and quate oxygen supply to peripheral tissues and Frappell, 2002; Duffy, 2010). cells (Lamkemeyer et al., 2003; Paul et al., Several investigations on the Hb protein 2004; Pirow and Buchen, 2004). If oxygen level have thus far revealed that changes in the shortage persists for a prolonged period of oxygen-binding affinity of the multimeric Hb time (i.e. a few hours) (see e.g. Zeis et al., 2004; molecule are a direct consequence of modifica- Becker et al., 2011a), then the microcrusta- tions in the Hb subunit composition cean’s oxygen transport capacity is improved (Kobayashi and Takahashi, 1994; Kimura et al., by the induction of Hb and even a modifica- 1999; Lamkemeyer et al., 2003; Zeis et al., 2003; tion of the Hb structure (Fox et al., 1951; Gorr et al., 2004; Gerke et al., 2011). Compared Kobayashi and Hoshi, 1982; Tokishita et al., to vertebrates, invertebrate Hbs are known to 1997; Pirow et al., 2001; Zeis et al., 2003; Gorr exhibit a much broader variability in their et al., 2004; Pirow et al., 2004; Gerke et al., molecular structures (Weber and Vinogradov, 2011; Zeis et al., 2013). In response to severe 2001), presumably indicating adaptations to hypoxia (approximately 2À3 kPa) (e.g. Zeis the wider ranges of environmental conditions et al., 2003; Gerke et al., 2011), Daphnia Hb to which these animals are subjected in nature. levels drastically rise by a factor of 15À20, thus Studies on the molecular structure of Hb in notably coloring adult animals red within a daphniids provide evidence that the extracel- single molting cycle (Kobayashi and Hoshi, lular protein is composed of multiple 1982; Pirow et al., 2001; Zeis et al., 2003; Gerke 31À37 kDa di-domain subunits (Dangott and et al., 2011). Other branchiopods, such as Terwilliger, 1980; Ilan et al., 1982; Peeters et al., Triops, are known to show a similar response 1990; Kimura et al., 1999; Lamkemeyer et al., to hypoxia by modifying their Hb structure or 2003; Zeis et al., 2003; Gerke et al., 2011). As elevating hb gene expression (Guadagnoli previously demonstrated (Tokishita et al., et al., 2005). Earlier investigations on the de novo 1997), there is remarkably low similarity synthesis of the respiratory protein in D. magna between the amino acid sequences of the first provided evidence of highly controlled and and second Hb domains (approximately 24%). localized gene expression, with Hb synthesis This suggests that Daphnia’s di-domain Hb sites restricted to the fat cells and the epipodite subunits derive from an ancient duplication epithelia cells (Goldmann et al., 1999). via unequal crossing over of two single- Oxygen acquisition in the hemolymph of domain globin genes, which has been fre- Daphnia is not only adjusted by changes in the quently shown for members of the Hb gene Hb concentration but also arises from an family (see Weber and Vinogradov, 2001). altered oxygen-binding affinity of the respira- Each subunit consists of two heme-containing tory protein. Expression of higher-affinity Hbs globin domains (Dangott and Terwilliger, in animals facing environmental change is 1980) and has a characteristic structure of a assumed to reduce Hb synthesis costs due to pre-A segment, eight alpha helices and five an enhanced oxygen transport efficiency of interhelical regions (Tokishita et al., 1997; Kato these molecules in comparison to low-affinity et al., 2001). So far, 12 (D. pulex)to16(D. mag- variants (Kobayashi et al., 1994; Pirow et al., na) subunits are known to aggregate, forming 2001). In the long term, adjustments in the cel- functional macromolecular multimers (Dangott lular Hb repertoire allow daphniids to inhabit and Terwilliger, 1980; Peeters et al., 1990; oxygen-poor water layers, thereby providing Lamkemeyer et al., 2006) that are freely dis- access to their respective food resources, which solved in the hemolymph (Pirow et al., 2001).

PHYSIOLOGY OF THE CLADOCERA 18.5 THE GENETIC BASIS OF PHYSIOLOGICAL PLASTICITY: A CASE STUDY 245

Recent proteomic analyses of D. pulex that prolyl hydroxylase homologue, EGL-9, plays were acclimated at different temperature and an important role in regulation of stability of oxygen conditions revealed the differential the HIF-1α homologue in response to expression of a set of seven different Hb subu- hypoxia (Epstein et al., 2001). A proline resi- nits, each encoded by a separate gene locus due within the conserved LXXLAP motif (Gerke et al., 2011). Similar results were within the oxygen-dependent degradation obtained from studies on D. magna (Zeis et al., domain (ODDD) of HIF-1α is hydroxylated by 2003; Lamkemeyer et al., 2006). As demon- HIF-PHD in response to increased oxygen con- strated by the Gerke et al. (2011), oxygen and centration in mammals. Hydroxylation of this temperature acclimation mainly caused similar proline residue results in degradation of changes in the Hb subunit composition. HIF-1α mediated by the ubiquitin-proteasome However, further investigations under differ- pathway (Jaakkola et al., 2001). HIF-1α and its ent experimental or methodical conditions are oxygen-dependent regulation are conserved in likely to facilitate the identification of further various organisms such as nematodes, insects, Hb subunits, which may be only expressed in fishes, amphibians, and mammals. HIF-1α a condition-specific mode. recognizes cis-acting elements called HREs in Whereas the presence of multiple genes the regulatory region of target genes; the T/G/ encoding the different Hb subunits provides CACGTG hexanucleotide is a core sequence of the basis for alterations in the protein’s struc- HREs (Wenger et al., 2005). As to crustaceans, tural and functional characteristics, the differ- cDNAs encoding HIF-1α homologues were ential gene expression of specific sets of Hb isolated more recently from grass shrimp isoforms is regulated via the binding of dis- Palaemonetes pugio, Dungeness crab Cancer mag- tinct transcription factors to the promoter ister, and D. magna (Li and Brouwer, 2007; regions (Kimura et al., 1999) of the respective Tokishita et al., 2006). These HREs are also hb genes. In response to hypoxia, binding of found upstream of the D. pulex hb genes the transcription factor HIF (hypoxia-inducible (Colbourne et al., 2011; Gerke et al., 2011), with factor; a protein) to hypoxia-response elements analogue roles for differential isoform expres- (HREs) in the upstream regulatory (promoter) sion in response to disturbed oxygen homeo- regions of genes was shown to have a direct stasis. By employing heterologous transfection impact on the induction of multiple Hb iso- studies, Gorr et al. (2004) provided evidence forms in D. magna (Gorr et al., 2004). HIF-1 is a that the extent of hypoxia-induced differential heterodimer comprised of two bHLH-PAS fac- gene expression is not only governed by the tors, named HIF-1α and ARNT (aryl hydrocar- number of HREs in the intergenic regions bon nuclear translocator), which are members (Kimura et al., 1999; Nunes et al., 2005) but of the family of basic helix loop helix-Per/ may also involve interfering effects of other ARNT/Sim (bHLH-PAS) transcription factors regulating elements. Thus, the role of HIF (Wang et al., 1995). Stability of HIF-1α is con- binding, the position of binding sites, and pos- trolled by ambient oxygen conditions, while sible interactions with other transcription fac- ARNT is a partner of various other bHLH-PAS tors still need to be addressed. proteins and is stable regardless of ambient It is therefore clear that hb gene expression oxygen conditions (Salceda and Caro, 1997). is a complex mechanism that includes numer- Stability of HIF-1α is regulated by a mem- ous regulatory components and processes, ber of 2-oxoglutarate-dependent dioxygenase which contribute to a highly fine-tuned super family, named HIF prolyl hydroxylase cellular response. Besides HIF-1-induced Hb (HIF-PHD). It was reported that the C. elegans expression, further transcriptional control

PHYSIOLOGY OF THE CLADOCERA 246 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY elements were shown to regulate the Hb con- expression profiles of all di-domain hb genes centration in daphniids. Under normoxic con- revealed a significant age-related trend in the ditions, Gorr et al. (2006) revealed Hb levels in functional divergence among the paralogues D. magna to be predominantly under the regu- recent duplicates, which are most similar in latory control of methyl farnesoate or related their gene sequences, generally showed indis- juvenoid hormones, which bind to their tinguishable gene expression patterns for the respective response elements [i.e. juvenoid tested conditions, while more ancient dupli- response elements (JREs)]. However, a recent cates within Daphnia’s hb gene family were study (Zeis et al., 2013) using D. pulex discov- more divergent in their expression patterns ered that Hb transcript and protein levels only (Colbourne et al., 2011, Fig. S29). Consequently, poorly correlate, suggesting that Daphnia’s Hbs duplication followed by functional divergence are also posttranscriptionally regulated. By in the hb gene family of Daphnia represents an exploiting the exceptional knowledge available impressive evolutionary mechanism, which on Daphnia’s Hbs, further studies on this mul- enables these animals to express a selective tigene family in particular, will allow the iden- suite of functionally distinct Hb isoforms. tification of major regulatory networks linked These isoforms mediate adequate oxygen trans- to plastic responses. port capacities to maintain or restore cellular The remarkable flexibility of Daphnia at integrity in response to the environmental con- responding to altered oxygen availabilities and ditions faced (Tokishita et al., 1997; Zeis et al., demands, by adjusting both the quantity and 2003; Gerke et al., 2011). the quality of Hb, is assumed to be a direct Although many duplicates tend to diverge consequence of their exceptional genomic char- in sequence and function over time, paralo- acteristics. Although a number of investiga- gous genes arranged within tandemly dupli- tions in the late 1990s (Tokishita et al., 1997; cated gene clusters are predisposed to Hebert et al., 1999; Kimura et al., 1999) identi- homogenization events by unequal crossing fied multiple hb genes encoding the different over and unidirectional GC due to their high Hb subunits in several species of Daphnia (i.e. degree of sequence homology (Hoffmann in D. magna and D. exilis), our understanding et al., 2008). In the genus Daphnia, GC events has been significantly improved by genome are a common feature, and one example of sequencing of D. pulex and D. magna widespread GC is found in the di-domain hb (Colbourne et al., 2011). The draft D. pulex genes (Hebert et al., 1999; Sutton and Hebert, genome sequence assembly and annotation 2002; Colbourne et al., 2011). By shuffling revealed 11 di-domain hb genes (hb1Àhb11); 8 nucleotide variation among related genes, of these (hb1Àhb8) are found arranged within a these genetic exchanges result in a substantial tandem gene cluster on chromosome 7 reduction in divergence among duplicates as (Colbourne et al., 2011). Gene duplication their sequence evolution becomes concerted. events are considered important for shaping Multigene families that undergo such a con- genomes and are significant sources of evolu- certed evolution are characterized by a higher tionary novelty (for reviews, see Innan and than expected sequence similarity among para- Kondrashov, 2010; Proulx, 2012). Daphnia’s logous genes within a species, while there is di-domain hb genes thus provide a vivid illus- distinct divergence from the orthologous gene tration of how paralogues can evolve context- family in other species. dependent functional specifications. Exposing Cladocerans are an attractive target for com- D. pulex to eight different environmental con- parative studies of Hb evolution (Hebert et al., ditions and investigating the respective gene 1999). Several investigations have explored the

PHYSIOLOGY OF THE CLADOCERA 18.5 THE GENETIC BASIS OF PHYSIOLOGICAL PLASTICITY: A CASE STUDY 247 origin and evolution of duplicated di-domain populations are scarce (but, see Schwerin et al., hb genes and the consequences of their struc- 2010). However, different clones and popula- tural arrangements in different genera of the tions of Daphnia have been shown to vary in Daphniidae (Dewilde et al., 1999; Hebert et al., their ability to synthesize adequate levels of 1999; Kimura et al., 1999; Sutton and Hebert, Hb in response to distinct oxygen concentra- 2002; Colbourne et al., 2011), yet virtually tions in the ambient environment (Carvalho, nothing is known from other cladoceran fami- 1984; Weider and Lampert, 1985; Duffy, 2010). lies. Comparative analyses of the hb gene clus- Since spatial and temporal changes in dis- ters in two Daphnia lineages, D. pulex and solved oxygen concentration may be an impor- D. magna, revealed nearly identical genomic tant selective force influencing the clonal arrangements within a 23.5-kb interval (genotypic) composition of natural popula- (Colbourne et al., 2011). However, D. pulex tions, investigating Hb variations and tracing possesses an extra hb gene (i.e. hb6) within the the variation of individual globin genes within tandem gene cluster, and three additional, and among species allows the exploration of non-clustered hb genes (i.e. hb9Àhb11) are regulatory mechanisms that enable animals to found in other genomic regions, with hb9 being adapt to different environmental conditions located on the antisense strand (Gerke et al., (see e.g. Storz et al., 2007; Storz et al., 2009). As 2011). All clustered hb genes, plus hb9 in this case study illustrates, organisms can sur- D. pulex, are composed of seven exons that are vive and function under a wide range of con- separated by six introns. In contrast, hb10 and ditions in nature owing to a distinct set of hb11 in D. pulex consist of six exons, with the adaptive responses. The remarkable plasticity, second intron deleted from the ancestral gene characterized by differential Hb subunit structure (Colbourne et al., 2011). Similar expression in response to altered oxygen con- intron losses in di-domain hb genes have also ditions, is not only found in cladocerans but been reported (Kato et al., 2001) in another also occurs in other branchiopods such as cladoceran species, Moina macrocopa.Other Triops (Notostraca) (e.g. Guadagnoli et al., than the obvious absence of hb6 from the 2005). As these authors show, Hb subunit D. magna, cluster, elements in synteny expression is not reversed in Triops when con- between the two species are seemingly pre- ditions return to normoxia, which contrasts served from a duplication history that pre- with hitherto studied cladocerans. In combina- dates the split between the Ctenodaphnia and tion with the differences in Hb domains of the Daphnia subgenera (Colbourne et al., 2011), two Daphnia subgenera mentioned above, this including a non-coding RNA gene that inter- shows that genetic expression mechanisms rupts the cluster between hb4 and hb5. may differ significantly within the Although the hb gene clusters in both species Branchiopoda, and even within the Cladocera, are homologues due to ancestral gene dupli- depending on the lineage. cation events, phylogenetic reconstructions of The structural and regulatory polymorph- all di-domain hb genes in D. pulex and D. mag- isms of cladoceran hbs represent a superb na provided evidence that GC tracts have model for studying oxygen-responsive genes heavily homogenized the protein-coding and the mechanisms involved in their regula- regions, whereas the intergenic regions of all tion. Hence, they highlight the relationship hbs show less divergence between the two between environmental stimuli and down- Daphnia lineages (Colbourne et al., 2011). stream functional molecular responses. The In contrast to laboratory studies, investiga- detailed knowledge available on Hb structure- tions into Daphnia’s Hb function in natural function relationships and its key role in

PHYSIOLOGY OF THE CLADOCERA 248 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY oxygen homeostasis consequently promote a the discovery of previously unknown compo- deep understanding of the cellular mechanisms nents important to physiological functioning. that control a divergent and fine-tuned expres- Hierarchical cluster analysis of genome-wide sion of context-dependent suites of molecules expression patterns reveals the coordination of under environmental constraints, and ulti- gene function within pathways and provides mately provides genuine insight into the nature insight into the interaction of pathways of acclimation and adaptation processes. through regulatory networks. A network perspective makes it possible to infer the functional roles of unannotated genes if they 18.6 HUNTING FOR share expression profiles with annotated PHYSIOLOGICALLY RELEVANT genes. Principal component analysis of gene GENES AND REGULATORY expression data can also reveal underlying NETWORKS similarities and differences in physiological responses to different kinds of perturbations 18.6.1 Linking Gene Expression Profiles (Gibson and Muse 2009). Global gene expres- to Physiological Traits and Responses sion studies are hypothesis generating, identifying genes that can most usefully be subjected Genome-wide expression profiling, also to the genetic and biochemical analysis neces- known as functional genomics or transcriptomics, sary to pinpoint the molecular basis of physio- is a powerful tool for understanding how logical functioning. Transcriptomics data can organisms develop, function, and respond to be integrated with other genome-wide data environmental factors. Although posttranscrip- sets, such as those generated by comparative tional processing of mRNA and downstream phylogenomics, proteomics, epigenomics, and regulation of protein degradation and transla- metabolomics, for a systems biology approach tion can modify the effects of gene expression that works to understand holistically how on organism physiology and cell function, (1) organisms develop, function, and respond gene expression profiling still provides impor- to their environments and (2) the basis of tant information about the genetic control of physiological and phenotypic diversity within biological processes and how variation in gene and between lineages. These datasets can also expression affects phenotypic variation among yield insights into the emergent properties of individuals and among taxa (Gibson and gene regulatory networks, thus furthering our Muse, 2009). understanding of the sorts of gene regulatory In the absence of global gene expression network architectures that exist and how dif- studies, the expression of candidate genes ferent expression patterns have evolved known or suspected to be involved in the bio- (Scoville and Pfrender, 2010; Latta et al., 2012; logical process of interest can be assayed by Whitehead, 2012). northern blot or quantitative polymerase chain Currently, work on cladoceran genome- reaction (qPCR) methods, allowing targeted wide gene regulation utilizes microarray plat- questions about gene function under different forms developed for D. pulex or D. magna, environmental conditions, mutational pertur- particularly cDNA or long oligonucleotide bations, or across different taxa (e.g. Scoville microarrays that permit competitive hybridiza- and Pfrender, 2010; Schwarzenberger et al., tion of fluorescently labeled, amplified cDNA 2012). Assaying genome-wide patterns of gene to a set of probes representing expressed genes, expression frees researchers from the require- predicted genes, and other transcriptionally ment of a priori genetic knowledge, and allows active genome features. Hybridization of two

PHYSIOLOGY OF THE CLADOCERA 18.6 HUNTING FOR PHYSIOLOGICALLY RELEVANT GENES AND REGULATORY NETWORKS 249 samples per array (e.g. control vs. treatment, Finally these data must be further analyzed adult vs. neonate) allows a direct comparison for biological interpretation and visualization of of gene expression; many experimental designs significant genes within pathways and regula- also permit indirect comparisons of samples tory networks. Functional analysis is based on across arrays, e.g. comparisons among different homology to annotated genes in databases such treatments that have each been directly com- as the Gene Ontology (GO) database, which pared to a common reference. As sequencing assigns each gene to an inferred biological pro- costs decrease and better bioinformatics tools cess, molecular function, and cellular compo- are developed, inferring gene expression nent. Other databases infer membership in gene from direct sequencing of amplified cDNA pathways [e.g. the Kyoto Encyclopedia of Genes fragments via next-generation sequencing and Genomes (KEGG)] (reviewed in Tipney and (NGS) platforms, or RNA-Seq, will become Hunter, 2010). It is worth noting that about 36% more attractive and has the potential to over- of Daphnia genes show no homology to other come some of the limitations of oligonucleotide sequenced organisms (Colbourne et al., 2011); microarray platforms, such as incomplete these lineage-specific genes are therefore probe sets and a limited ability to capture dif- excluded from homology-based analyzes. After ferential transcription of splice variants and the categorization of significant genes, a variety allele-specific transcription. of methods exist to test whether sets of signifi- An early and crucially important step in cant genes are enriched for GO categories (e.g. genome-wide expression studies is developing Blast2GO), KEGG pathways [Database for the experimental design that will best answer Annotation, Visualization and Integrated the research question with the limited Discovery (DAVID)], or previously identified resources available: which genotypes (e.g. spe- gene lists [e.g. Gene Set Enrichment Analysis cies, populations, mutants, or wild-type indivi- (GSEA)]. These higher order analyses allow the duals); life stages (neonate, juvenile, or biologist to make sense of and visualize the mil- reproductive adult); and environments (e.g. lions of data points that comprise a microarray chemical exposure, chemical concentration, experiment (Tipney and Hunter, 2010). and the duration of exposure, as well as basic culture decisions about light, temperature, and feeding regimes); and whether to collect time 18.6.2 Case Studies in Environmental series data as animals undergo physiological Functional Genomics changes or to assay organisms in physiological equilibrium. Finally, decisions about replica- The study of genome-wide responses to tion (technical vs. biological), and, if microar- environmental conditions is a young but rap- ray platforms are used, which samples will be idly growing field and many of the early appli- directly compared and which will be compared cations involve exposure to environmental indirectly, have important implications on the stressors and toxicants. Environmental biolo- power to detect differential gene expression gists are exploring ways to incorporate the (Gibson and Muse, 2009). After exposures, sac- results of genome-wide expression studies in rifice of animals, RNA extraction, preparation biomonitoring efforts, seeking to identify gene of labeled cDNA, microarray hybridization, expression patterns specific to conditions, and and collection of fluorescence intensity data, using functional genomics to illuminate the the data must be analyzed with appropriate physiological mechanisms underlying these statistical methods to identify those genes with responses so that better estimates of individual differential expression across comparisons. effects and better predictions about the

PHYSIOLOGY OF THE CLADOCERA 250 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY population and community impacts of expo- that also compared the sensitivity of gene sures can be made. Environmental stressors expression profiling to the commonly used often do not affect just a single gene or gene comet assay for DNA damage. D. magna neo- pathway, but can instead have cascading nates and 7-day-old adults were exposed to a effects, ranging from a highly specific response mixture of benzo(a)pyrene and sodium dichro- (e.g. to lowered oxygen concentrations) to gen- mate. Gene expression was found to be a more eral stress responses (e.g. to altered food qual- sensitive indicator of genotoxicant effect than ity and quantity), while simultaneously the comet assay even though the comet assay involving many aspects of organismal func- was negative, significant differential gene tioning. Therefore, such stressor exposures can expression was detected. Gene expression dif- provide a suitable model for exploring the fered in neonate and adult Daphnia, and adults intersections of multiple physiological path- showed a stronger transcriptional response to ways using a systems biology approach (Eads genotoxicants, with three times as many genes et al., 2008; Shaw et al., 2008). Daphnia is often showing differential expression (106 genes in used as a surrogate species to understand the adults vs. 34 genes in neonates). Adults had genomic responses to environmental stressors higher constitutive expression of DNA repair that are important factors in human health and and oxidative stress genes than neonates under well-being. So far, tolerance limits and regula- unexposed conditions, suggesting that adults tory processes that allow organisms (including are better prepared to deal with DNA damage humans) to cope with pollutants in the short- and, perhaps, that adults activate these repair term span of an individual’s lifetime and in and protective pathways in response to their the longer time frame of evolutionary change, higher metabolic rates and the associated pro- are still poorly understood. Daphnia constitute duction of reactive oxygen species. an excellent model for physiological functional Exposed adults also showed induction of genomics because a long history of environ- DNA repair genes, while neonates did not. mental and ecological research has provided a Additionally, in adults oxidative stress genes wealth of physiological and toxicological infor- and hb genes were induced, but chitinase and mation that can be exploited when designing other proteases were downregulated upon genomics experiments. Its importance as an exposure. Since these catabolic genes are impli- ecotoxicological model make it imperative that cated in molting associated with reproduction, the research community rapidly and collabora- suppressed gene expression is consistent with tively use these relatively new genomic reproductive deficits and population level con- approaches to understand Daphnia’s baseline sequences after genotoxicant exposure. physiology and the ways that individuals and Unexposed neonates showed a greater expres- populations function in the face of environ- sion of genes involved in polysaccharide bind- mental perturbations (Shaw et al., 2008). In rec- ing, pattern binding, and chitin binding than ognition of its relevance to understanding unexposed adults, consistent with greater cuti- basic biological processes and as a sensitive cle formation in developing neonates. While organism for environmental monitoring, just neonates did not show induction of DNA recently D. pulex was listed as one of the few repair pathways, they did show induction of selected model organisms for biomedical oxidative stress response genes. This study research at the National Institute of Health demonstrated the potential of gene expression- (http://www.nih.gov/science/models/). based biomarkers for genotoxicant exposure David et al. (2011) explored the global tran- and showed that gene expression assays can scriptional response to genotoxicants, a study be more sensitive than the comet assay

PHYSIOLOGY OF THE CLADOCERA 18.6 HUNTING FOR PHYSIOLOGICALLY RELEVANT GENES AND REGULATORY NETWORKS 251 following nonlethal exposure levels that never- genome, with low homology to any previously theless can induce reproductive effects. It also known metallothionein genes. This paper showed that gene expression profiles can differ highlights the utility of microarray studies, dramatically between adult and neonate not only for determining environmental toxi- Daphnia. Adults were more transcriptionally cogenomic signatures and exploring physio- responsive to the compounds tested and may logical pathways involved in stressor response therefore be more appropriate subjects for but also for gene discovery. bioassays than the neonates traditionally used A more recent study of the Cd response in in toxicology testing. D. magna is notable for combining microarray- Another functional genomics study explored based transcriptional profiling with metabolo- the effects of ibuprofen on gene expression in mics data to investigate the role of nutrient D. magna (Heckmann et al., 2008). The observed uptake, metabolism, and decreased energy effects of ibuprofen had parallels to its effects reserves in chronic toxicity (Poynton et al., on invertebrates, including early disruption of 2011). Mass spectrometry of hemolymph of eicosanoid metabolism. In Daphnia, there were Daphnia exposed for 24 h to sublethal Cd con- also effects on the endocrine system, juvenile centrations revealed disruptions in both amino hormone metabolism, and oogenesis, thus pro- acids and fatty acids, while differential gene viding strong evidence for a mechanism under- expression analysis showed reduced expres- lying observed reproductive effects in sion of genes encoding digestive enzymes ibuprofen-exposed daphniids. By comparing the (consistent with a role for impaired nutrient expression profile of ibuprofen exposure to that absorption in Cd toxicity), as well as upregula- of exposure to a very different stressor, cad- tion of genes for cuticle proteins and oxidative mium (Cd) (Poynton et al., 2007), the authors stress response. This study focused on the ini- were also able to identify common transcrip- tial phase Cd response but revealed metabolic tional patterns associated with a general stress changes that could lead to decreased fitness response in D. magna. These patterns included during prolonged exposure. Assaying gene induction of glycolytic, proteolytic, homeostatic, transcription and metabolite composition in and heat shock protein genes, and downregula- parallel has allowed researchers to explore the tion of genes involved with energy metabolism hypothesized roles of gene expression changes and translation. The authors suggested that the and more fully describe the physiological ibuprofen-specific transcriptional response mechanisms of Cd toxicity. could be useful as an early indicator of signifi- Another recent paper addressed the effects cant population ibuprofen exposure with risk to of the algal toxin microcystin on D. pulex Daphnia reproductive biology. (Asselman et al., 2012). Microcystins are pro- The Daphnia transcriptional response to Cd duced by the harmful cyanobacteria has also been investigated in D. pulex.Shaw Microcystis aeruginosa, an organism associated et al. (2007) used a first-generation cDNA with toxic algal blooms. Microarray analysis of microarray to identify transcriptional gene expression supports a characteristic stress responses in adult animals exposed for 48 h to response and a role for energy budget distur- sublethal concentrations of Cd. The study bances in microcystin toxicity. Daphnia fed a revealed many Cd-responsive genes to be diet contaminated with Microcystis for 16 days associated with molting and metal detoxifica- show differential gene expression of ribosomal tion, including the metallothionein gene. genes, oxidative phosphorylation genes, pro- Importantly, this led to the discovery of addi- tein export genes, and genes associated with tional novel metallothionein genes in the mitochondrial function. Gene expression

PHYSIOLOGY OF THE CLADOCERA 252 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY patterns suggest that microcystin toxicity may induced impacts such as climate change, eutro- involve upregulation of protein synthesis and phication, landscape changes, and pollution. energy production pathways. Microcystis also As Hairston and De Meester (2008) noted in a affected expression of several paralogous gene recent review “The animals hatched from clusters, and some paralogous genes were var- decades-old sediments are living organisms iably regulated. Taken together, these results with genotypes representative of past popula- suggest physiologically important roles for tions. Evolutionary changes in plastic charac- duplicated genes and indicate that paralogues ters such as physiology and behavior can thus can assume different functional roles. The be reconstructed by comparing the perfor- authors caution that statistical methods tai- mance of genetic lineages obtained from differ- lored to analyze the overabundance of dupli- ent sediment layers in a common set of cated genes in Daphnia genomes might be environmental conditions. This approach has necessary to accurately assess functional gene successfully been applied for a growing range and pathway enrichment in these species. of traits and environmental changes.” As all branchiopods can produce dormant stages, the genetic responses to environmental 18.7 PALEOGENOMICS change over time can be studied for a wide range of taxa in the form of genetic archives in An emerging area of research, with a huge freshwater lake sediments deposited as egg potential for the study of cladoceran physiol- banks (Brendonck and De Meester, 2003). Yet, ogy, is the field of paleogenetics and, more despite its wide occurrence over a wide phylo- recently, paleogenomics. Diapause in the genetic range, nearly all the research on Cladocera allows the study of populations cladoceran resurrection ecology and paleoge- through time and an assessment of historical netics gravitates around Daphnia, in which the changes to fitness and genes. Paleogenomics, dormant embryos are encased in ephippia (as i.e. the study of extinct genomes, can be directly for all Anomopoda, but not for all Cladocera). applied to extinct populations through the use A few studies exist on other genera, e.g. of cladoceran resting stages. The method can Ceriodaphnia, which is still within the daphniid be combined with other techniques that have family (Reinikainen and A˚ hle´n, 2012). The been used to study adaptability of populations extent of the time dimension in which the through time. For example, the term resurrec- genes can be studied through resurrection tion ecology is widely used (Kerfoot et al., 1999) ecology is determined by the viability of the for the study of reviving specimens from dia- ephippia, which mostly stretches to decades pausing or dormant stages and comparing and rarely to centuries (Frisch et al., unpubl. them to present populations in order to exam- data), yet paleogenomics allows us to examine ine fitness and gene responses under certain traits (through genes) even further, to centuries (experimental) conditions. This approach and even millennia (e.g. Frisch et al., subm.; allows an analysis of neutral versus adaptive Mergeay et al., 2007; Morton et al., 2012). variation of the genes and an increased under- Besides being ecologically relevant, the impor- standing of evolutionary processes, particu- tance of such techniques lies in the possibility larly those operating on adaptive genetic to examine the genetic adaptivity in plastic variation (see Orsini et al., 2013). The ecologi- responses, the evolution of phenotypic plastic- cal significance lies in the fact that microevolu- ity (physiological and behavioral), and rapid tionary changes at the population level can be evolutionary changes under strong selection studied in response to a wide range of human- pressures induced by humans or biological

PHYSIOLOGY OF THE CLADOCERA 18.7 PALEOGENOMICS 253 interactions (e.g. Decaestecker et al., 2007; genetic adjustments in D. magna, even over Hairston and De Meester, 2008). Hairston and very short time spans (months to years). De Meester (2008) reviewed two case studies Human-induced impacts on freshwater ecosys- on human-induced selection pressures that tems might (irreversibly) change cladoceran strongly alter aquatic ecosystems (eutrophica- physiology by driving selection. In a recent tion and fish introduction) and elicit rapid study, Frisch et al. (unpubl. data) were able to genetic responses in Daphnia. Such studies pro- hatch live D. pulicaria from ephippia derived vide hard evidence for the evolution of cladoc- from a core taken in South Central Lake (U.S.) eran physiological traits, as well as the range from as far back as 1418 AD, the oldest resur- (plasticity) and limits of acclimation and adap- rected cladoceran to date. The authors com- tation (evolutionary constraints). pared the genetic structure of populations Therefore, resurrection ecology and paleoge- through time in relation to the lake’s eutrophi- nomics provide a powerful tool for under- cation, using a combination of resurrection standing the genetic basis of phenotypic ecology, paleogenomics, and historical records plasticity (Orsini et al., 2013). Using a combina- to investigate the response of extinct versus tion of these methods, Orsini et al. (2012) extant populations in relation to eutrophication examined the genome of D. magna in space levels (throughout the lake’s history). As De and time, and correlated the response of genes Meester et al. (2011) illustrated for tempera- clusters to three major selection pressures ture, the work by Frisch et al. (unpubl. data) (land-use, fish predation, and parasitism) that suggests that cladocerans can adapt quickly to are known to elicit a rapid (i.e. microevolution- environmental conditions and that such ary) genetic and phenotypic response in clado- changes can be irreversible. Loci with a signa- cerans. Such studies illustrate that the genetic ture of directional selection were related to his- background of cladoceran physiology remains toric phosphorus levels in the lake and an a vast terra incognita. “Of the 164 genes identi- adaptive shift occurred to higher phosphorus fied, only 88 (54%) could be annotated and 86 concentrations. Resurrected clones from the fif- of these have a putative function based on the teenth century grew faster under low phos- homology with other organisms. 109 genes phorus conditions and showed higher were linked to general stress response; of phosphorus retention when compared to the these, 53 (49%) returned unknown function” twenty-first century population. Therefore, (Orsini et al., 2012). The authors found after agricultural expansion around the lake repeated local adaptation by the populations (around the 1920s), D. hyalina lost the physio- and also identified the mechanisms for rapidly logical ability to retain phosphorus at low responding to the three selection pressures levels due to clonal selection and genetic ero- (through homology) as genes involved in pro- sion, in contrast to the “extinct” populations teolysis, metabolic processes, neuronal devel- from before eutrophication. This shows that opment, and transcriptional and translational over the course of a few hundred years (which regulation. This provides hard evidence that is a very short geological time, considering the even human impact can elicit a strong and age of the lineage), a cladoceran species can rapid genetic response in cladocerans and that undergo significant and rapid genetic shifts genotypes in the industrialized world have that alter its tolerance to the environment. gone through this selection and are not repre- The key to using paleogenomics to its full sentative of the original “wild” genotypes. potential lies in identification of the genes that Similarly, De Meester et al. (2011) have shown determine ecologically relevant trait variation that temperature changes can quickly result in (Miner et al., 2012). Identifying the large number

PHYSIOLOGY OF THE CLADOCERA 254 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY of unknown genes in cladocerans (Daphnia; of cladocerans as models for physiological Colbourne et al., 2011) is a task for the future, genomic research. Importantly, working in spe- and would help to identify the important genes cies with well-characterized ecologies will involved in determining plasticity. Other issues allow us to ask how physiologies and genetic also remain challenging, such as the extraction mechanisms vary with environmental factors. and sequencing of ancient DNA from ephippia, Comparative functional genomics can test and the use of homologies to identify unknown whether or not similar physiologies have been genes and gene functions. Furthermore, all res- achieved by regulating the same genetic path- urrection ecology and paleogenomics is cur- ways in the same way or through the indepen- rently focused on Daphniidae and therefore on dent evolution of substantially different the planktonic filter feeders in the aquatic eco- mechanisms, and can help to unravel the basis system. No studies have yet been carried out on of qualitative and quantitative differences in benthic/littoral (e.g. Chydoridae) or predatory physiological responses (Whitehead, 2012). cladocerans (e.g. Leptodoridae). Well studied in Ultimately, researchers will want to know paleolimnology and better preserved in the sedi- the genetic and genomic differences responsi- mentary record (e.g. Frey, 1962), benthic/littoral ble for the physiological variation and associ- chydorids have a particular potential for paleo- ated gene expression variation that we genomics, yet the level of genetic knowledge is observe. Gaining this knowledge will require nowhere near that of daphniids. In time, our the incorporation of evolutionary genetics and knowledge in all of the aforementioned fields genomics [e.g. quantitative trait locus (QTL) will undoubtedly expand (vide infra). For further mapping, expression QTL (eQTL) mapping, reading on paleogenomics or resurrection ecol- genome-wide association studies, and compar- ogy, we refer the reader to recent reviews by ative genomics], molecular genetics (e.g. Brendonck and De Meester (2003), Hairston and RNAi), and other molecular and bioinformat- De Meester (2008), and Orsini et al. (2013). ics methods that can identify variation in regulatory elements associated with gene regulation (e.g. experimental identification of 18.8 FUTURE DIRECTIONS: transcription factor-binding sites and sites of EXPLORING PHYSIOLOGICAL epigenetic modification, and computational VARIATION WITH FUNCTIONAL searches for conserved regulatory motifs and GENOMICS their variation within and among populations). The integration of physiology and functional In order to understand physiology in an genomics with ecological and evolutionary evolutionary and ecological context, the simi- genomic analysis has the potential to enrich larities and differences among individuals many aspects of our understanding of Daphnia. within and among populations and among spe- As outlined in this chapter, the field of cies must be understood and exploited. (physiological) genomics is rapidly advancing Physiological responses can vary, but little is and Cladocera are on the frontline. Studies on known about the mechanisms underlying vari- Daphnia genomics facilitate the identification of ation or the evolutionary forces generating, ecologically relevant genes that enable context- maintaining, or constraining such variation. dependent responses. By linking different Comparative approaches to identify apparently research disciplines, such as evolutionary ecol- (i.e. phenotypically) universal physiologies and ogy, physiology, molecular biology, and geno- those that vary among populations and species mics, current research on Daphnia aims to will be facilitated by the further development increase our understanding of organisms as

PHYSIOLOGY OF THE CLADOCERA 18.8 FUTURE DIRECTIONS: EXPLORING PHYSIOLOGICAL VARIATION WITH FUNCTIONAL GENOMICS 255 integrated units of biological organization 18.8.1 Trends and Suggestions for shaped by the context of the ecological and the Future Research history of evolution. Studying adaptive traits using such a systems-level approach will ulti- First, new state-of-the-art high-throughput mately provide a radically new perspective on techniques such as NGS and RNA interference the nature of genetic constraints and the evolu- (RNAi) have now become more accessible. tion of adaptation and speciation. Conse- Promising new methods include advances in quently, unraveling key adaptive responses in bioinformatics, which allow the manipulation Daphnia will positively trigger a cascade of of large datasets, such as gene expression pro- subsequent studies (in Daphnia and other files. RNAi is a powerful tool to investigate organisms) that may have a great impact on gene function by knocking down distinct target forecasting the fate of keystone species, and genes. Recently, RNAi has been developed for therefore ecosystem stability, under growing cladocerans (i.e. the Dll gene in D. magna) environmental challenges. But where do we go (Kato et al., 2011). But, there is more. Cladocera from here? There can be no doubt that our also appear to be promising model systems for knowledge of the genetic mechanisms of cla- epigenetics (D. magna; Vandegehuchte et al., doceran physiology has rapidly increased since 2010a,b; Harris et al., 2012; Robichaud et al., the late twentieth century. With the first cla- 2012). Sex determination and sexual reproduc- doceran genome available (Colbourne et al., tion in cladocerans may be epigenetically con- 2011), we are now on the brink of a new era. trolled and DNA methylation patterns are Because of their ecological and genetic charac- responsive to toxicants and heavy metals teristics, cladocerans can play an important (Harris et al., 2012). However, this field is still role in exploring relevant biological questions. young, especially with regards to the potential Containing model organisms with well- of cladocerans as model systems. Examining studied ecologies and a major role in ecosys- protein expression profiles under different tems, future study of the Cladocera has the environmental conditions falls under the area potential to “revolutionize genomic research of proteomics, and the D. pulex genome provides and enable[s] us to focus on a number of out- a major step forward in this area of research as standing questions” (De Meester et al., 2011). well (Fro¨hlich et al., 2009; Schwerin et al., 2009; For example, by approaching cladoceran phys- Zeis et al., 2009). The same can be said for meta- iology and biology from a functional genomics bolomics, which examines cladoceran metabo- perspective, we can advance our understand- lites in response to different environmental ing of gene evolution processes and the genetic conditions or stressors (e.g. toxins; Taylor et al., basis of phenotypic plasticity. Research on cla- 2010). For paleogenomics, new techniques are doceran genomics is also relevant in the con- expected to increase the molecular resolution text of biodiversity and conservation because of ancient DNA samples (Orsini et al., 2013). of the presence of human-induced, irreversible Second, a major challenge for the future genetic changes in natural populations. As cla- is the characterization and exploration of docerans play a pivotal ecological role, their function (and structure) of the circa 13,000 system functioning and fitness are important (forming approximately 36% of the genome) indicators for freshwater ecosystem health (e.g. unknown “Daphnia-specific ”protein-coding genes Rapport et al., 1998). Therefore, genetic meth- (Colbourne et al., 2011). With more crustacean ods combined with physiology provide a pow- genomes expected to be sequenced in the near erful tool for monitoring (and perhaps future (Stillman et al., 2008), the evolution and forecasting) ecosystem stability. the function of these genes will become clearer.

PHYSIOLOGY OF THE CLADOCERA 256 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY

At present, these still-to-be-annotated genes are cDNA library The complete mRNA collection not known to occur in other crustacean or cla- of an organism or sample, which is stored in a doceran lineages. Until now, nearly all the host (e.g. bacterial plasmid, yeast, and bacterial genetic research has focused on Daphniidae (in artificial chromosomes) as cDNA. See also fact, on Daphnia), leaving the majority of transcriptomics. Cladocera untouched. Other groups take up Enrichment analysis A statistical test of equally important, yet different roles in aquatic whether a set of genes (e.g. a set of differen- ecosystems, such as the predatory haplopods tially expressed genes) contains more members and the benthic/littoral chydorids (the most of a genetic pathway or gene class than would speciose group in the Cladocera) (Forro´ et al., be expected by chance. 2008). Epigenetics Modification to the genome pro- Third, and finally, the majority of physio- duced through environmental effects (e.g. logical studies in cladocerans have hitherto DNA methylation) that in some cases comprise focused on species acclim(atiz)ation and adap- inheritable factors. tation processes in response to (natural or eQTL mapping A quantitative trait loci map- human-induced) environmental stimuli. In ping study for which the continuous variable of order to understand the mechanistic basis of interest is the expression level of one or more physiological responses (which include pheno- genes; eQTLs are regions in the genome (loci) typic plasticity), holistic approaches are that regulate the expression of other areas in the needed. This integration can only be achieved genome. See also QTL mapping, gene expression. by analyzing physiological traits in a functional genomics context, and Daphnia is an Expression profiles See gene expression. ideal model to do so. Functional genomics The study of genomes through the characterization of the effect of sequences on the phenotype at any level GLOSSARY TERMS (molecular, cellular, tissue, and whole organism). See also transcriptomics. Acclimation Phenotypic change in an individ- Gene cluster A set of genes located in the ual organism as a physiological response to its same genomic region. See also synteny. environment. Gene conversion (GC) Replacement of one Adaptation Phenotypic change in a popula- allele by another through non-homologous tion due to natural selection favoring alleles recombination. that confer increased fitness. Gene duplication The duplication of a gene, Allele Any variant of a gene at a particular resulting in the presence of two or more locus. copies in the same genome. It can be caused by Annotation (of genes/genomes) The process of errors in replication, by recombination, or by assigning biological information, such as the retrotransposons. The Daphnia genome is char- identity and function of genes and other genetic acterised by a high number of gene duplica- elements, to positions in a genome sequence. tions, resulting in many paralogues of the same See also functional genomics. gene, as in the hb gene cluster for example. Complementary DNA (cDNA) DNA synthe- See also paralogues and tandemly repeated genes. sized from an mRNA template by reverse tran- Gene-environment interaction Environmental scription of RNA to DNA followed by DNA effects on genetic function that contribute to polymerization. phenotype.

PHYSIOLOGY OF THE CLADOCERA GLOSSARY TERMS 257

Gene expression The conversion of the infor- Isoforms Variants of a protein expressed by mation encoded in genes (as a DNA sequence) the same gene or closely related genes. Can be into RNA. Examination of gene expression pat- the result of alternative splicing. terns and profiles relates to the study of genes Metabolomics The study of all metabolites in that are differentially regulated and tran- an organism and the chemical reactions they are scribed under certain conditions of interest involved in (structures, pathways, etc.). (e.g. metallothionines under metal pollution Microarray Techniqueforthequantificationof stress in Daphnia). whole-genome transcription levels. All nucleic Genetic linkage The probability of two genes acids extracted from a sample are labeled with being inherited together in a non-random fash- fluorescent dyes and hybridized to a microarray ion due to their physical proximity on a chromo- chip. The targets for binding on the chip are short some, or patterns of linkage disequilibrium. oligonucleotides matching unique regions of the Genome All inheritable genetic material that genome. Fluorescence levels are proportional to is present in an organism (i.e. DNA). RNA abundance and gene expression. Genome assembly The process of uniting Messenger RNA (mRNA) Molecules that con- individual DNA sequences generated by a vey the genetic sequence of an actively tran- sequencing project into long (ideally chromo- scribed gene from the nucleus to the ribosome, some-length) DNA sequences. where it specifies the sequence of amino acids Genomics The study of the structure and to be synthesized into a protein. function of genomes. Model organism An organism that is amenable Genotoxicants DNA-damaging mutagenic to scientific investigation and of which the biol- agents (e.g. UV rays and chemicals). ogy can provide insights on other species. Heterologous transfection Insertion of an Daphnia can reproduce clonally, is easy to cul- extraneous DNA fragment into a genome. ture, and is a model organism for the study of Often used to study gene expression and the genotype-environment interactions, epigenetics, functional consequences of genetic variants in phenotypic plasticity, and ecological adaptation. a non-native genetic background. Next-generation sequencing (NGS) A variety Hypoxia-inducible factor 1 (HIF-1) Oxygen- of methods of high-throughput, low-cost responsive hetero-dimeric transcription factor sequencing, which works by generating thou- that mediates cellular responses to tissue hyp- sands of short sequencing reactions in parallel. oxia by the regulation of distinct gene expres- These methods produce a high number of sion events. short (length depends on the platform, e.g. À Homologue Identity by descent; homologous 50 400 bp) sequences (or “reads”). genes have the same origin but have evolved Orthologue (or orthologous gene) Genes that independently in different lineages through share a common ancestor and are present in time. See also paralogue, orthologue. different species (not in the same genome). See Hypoxia-response element (HRE) Located in also homologue, paralogue. the regulatory region (i.e., promoter region) of Paleogenomics The study of ancient/extinct hypoxia-responsive genes; binding to transcription genomes. In the case of cladocerans, paleoge- factors such as HIF-1 induces gene expression of nomics is carried out on embryos from ephip- distinct target genes. pia that are stored in lake sediments. Intergenic Regions of the genome that fall Paralogue (or paralogous gene) Genes that outside protein-coding regions. share a common ancestor and are present in

PHYSIOLOGY OF THE CLADOCERA 258 18. THE GENOMICS OF CLADOCERAN PHYSIOLOGY the same genome. The result of gene duplica- ephippia that are stored in lake sediments). tion events. See also gene duplication, homologue, See paleogenomics. orthologue. RNA interference (RNAi) RNA-directed post- Phenotypic plasticity Condition-dependent transcriptional mechanism for gene downregula- development that allows an organism to tion. Short (approximately 20-nt long) RNA respond to environmental change by altering fragments guide a multiprotein complex towards its phenotype specific transcripts, causing their degradation. Phylogeny A hypothesis about the evolution- RNA sequencing (RNA-Seq) Technique for ary relationships among species or populations the identification and quantification of whole- Polygenic trait (quantitative trait) A trait that genome transcription levels. The total RNA is affected by several to many genes extracted from the sample is sequenced using Proteomics The study of the complete set of high-throughput methods. The number of proteins present in a given sample (cell, tissue, times that a sequence is read is proportional to organism). its expression level. Can identify polymorph- Quantitative polymerase chain reaction isms and splice variants with base-pair resolu- (qPCR) Quantitative or real-time PCR is a tion. See cDNA, NGS. technique that allows the quantification of the Synteny Conservation of the position of genes abundance of selected DNA sequences in a along the chromosome in different lineages of sample. Can be used on cDNA to quantify organisms. For example, in multiple Daphnia gene expression levels and on genomic DNA species, genes in the hb gene cluster are to measure gene copy number variations. arranged in the same order. See gene cluster. QTL mapping Quantitative trait loci mapping, Tandem gene cluster Gene cluster generated a statistical association of genetic polymorph- by a series of tandem duplications. For exam- isms (markers) in a genome and variation of a ple the hb genes in Daphnia. See tandemly quantitative phenotypic trait. It allows quanti- repeated genes, gene cluster. fication of the relative genetic contribution of Tandemly repeated genes Duplicated genes each locus to the total observed variation in located in adjacent positions in the genome. the trait. See eQTL. Typically the result of small local duplications. Quantitative genetics The study of the See also gene duplication. effect of genetic variation on quantitative or con- Transcriptionally active regions Portions of tinuous traits (e.g. body size). See also QTL thegenomethatshowevidenceofbeingtran- mapping. scribed under certain conditions. Can be associ- Regulatory elements DNA elements that reg- ated with genes in the genome or with ulate or modify the expression of distinct transcription of non-coding sequences (e.g. genes or gene expression events. Includes tran- regions encoding short and long RNAs elements). scription factors, promotors, and transcription Transcriptomics The study of gene expression factor binding sites. through the measurement of all RNA species Resurrection ecology The study of the ecology [e.g. mRNA, small interfering RNA (siRNA), of populations or species revived from the past and microRNA, i.e. the transcriptome] in a by breaking dormancy (e.g. resurrection ecol- given sample. See also microarray and RNA ogy of Daphnia by hatching populations from sequencing.

PHYSIOLOGY OF THE CLADOCERA Conclusions: Special Traits of Cladoceran Physiology

Some representatives of the Cladocera occur excessive food consumption (i.e. luxury con- in enormous quantities, but other species are sumption), the proportion of ingested food that rare and confined to narrow ecological niches, is digested may be low. Cladocera consume a potentially partly due to their physiological lot of chlorophyll; during digestion, only a lit- adaptations. tle is slightly reduced to pheopigments and it Cladocera consume small algae, bacteria, is not used in the construction of Hb. and detritus, thus forming a link between pri- Starvation is accompanied by profound mary production and the predators that eat changes in the chemical composition of the them. A high intensity of feeding is character- body. istic of Cladocera: the ingested food stays in Respiration occurs through the body surface the intestine for several minutes up to about of cladocera. Littoral, and especially bottom- half an hour. During this time, the Cladocera living species, exist under oxygen stress but extract sufficient material to support their are surrounded by abundant food resources, intensive reproduction. They also synthesize whereas planktonic species usually enjoy a abundant chitin. good oxygen environment, but their food Particular body constituents of Cladocera resources are periodically either abundant or are dynamic in relation to the season, the scarce. chemical composition of their food and of the Littoral and bottom-dwelling species live environment, and starvation. Data are now under conditions of hypoxia and anoxia. available on the quantity of glycogen, chitin, Cladocera frequently contain Hb but can man- lipids, slimes, and pigments [e.g. carotenoids, age well without it. Studies on the impact of melanin, hemoglobin (Hb)] consumed and xenobiotics on respiration have been reviewed. their metabolism, and on the dynamics of par- The heart is myogenic and acetylcholine is ticular elements (including phosphorus, cal- the inhibitory transmitter substance (Postmes cium, strontium, sodium, and iron). Cladocera et al., 1989). Two kinds of blood cells have may accumulate physiologically important now been identified (with reference to substances, those of no such importance, and Daphnia), one of which performs phagocytosis. toxicants. The heart rate is similar for all Cladocera spe- Cladocera consume foods that have low cies, except for Ilyocryptus, in which the heart quantities of nitrogen and phosphorus and an rate is 2À3 times lower. The heart rate notice- excess of carbon. Thus, they have to get rid of ably increases with various disturbances and excess carbon. Only a very small amount of drops only before death. Heart arrest occasion- the consumed lipids are transformed. With ally occurs without harming the individual

Physiology of the Cladocera. DOI: http://dx.doi.org/10.1016/B978-0-12-396953-8.00029-X 259 © 2014 Elsevier Inc. All rights reserved. 260 CONCLUSIONS: SPECIAL TRAITS OF CLADOCERAN PHYSIOLOGY and it can resume beating without an obvious immobilization, fatigue, stress, and the impact reason. Adhesion of blood cells (which nor- of xenobiotics on locomotion. mally drift in the blood) to the surface of Cladocera can discern polarized and colored organs may occur for unknown physiological light, can orientate themselves in space, and reasons. manifest endogenous rhythms. Excretion is principally carried out by Cladocera also exhibit complex behavior, paired maxillary glands. The main final prod- which differs in particular species. This uct of protein metabolism is ammonia (NH3), includes migration, swarming, akinesis, and accompanied by smaller amounts of urea. escape behavior. Published data are available Depending on their structure, xenobiotics are on disturbances to their behavior by transformed within the body and may be xenobiotics. passed into the next generations. Within cer- Ecological aspects considered include the tain limits, Cladocera can support homeostasis physiological background of limits to physio- of various processes within a dynamic envi- logical factors, synergism and antagonism ronment, including homeostatic maintenance among factors, lipid pathways from algae via of chemical constituents and osmotic pressure. Cladocera to fish, and environmental condi- The principal types of osmotic regulation tioning by Cladocera. are hyperosmotic regulation (in fresh water), In Cladocera, some functions are facultative, hypoosmotic regulation (in marine Penilia), such as the presence and formation of Hb, and amphiosmotic regulation. Xenobiotics beating of the heart, and vision by means of affect osmotic regulation. their eyes. Nevertheless, without the latter, Cladocera live for a week up to several some cladocerans do quite well. Hb generally months, depending on the species and the contributes to the activity of cladocerans. environmental conditions. Body length incre- However, it may be absent or its function may ments occur between molts. Mechanical dam- be blocked by carbon monoxide, and such spe- age is repaired by regeneration, which cimens continue successful life activities. It produces either normal or abnormal struc- must be remembered that in vertebrates the tures. The current status of investigations into role of Hb is obligate (with the exception of senescence and mortality is described. one group of fish: the Chaenichtyids). Cladocera are remarkable for their predomi- Cladocera cannot be considered to be physi- nantly parthenogenetic reproduction, which is ologically primitive. sometimes interrupted by bisexual reproduc- In Cladocera, the absolute value of particu- tion. The appearance of males depends on lar parameters is not species specific but environmental conditions, food composition, depends on clonal composition and on previ- and hormonal regulation. With the exception ous acclimation. of Leptodora, there are no larval stages in the Seemingly chaotic (although within certain free-living period of their life cycle. limits), dynamic combinations of dozens of The trajectories of littoral and pelagic spe- Cladocera species live in an environment con- cies differ greatly but little is known about taining hundreds of dynamic multidirectional this. The complete muscle system has only factors. Each specimen simultaneously per- once been described, with reference to ceives all of these factors with its sense organs. Daphnia. Therefore, comparative investigations Due to such a dynamic environment and their of the muscles of littoral species are urgently physiological individuality, particular species needed. Published studies are available on can form resultant population peaks, whose

PHYSIOLOGY OF THE CLADOCERA CONCLUSIONS: SPECIAL TRAITS OF CLADOCERAN PHYSIOLOGY 261 position in time and their absolute value may addition, some issues of cladoceran physiology differ greatly in different years. are well studied (e.g. some aspects of respira- Initially, toxic and inhibitory pollutants are tion, osmoregulation, and neurosecretion), diluted and may stimulate various vital pro- while others are still awaiting investigation. cesses. Their combination with sex hormone-like Cladocera exhibit physiological radiation, as pesticides can cause multidirectional effects and seen in their various functions. Planktonic lead to disturbances in the natural balance. Cladocera are normoxic; bottom-living ones The available data on cladoceran physiology prosper in hypoxic or anoxic conditions. mostly concern daphnids living in open water; Sometimes, physiological radiation is formed bottom-living and littoral Cladocera have been in the background of conservative morphol- little studied. The latter species live under ogy, for example in species of the genus conditions of hypoxia and anoxia with an Moina, some of which are freshwater species excess of food (organic debris at all stages of and some of which live in saline lakes. decomposition); thus, their investigation may reveal specific adaptations that differ from Let everything that has breath praise the Lord. those characteristic of open-water species. In Psalm 150.6.

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Note: Page numbers followed by “f ”, and “t ” refers to figures and tables respectively.

A Bythotrephes cederstroemi, 13, 76À77 D Acantholeberis curvirostris,2f,52f, Bythtotrephes longimanus,14t,15t, 108, Dadaya, 26, 34, 166À167, 182 65À66, 110, 190 110, 144, 207t, 216À217 Daphnia,1,6f,9À11, 13, 17À18, 18f, Acroperus, 48, 52, 77, 88, 96, 99, 125, 21À22, 24, 26À29, 30t,35À37, 39, 144, 147, 167, 182À183 C 42À54, 53f,56À71, 57f,64f,73À74, Acroperus harpae, 79, 132, 220t Camptocercus, 52, 77, 95À96, 152, 77À85, 83f,88À97, 91f,92f,94f,95f, Alona,6À7, 29, 77, 82, 85, 146, 157 183À184 99À105, 100f, 106f, 107, 109, Alona affinis, 79, 144, 156, 167, 176f, Camptocercus fennicus, 153 113À115, 117À123, 124f, 125À126, 220t Camptocersus lilljeborgi, 156, 220t 130À139, 142À149, 151À157, Alona cambouei, 188 Ceriodaphnia, 1, 13, 29, 35, 46, 48, 51, 159À163, 160f, 161f, 162f, 165f, Alona costata, 119 99, 101, 108À109, 121, 134À136, 139, 166À175, 177À178, 179f, 182À183, Alona guttata, 144 148, 168, 171À172, 183, 188, 190, 185, 187À197, 199À200, 202À206, Alona intermedia,52 207t, 208, 252À253 207t, 210t, 211À219, 224À226, 228, Alona labrosa, 183À184 Ceriodaphnia affinis,14t,30t, 103, 114, 230, 232À233, 236À243, 246, 249, Alona rectangula, 147 127, 148, 195 251À255, 259À260 Alona setosocaudata, 183À184 Ceriodaphnia cornuta, 73, 158 Daphnia ambigua, 7, 70, 123, 195À196, Alonella,6À7 Ceriodaphnia dubia, 31, 51, 74, 104, 143, 220t Alonella excisa, 154À155, 190 157, 173, 192, 195, 220t, 224 Daphnia carinata, 46, 60, 84, 147, 172, 195 Alonella exigua, 154À155 Ceriodaphnia lacustris, 105 Daphnia catawba, 174, 190, 195, 207t Alonella nana, 167 Ceriodaphnia laticaudata,75À77, 190, Daphnia cristata,82 Alonopsis, 34, 141f, 142 220t Daphnia cucullata,14t, 18, 23t,43À44, Alonopsis elongata, 17, 52À53 Ceriodaphnia megalops,26t, 220t 48, 50, 63, 121, 123, 135, 188, 197 Anchistropus,35 Ceriodaphnia pulchella,14t, 190, 207t, Daphnia curvirostris, 147, 206, 220t Archepleuroxus, 131 208 Daphnia dentifera, 70, 220t, 224À225, Ceriodaphnia quadrangula,14t,15t, 29, 230À231 B 47, 50, 79À80, 85, 155, 189 Daphnia galeata,17À18, 36À39, 43, 46, Bosmina,1,7,13,22t,28À29, 36, 43, Ceriodaohnia reticulata, 16, 101, 133, 48, 61À64, 68, 72, 78, 82, 101, 105, 46À47, 59, 68, 70, 73À74, 82, 85, 155, 173, 183, 203À204, 220t 111, 122, 135, 173 99À101, 108À109, 125, 139, 143, 155, Ceriodaphnia setosa, 190 Daphnia hyalina, 7, 13, 14t, 18, 28t, 157, 171À172, 176À177, 183À184, 195 Chydorus,6À7, 27, 38À39, 47, 52, 50À51, 62, 108, 120, 132, 193, 220t, 253 Bosmina coregoni, 13, 35, 170 77À78, 121, 133, 146, 149, 157, 176, Daphnia longiremis,82 Bosmina longirostris,14t,16t, 17, 23t, 188, 190 Daphnia longispina,3f,14t,23t,26t, 36, 47, 66f, 82, 124, 132, 155, 188, 203, Chydorus ovalis, 77, 107, 148, 191 43À44, 46À49, 59, 68, 73, 77À78, 220t Chydorus piger ( 5 Paralona pigra), 34, 80À82, 119À120, 130, 140, 142, 147, Bosmina longispina,14t 154À155 173, 188À189, 195À196, 224 Bosmina meridionalis, 173 Chydorus sphaericus, 77, 85, 114, 121, Daphnia lumholtzi, 123, 211, 225 Bosmina obtusirostris,21t, 146, 149 131, 157, 167, 181, 184À185, 220t Daphnia magna,4f,9À10, 13, 14t, 16, Bosminopsis,1 Ctenodaphnia, 158, 203t, 206, 207t, 18, 23t, 24, 28À29, 30t,31À33, 31f, Bryospilus, 182 212À213 41À42, 44, 46À51, 54f,55f,58À74,

321 322 INDEX OF LATIN NAMES OF CLADOCERA

76f,77À89, 90f,91À94, 96À97, E Megafenestra aurita,21À22 100À105, 107, 110À121, 111f, Eurycercus,1,6À7, 7f, 10, 17, 26t, Moina, 1, 13, 14t,15t, 17, 31, 46À47, 51, 124À127, 130À133, 134f, 135À140, 33À34, 39, 39f, 46, 52À53, 62À63, 77, 68, 74, 79, 99, 103, 107À109, 127, 140f, 142, 144, 146À149, 153, 79, 88À89, 88f,89f,95À96, 99, 125, 130, 132À133, 136, 139, 142À145, 155À159, 162À163, 164f, 168À175, 133, 143, 151À152, 156, 167, 170, 149, 157, 159, 166À168, 171À172, 177, 179, 183, 185À186, 188À191, 177À178, 182À184 176À177, 183, 190, 199, 206, 207t, 193À197, 202, 206, 209À210, Eurycercus glacialis, 110, 203À204 208, 217 215À216, 219, 220t, 224À227, 225f, Eurycercus lamellatus,14t, 21, 48À49, Moina brachiata,15t,19À20, 48, 68, 229À233, 240À241, 245À246, 58, 65, 68, 79, 121, 130À131, 108, 119, 140À142, 208 248À249, 251, 253, 255 154À155, 167, 175À176, 184, 220t Moina irrasa, 148 Daphnia mendotae, 7, 50, 78, 111, Eurycercus longirostris, 143 Moina macrocopa,14t, 16, 27, 48À49, 144À145, 195 Evadne, 10, 108À109, 131, 135À136, 59À62, 72À74, 79À81, 105, 114, 119, Daphnia middendorffiana,76À77, 171, 218 127, 130, 132, 137À138, 140À142, 110À111, 173, 189, 207t 147À148, 175, 189, 194, 208, 220t, Daphnia nivalis, 154 G 246À247 À Daphnia obtusa, 43, 71, 78, 125, 143, Graptoleberis,2À4, 52, 151 Moina micrura, 16, 24 25, 26t, 70, 84, 157, 207t, 220t 167, 207t, 208 À Daphnia pamirensis, 135 136 H Moina mongolica,16t, 74, 146 Daphnia parvula, 7, 147, 226À227 Moina rectirostris ( 5 Moina brachiata), À Daphnia projecta, 154 Holopedium,1 2, 10, 17, 22t,25, 19À20, 48, 119 À À À Daphnia pulex, 7, 9, 13, 14t,15f,15t, 28 29, 42 43, 47 48, 50, 52, Moinodaphnia macleayi,91 À 16À17, 16t,19À20, 26t,28À29, 28t, 88 89, 103, 115, 165, 170, 190, 193, Monospilus, 125 30t,31À32, 36, 43, 46, 50, 59, 61À63, 195, 211 63f,67À68, 72À73, 78, 82, 83t, 84, 87, Holopedium gibberum, 13, 35, 50 O 89, 91À93, 100À101, 110, 121À124, Ophryoxu,52 127, 130À131, 134À136, 140, 142, I Ophryoxus gracilis,35 144À145, 147, 151, 163, 168, Ilyocryptus, 1, 79, 89À91, 95À96, 125, Oxyurella, 125 170À174, 176À177, 179, 182, 186, 149, 153, 181, 184, 259À260 188À191, 193À195, 197, 199À200, Ilyocryptus agilis,89À91 202À204, 202t, 206, 207t, 208À210, Ilyocryptus sordidus, 220t P 209t, 213, 215À216, 220t, 224, Paralona pigra, 154À155 226À229, 233, 238, 240À242, 244, K Penilia, 47, 99, 100f, 107, 109À110, 167, 246À249, 251, 255 193, 260 Kurzia, 168, 171À172 Daphnia pulex var. pulicaria,20f, Penilia avirostris, 108À109 5 28À29, 32, 46, 50, 70, 72, 76, L Peracantha truncata ( Pleuroxus 136À137, 184, 202 truncatus), 168, 171À172, 174 Lathonura, 34, 95À96, 153, 181, 184 Daphnia pulicaria,20f Picripleuroxus laevis, 167 Lathonura rectirostris,84À85, 95À96 Daphnia retrocurva, 123, 169 Picripleuroxus striatus, 33, 79 Latona parviremis,52 Daphnia rosea,45À46, 50, 63, 68, 227 Pleuroxus, 1, 29, 52, 77 Latonopsis, 132, 188 Daphnia schefferi,87À88 Pleuroxus denticulatus, 119, 135, 142, Latonopsis cf. australis, 132 Daphnia schødleri,61 146À147, 172À173 Leptodora,1,6,22t,25À26, 28À29, 35, Daphnia spinulata, 186 Pleuroxus procurvus, 142, 172À173 48, 62, 83, 87, 96, 102, 108À109, Daphnia tenebrosa, 114, 174, 202À203, Pleuroxus trigonellus, 79, 220t 129À130, 133, 142, 159À160, 165, 202t Pleuroxus truncatus, 33, 144, 149, 158, 170À172, 211, 213, 217, 260 Daphniopsis tibetana,16t,22t, 208 167À168, 171À174, 183 Leptodora kindtii, 13, 14t, 28, 144 Diaphanosoma,1,28À29, 46À47, 50, 52, Podon, 108À109, 171, 218 Leydigia,1,45À46, 52 62, 73, 102, 108, 157, 185 Podon intermedius, 162À163, 213À214 Limnosida,52 Diaphanosoma celebensis, 120 Podonevadne, 131, 135À136 Diaphanosoma leuchtenbergiana,62 Polyphemus, 1, 27, 35, 59, 133, 155, 183, Diaphanosoma sarsi,73 M 185, 218 Disparalona rostrata, 131, 158, 167 Macrothrix,1,6À7, 73, 99, 146À147, Polyphemus pediculus,14t, 28, 183À184, Drepanothrix, 34, 153, 177, 181 188 194, 207t, 217 Drepanothrix dentata, 79, 177 Macrothrix triserialis, 147 Pseudevadne, 10, 131 INDEX OF LATIN NAMES OF CLADOCERA 323

Pseudochydorus, 6, 21, 77, 171À172, 183 Scapholeberis mucronata,5f, 28, 158, 126, 130À134, 142, 149, 157, 159, Pseudochydorus globosus, 21, 35, 45À46, 168, 188, 220t 163, 166À168, 171À172, 175, 77, 121, 183 Sida, 1, 34, 47À48, 52, 58, 65, 109, 114, 177À178, 185, 189, 207t, 208 Pseudosida,1,59 121, 125, 149, 159À160, 168, 172, Simocephalus exspinosus,84À85, 207t Pseudosida bidentata,65À66 177, 185, 211À212, 217 Simocephalus serrulatus,58À59, 65, 69 Sida crystallina,14t, 50, 87À88, 160f, Simocephalus vetulus,14t,15t,16t, 17, S 181, 196, 220t 23t,26t, 27, 48, 59, 68, 76À77, 80, 85, Scapholeberis,1,6À7, 34, 82, 101, 133, Simocephalus,1,6À7, 10, 17, 21À22, 87À88, 105, 116, 133À134, 191, 196, 149, 153, 170À172, 174, 182À183 21f, 24, 27, 29, 34, 39, 46À48, 50, 52, 207t, 214À215, 220t Scapholeberis armata, 140À142 58, 67, 69, 73, 77, 79, 82À83, 85À86, Streblocerus serricaudatus,145À146, Scapholeberis kingi, 73, 173 88, 109À110, 114À115, 118À120, 190 Index of Chemical Substances

Note: Page numbers followed by “f” and “t” indicates figure and tables repectively.

A AMP, 61 Carbon monoxide, 80, 132À133, 155, Acetylcholine, 58, 92À93, 96, 113, 161, Androsterone, 41, 136, 140 260 259À260 Anthracene, 30t, 32, 174 Carotene, 24À25, 27 Acid Aprophen, 93 Carotenoids, 13À14, 20À27, 41, 73, acetylsalicylic acid, 196 Arsenic (As), 102À103, 195 126, 133, 146À147, 259 amino acids, 16À17, 26, 35, 39À40, Asparagine, 16 Chitin, 13À14, 17À19, 55, 103, 40f, 101, 115, 157, 194, 251 Astacene, 24À25 108À109, 121, 229, 250À251, 259 arachidonic acid, 43, 62, 64, 189, Astaxanthin, 24À25 Chloretone, 157 192À193 ATP, 61, 116, 161 Chlorine (Cl), 102, 109À110 butyric acid, 42 Atrazine, 32, 139, 139f Chloroform, 157 caffeic acid, 93 Atropine, 58, 93, 123, 163 Chlorophyll, 39, 71À72 docosahexaenoic acid, 43, 62, Chlorpyrifos, 116, 196 192À193 B Cholesterol, 21t,36À37, 40À42, 65À66, docosapentaenoic acid, 41 Bacitracin, 186 72 eicosapentaenoic acid, 36, 41, 45f, Banlen, 124, 148 Cholesterol oleate, 42 122, 174, 189 Benzo(a)pyrene, 30t,104À105, 118, Cholesterol stearate, 42 eicosatrienoic acid, 41 250 Choline, 41, 93, 114 eicosenoic acid, 41 Benzo(a)quinoline, 116 Chromium (Cr), 102À103 fatty acids, Biliverdin, 27 Cobalt (Co), 147 lactic acid, 83, 83f,85 Biphenyls, 105, 158 Congo red, 10, 58 linoleic acid, 41À43, 62À64, 70 Bismarck brown, 10, 17, 99 Copper (Cu), 28À29, 65, 86, 102, 106, linolenic acid, 41, 43À45, 62, 70 115À117, 124, 135, 147, 158, myristic acid, 70 C 191À192, 195 nucleic acids, 17, 61, 201 Cadmium (Cd), 31, 66, 74, 102À103, Copper sulfate, 97, 115, 117 octadecatertraenoic acid, 41, 45 117, 135, 147, 158, 195, 251 Coumarin, 117 À oleic acid, 41, 70 Calcium (Ca), 28t, 29, 32, 58, 61, Cryptoxanthin, 24 25 palmitic acid, 41, 45, 62, 70 66À67, 91À92, 102, 111, 122, 125, Curcumin, 94 palmitoleic acid, 41, 45, 70 131, 174, 191, 259 Cyclophosphane, 157 phosphoric acid, 41, 193 Carbaminoylchloride, 58, 79 Cypermethrin, 51, 72 stearic acid, 41À42, 70, 194 Carbohydrates, 13À14, 15t,17À20, Cytochrome, 27, 77, 81, 83, 116, 118 stearidonic acid, 41, 70 35, 38, 41À42, 44, 56, 57f,59, Acridine, 116 65À66, 73, 82, 84À85, 101, D ADP, 61, 116 133À134, 194 2,4-D, 85À86, 97 Adrenaline, 69, 79, 87, 93, 156, 163 aliphatic carbohydrates, 41 DDT, 30t, 105, 140 Alanine, 16, 16t Carbon (C), 28, 37À39, 41, 64À66, 66f, Digitalin, 93 Amiloride, 112 68, 70, 101, 125, 132, 193, 259 Diglycerides, 41, 45 Aminasin, 93 Carbon dioxide, 49, 66, 81À82, Dihydroergotoxin, 93 Ammonia, 60, 99À101, 193, 197, 260 99À101, 157 Dimethoate, 148, 186

325 326 INDEX OF CHEMICAL SUBSTANCES

DNA, 14À15, 17, 174, 200À201, 203, Isadrin, 93 Nitrogen (N), 1, 13, 28, 60, 100À101 205, 209, 215À216, 228À229, Isocryptoxanthin, 24À25 4-nonylphenol, 126, 135À137, 140 236À238, 243, 250, 255 Isoflurane, 157 Noradrenaline, 79, 93, 163 Dopamine, 94, 163 Isozeaxanthin, 24À25 O E K Oxygen (O), 10À11, 49, 75À80, 82, Ecdysteroid, 41, 125À126, 135 Ketocarotenoid, 24À25, 26t 85À86, 190, 243À244 Echinenone, 24, 26t Eosin, 10, 58 L P EPA, 36, 42, 45, 62À65, 189, 192 Lactose, 93 Polychlorinated biphenyls (PCB), 105, Epinephrine, 49, 58, 93À94, 155À156 Lead (Pb), 29, 32, 102, 114, 117, 127, 158 Ergotamine, 93 192 Phenantridine, 116 Eserinum, 157 Lecithin, 42, 45 Phenazine, 116 Estradiol, 41, 136, 138 Leucine, 16, 16t, 113 Phenol, 51, 85À86, 97, 187À188, 196 Estron, 41, 136 Lincomycin, 186 Phenobarbital, 93, 113, 116 Ethanol, 148, 157 Lindane, 105, 186 Phenylurethane, 140 Ethynylestradiol, 94 Lipids, 19À20, 23t, 35, 38t,40À45, 59, Pheophorbide, 72 61, 73, 192À193, 259 Pheophytin, 72 F Lipoids, 41 Phosphatak, 97 Fenoxycarb, 135, 138À139 Lipopolysaccharides, 41 Phosphatides, 41 Fenvalerate, 105 Lipoproteins, 41 Phosphatidylethanolamine, 23t, 45, Fipronil, 196 Lithium (Li), 28 64À65 Formalin, 27, 114, 157 Lutein, 24, 26t Phospholipids, 23t,41À42, 61, 189 Lutein epoxide, 24À25 Phosphorus (P), 13, 17, 28À29, 39, 49, G 60À61, 68, 70, 102, 116, 253, 259 Glutamine, 16 M Physostigmine, 58, 93, 123 Glutathione, 67, 84, 114, 116, 158, 174 Magnesium (Mg), 29, 72 Physostigmine salicylate, 157 Glycine, 16, 94, 189À190 Malathion, 105, 116 Pilocarpine, 58, 93, 155À156 Glycogen, 14À15, 17, 55, 73À74, 85, Manganese (Mn), 28, 30t, 102À103, Pituitrin, 93 103, 124, 134, 215, 259 115 Potassium (K), 58, 91À93, 191, 195 Glycolipids, 41 Mecholyl, 58, 93, 155À156 Potassium cyanide, 140 Glyphosate, 51, 116À117, 192 Mallory’s stain, 10, 52À53 Potassium dichromate, 114, 148, 195 Guanidine, 58 Mercury (Hg), 77, 102À103, 135, 148, Pirimicarb, 30t, 148, 186 195 Progesterone, 41, 136À137 H Metallothioneins, 103, 118 Propranolol, 93À94 Halothane, 157 Methyl farnesoate, 135, 138, 245À246 Propylene phenoxetol, 157 Hematin, 81 Methylmercury, 103, 148, 195 Prostigmine, 58, 161 À À À Hemoglobin (Hb), 10À11, 20À21, Methylparathion, 51 PUFA, 36 37, 41, 43 45, 62 64, 192 À 26À27, 55, 72, 78À81, 87, 89, 101, Methyl red, 10, 58 Piperonyl butoxide, 37 38, 116, 118 117, 132À133, 155, 191, 216, Methoprene, 116, 136, 138À140 Pyrene, 105, 116 À 242À248, 259 Methoxychlor, 140 Pyriproxyfen, 135, 138 139 Heptachlor, 105, 106f Metrazol, 49, 58 HUFA, 62À64, 70À71 Monoglycerids, 41 Q Hydrocarbons, 104À105, 115À116, 245 Monosaccharide, 44 Quinidine, 58 Hydrogen (H), 15t,85 Mucopolysaccharide, 17 Quinine, 58 Hydrogen sulfide, 85 Myoglobin, 25f,27 Quinone, 26 I N R Iron (Fe), 27À29, 28t, 66, 79À81, 101, Neptunium (Np), 30t, 103 Rivanol, 36 125, 259 Nickel (Ni), 30t, 103, 124 RNA, 14À15, 17, 217 ferric iron, 81 Nitric oxide, 228À229 Roundup, 116À117 INDEX OF CHEMICAL SUBSTANCES 327

S T Uranium (U), 195À196 Saponin, 148 Testosterone, 41, 134f, 135, 137À138 Urea, 60, 100À101, 101f, 193, 260 Selenium (Se), 31À32, 67, 102À103, Tetradifon, 51, 105 Urethane, 157 148À149, 191À192 Tetrapyrrole, 72 Silver (Ag), 104, 195 Triacylglycerols, 23t, 42, 62À65, 133 W À À f Sodium (Na), 29, 42, 102, 109 112, 259 Tributyltin, 137 138, 137 Wax esters, 42 Sodium oleate, 42 Triolein, 42 Waxes, 40À41 Sterins, 41 Tristearin, 42 Sterols, 41, 72 Tyrosine, 16t,26 Strontium (Sr), 29, 67 Z Strophanthin, 93, 117 U Zinc (Zn), 28, 86, 102À104, 116, 124, Sulfolipids, 41 Uranine, 10, 58 191À192 Subject Index

Note: Page numbers followed by “f” indicate figure and those followed by “t” indicate table.

A Banlen, 148 C Abortion of parthenogenetic eggs, Behavior, 181 Cadmium (Cd), 31, 195 135À136 differences, among species, 181À182 bioaccumulation, 103 Acclimation, 256À258 emotional, 184À186 deltaaminolevulinic acid (ALA-D) Acetylsalicylic acid, 196 akinesis, 184 synthesis, 117 Acidity-alkalinity ranges, 190 escape behavior, 185À186 digestion, 74 Adaptation, 256 hydrostatic pressure, 183À184 reproduction, inhibitory effects, 147 Akinesis, 184 swarming, 183 transcriptional response, 251 defined, 184 vertical migration, 182À183 Cadmium chloride, 148 Algal food, 35À36 xenobiotics’ impact, 186 Calcium, 29 Algal lipids, 43À45 Benzo(a)pyrene, bioaccumulation, metabolism, 66À67 Allele, 256 104À105 Calorific value, 13, 14t Alonopsis elongata, slime glands in Bioaccumulation, 102À105 Carbohydrates, 17À19 thoracic limb IV of, 17 inorganic substances, 102À104 chitin, 17À19 Aluminum, 195 organic substances, 104À105 excretion, 101 Amino acids, 16À17, 16t Biochemical analysis, 201 glycogen, 17 Amphigonic eggs, oogenesis, 218 Biofiltration, 193À194 metabolism, 65À66 Amphiosmotic regulation, 110 Blood cells Carbon (C), 28 À Anal water intake, 108 109 adhesion, 96 Carbon dioxide (CO2), 82 Anesthesia, 157 anatomical background, 87À88 Carotenoid content, parthenogenetic Annotation, of genes/genomes, 256 Blood flow, 88À89 eggs, 133 Anomopoda, 1, 206À208 Body form modification, 120À123 Carotenoids, 22À26 diploid chromosome counts, 207t clot formation, 121À122 Carotenoprotein complexes, 16 gut passage time, 69 mechanical damage, 121 CDNA library, 256 thoracic limbs, 46 regeneration, 121 Ceriodaphnia Anoxia, 84À85 turbulence, 121 protein content, 13 Arsenic, 195 Body posture control, 177À179 Ceriodaphnia affinis bioaccumulation, 103 buoyancy, 178À179 senescence, 127 Assimilation of food, 67À72 Bombyx mori, 206À208 Ceriodaphnia reticulata digestion Bosmina glycogen, 17 efficiency, 68À69 akinesis, 184 vertical migration, 186 gut passage time, 68À69 escape behavior, 185 Cesium, bioaccumulation, 103 incomplete, 69À70 swarming, 183 Chemical composition feces, chemical composition, 72 temperatures, 189 amino acids, 16À17, 16t ingested chlorophyll, 71À72 Bosmina longirostris calorific value, 13, 14t unbalanced diet, 70À71 glycogen, 17 carbohydrates temperatures, 188 chitin, 17À19 B Bottom-living species. See Littoral glycogen, 17 Bacitracin, 186 cladocera essential and nonessential elements, À Bacterial food, 36À38 Buoyancy, 178 179 28À29

329 330 SUBJECT INDEX

Chemical composition (Continued) immobilization, 158 Daphnia galeata lipids, 19À20. See also Lipids Cost of resistance. See Resistance chitin, 18 moisture content, 13, 14t trade-offs, parasites RNA:DNA ratio, 17 phosphorus-containing substances, Crawling. See Locomotion Daphnia Genomics Consortium, 17 Crustacea, carbohydrate metabolism 206À208 pigments, 20À27 in, 19, 19f Daphnia hyalina carotenoids, 22À26 Cryoprotectants, dormant egg calorific value, 13 hemoglobin, 26À27 physiology, 146À147 chitin, 18 melanins, 26 Cultures, 9 clearance rate, 193 myoglobin, 27 C-value, 200. See also Genome size filtering rate, 193 slime, 17 ranges of estimates, 202t mortality, 120 salivary gland, 17 Cyanoacrylate glue, 10 water intake, 108 thoracic limb glands, 17 Cyclicity, reproduction, 129À130 Daphnia longispina vitamins, 20 Cyclophosphane, 157 anatomy, 3f xenobiotics, 29À32 Cytogenetics, 205À208 copper, 195 Chemical growth factors, 122À123 Anomopoda, 206À208 phenol, 196 chemomorphosis, 122À123 Onychopoda, 208 temperature, 188 Chemical immobilization, locomotion, overview, 205 vertical migration, 183 157 Daphnia magna Chemomorphoses, 122À123 D acetylsalicylic acid, 196 Chemoreception, 175À176 Daphnia. See also specific biological amino acids, 16 Chitin, 17À19 process and functions biochemical composition, seasonal arthropods, 19 amino acids, 17 variation, 13À15 dead Cladocera, 19 barrier defenses, 225 bioconcentration factors, 30t metabolism, 19 behavioral regions on compound cadmium, 195 Chitinases, 229 eye, 166f calorific value, 13 Chloroform, 157 chitin, 18 chitin, 18 Chlorophyll, 71À72 drinking water quality, 196 copper, 195 Chydorus gibbus, 181 ecoresponsive genome, 240À242 cultivation, 9 Chydorus sphaericus escape behavior, 185 ecological immunology, 232À233 temperatures, 188 genome estimates for comparison of esthetasc, 166f Circulation methods, 202t fatty acids, 23t blood cells glycogen, 17 filtering rate, 193 adhesion, 96 hemoglobin genes, 243À248 genotypes, 224 anatomical background, 87À88 hemoglobin (Hb) content, 11 hydrostatic pressure, 183À184 heart arrest, 95À96 immobilization, 10 hyperactivity, 186 heart rate, 89À92 nephridia position, 6f hyperglycemic hormone-reactive xenobiotics, 97 nervous system neurons, 164f heart regulation, 92À95 neurosecretory areas, 162f immobilization, 10 Cladocera. See also specific biological schematic, 165f muscles, 4f function orientation system, 178f neutral lipids, 23t morphological background, 1À6 negative feedback loops, 179f parasitic infection, 224 size characteristics, 6À7 parasitic infection, 224 pesticides, 197 systematic position, 1 pigments, 21À22 phenol, 196 weight characteristics, 6À7 protein content, 13 phospholipid, 23t Clearance rate. See Biofiltration RNA content, 17 phototaxis, 186 Clonal cultures, 9 salivary gland, 18f potassium, 195 Clot formation, 121À122 somersaulting, 185À186 priming, 233 Collection, of Cladocera, 9 temperatures, 189 radiolabelled strontium removal, Color, parthenogenetic eggs, 132 toxicity testing, 196À197 31f Colored light, perception, 171À172 human physiological liquids, silver, 195 Complementary DNA (cDNA), 256 197 somersaulting, 185À186 Copper (Cu), 29, 195 UV-B irradiation, 189 temperatures, 189, 191À192 SUBJECT INDEX 331

uranium, 195 biochemical analysis, 201 gaseous conditions, 193 vertical migration, 186 densitometry techniques, 201 infochemicals, 194 Daphnia pulex evaluation, 202À203 organic matter, 193À194 acidity-alkalinity ranges, 190 flow cytometry, 201 Environmental factors behavior, 182 flying spot, 201 conditioning. See Environmental DNA content, 17 scanning stage densitometry, 201 conditioning escape-like behavior, 186 DNA methylation patterns, 255 feeding, 50À51 filtering rate, 193 Docosapolyenoic acid, 189 suspended minerals, 51 lipids, 20 Dormant egg physiology, 143À147 lipids, 192À193 temperature, 189 cryoprotectants, 146À147 freshwater fish, 192À193 toxicity testing, 197 drying, 144À146 physiological background, 187À191 UVR, 194 freezing, 146 acidity-alkalinity ranges, 190 vitamins, 20 Down syndrome cell adhesion adaptation, 191 Daphnia pulicaria molecules (Dscams), 227À228 genotypes, 191 mating behavior, 182, 184 Drinking water, 108 illumination, 190 Databases, 249 Drinking water quality, 196. See also oxygen concentration, 190 Dead-man response. See Akinesis Water quality testing temperature, 188À190 Defecation, 58À59 Drying, dormant egg, 144À146 synergism and antagonism, 191À192 Deltaaminolevulinic acid (ALA-D) Dscams. See Down syndrome cell xenobiotics. See Xenobiotics synthesis, 117 adhesion molecules (Dscams) EPA. See Eicosapentaenoic acid (EPA) deltamethrin, 186 Epigenetics, 256 Densitometry techniques, 201 E Epipodites, 211À214 Density, parthenogenetic eggs, 132 Ecological correlates, genome size, EQTL mapping, 256 Detoxification, 117À118 203À205 Escape behavior, 185À186 Diaphanosoma brachyurum, 193 Eggs, parthenogenetic, oogenesis, Essential fatty acids, 41 Dichlorodiphenyltrichloroethane 216À218 Ethanol, 157 (DDT), 105 Eggs and embryos, parthenogenetic Eurycercus, 183À184 Digestion reproduction, 132À135 Eurycercus, slime glands in thoracic anatomical background, 51À53 carotenoid content, 133 limb IV of, 10, 17 glands, 52À53 chemical composition, 132 Eurycercus lamellatus assimilation of food color, 132 akinesis, 184 efficiency, 68À69 cultivation, 133 pigments, 21 gut passage time, 68À69 density, 132 Excretion incomplete, 69À70 environmental factors, 135 anatomical background, 99 defecation, 58À59 hatchability, 133 bioaccumulation, 102À105 fat body, 55À57, 56f hemoglobin, 132À133 inorganic substances, 102À104 histo-hemolymphatic intestinal membrane, 133À134 organic substances, 104À105 barrier, 58 respiration rates, 134 process, 99À102 metabolism, 59À67 RNA concentration, 134 carbohydrates, 101 calcium, 66À67 size, 132 nitrogen compounds, 100À101 carbohydrate, 65À66 xenobiotics, 135 phosphorus compounds, 101À102 lipid, 61À65 Eicosapentaenoic acid (EPA), 189 water, 102 phosphorus, 61 Electromagnetic fields, 175 xenobiotics protein, 60 Emotional reaction, 184À186 elimination route, 105À106 selenium, 67 akinesis, 184 transformation, 105 strontium, 67 escape behavior, 185À186 Extirpation, of eye and ocellus, peristalsis, 58 Enantiomers, 196 168À169 peritrophic membrane, 53À55 Endopolyploidy, 209À211 Eye. See also Vision xenobiotics’ impact, 74 Endoreplication. See Endopolyploidy extirpation, 168À169 Digital image processing, 10 Energy budget, 82 DNA, 17 Enrichment analysis, 256 F DNA estimation and measurement Environmental conditioning, 193À194 Fat body, 55À57, 56f techniques biofiltration (clearance), 193 Fat cell, 214À216 332 SUBJECT INDEX

Fat content, 13 Food consumption quantities, 48À49 gene expression profiles, 248À249 Fatigue, 157À158 littoral cladocera, 48 overview, 235À236 Fatty acids, 19À20, 22t,23t pelagic cladocera, 48À49 palaeogenomics, 252À254 Daphnia magna,23t Food resources physiological plasticity, 243À248 Feces, chemical composition, 72 algal food, 35À36 hemoglobin genes, 243À248 Fecundity, parthenogenetic bacterial food, 36À38 pre-genomics era, 236À238 reproduction, 130À132 organic debris, 38À40 Genotoxicants, 257 constraint at low food Freezing, dormant egg, 146 Global gene expression, 248 concentrations, 131À132 Freshwater cladocera, osmotic Glucosamine, 19 lag effect, 131 regulation, 109À110 Glycogen, 17 resource quality effect, 132 Freshwater fish, lipids in, 192À193 GNBP. See Gram-negative binding resource quantity effect, 131 Functional genomics, 248À252, 256 proteins (GNBP) xenobiotics, 132 Gram-negative binding proteins Feeding G (GNBP), 226À227 anatomical background, 33À35 Gamogenetic reproduction, 140À147 Growth environmental factors, 50À51 dormant egg physiology, 143À147 body form modification, 120À123 suspended minerals, 51 cryoprotectants, 146À147 clot formation, 121À122 food consumption quantities, 48À49 drying, 144À146 mechanical damage, 121 littoral cladocera, 48 freezing, 146 regeneration, 121 pelagic cladocera, 48À49 male physiology, 143 turbulence, 121 food resources chemical sensing, 143 chemical growth factors, 122À123 algal food, 35À36 touch, 143 chemomorphosis, 122À123 bacterial food, 36À38 Gaseous conditions, 193 life span, 119À120 organic debris, 38À40 Gene cluster, 256 mortality, 120 insufficient food, 49À50 Gene conversion (GC), 256 xenobiotics, 123À124 lipids, 40À43 Gene duplication, 256 Gut passage time, 68À69 algal, 43À45 Gene-environment interaction, 256 Gynandromorphism, 142 Cladocera, 42À43 Gene expression, 248À249, 257 essential fatty acids, 41 Gene Ontology (GO) database, 249 H luxury consumption (superfluous Genetic linkage, 257 Hatchability, parthenogenetic eggs, feeding), 49 Genetic specificity, parasites, 230À231, 133 mandible rolling, 45À46 230f Hearing, 177 morphological background, 50 Genome, 257 Heart arrest, 95À96 selectivity Genome assembly, 257 Heart rate, 89À92 labral rejection, 50 Genome size xenobiotics, 97 morphological background, 50 DNA estimation and measurement Heart regulation, 92À95 postabdominal rejection, 50 techniques Hemoglobin genes, 243À248 regurgitation, 50 biochemical analysis, 201 Hemoglobin (Hb), 10À11 thoracic limb strokes’ rate, 46 densitometry techniques, 201 parthenogenetic eggs, 132À133 water clearance rate, 46À48 evaluation, 202À203 pigments, 26À27 littoral cladocera, 46 flow cytometry, 201 respiration, 79À81 pelagic cladocera, 46À48 flying spot, 201 littoral cladocera, 79 xenobiotics’ impact, 51 scanning stage densitometry, 201 pelagic cladocera, 79À81 Female sex hormones, 136À137 ecological correlates, 203À205 Heterologous transfection, 257 Fenvalerate, 105 interspecific variation, 203À205 Histo-hemolymphatic intestinal Filtering rate. See Biofiltration overview, 200À205 barrier, 58 Fipronil, 196 Genome-wide expression, 248À249 Holopedium Fish, lipids in, 192À193 Genomics aluminum, 195 Flow cytometry, 201, 203 Daphnia. See Daphnia Homogenates, 11 Fluorescence analysis, 10 defined, 257 Homologue, 257 Fluorescence in situ hybridization functional genomics, 248À252 Hormesis, 148 (FISH), 206À208 future directions, 254À256 HRE. See Hypoxia-response element Flying spot densitometry, 201À203 trends and suggestions, 255À256 (HRE) SUBJECT INDEX 333

Hydrostatic pressure, 183À184 Inorganic xenobiotics, 194À196 oxygen consumption, 77 Hyperosmotic cladocerans, sodium aluminum, 195 swarming, 183 exchange in, 110À112 arsenic, 195 swimming, 154À155 Hypoxia, 82À84 cadmium, 195 water clearance rate, 46 Hypoxia-inducible factor 1 (HIF-1), copper, 195 Locomotion 257 mercury, 195 anatomical background, 151À152 Hypoxia-response element (HRE), 257 potassium, 195 environmental background, silver, 195 152À153 I uranium, 195À196 fatigue, 157À158 Ibuprofen Interspecific variation, genome size, immobilization, 157 gene expression, 251 203À205 chemical, 157 Ilyocryptus Intravital staining, 10 littoral and bottom-living species, akinesis, 184 Iron, 29, 81 181À182 movement, 181 Isoforms, 257 movement trajectories, 153À155 Immobilization, locomotion, 157 littoral cladocerans, 153 chemical, 157 J pelagic cladocera, 153À154 Immune regulation, 228 Joints, muscles, 156 swimming parameters, 154À155 Immunology muscle physiology, 155À156 infections K joints, 156 barrier defenses, 224À226 KEGG. See Kyoto Encyclopedia of levers, 156 overview, 219À223 Genes and Genomes (KEGG) pelagic cladocera, 182 parasites, avoiding, 223À224 Kyoto Encyclopedia of Genes and surface film, 182 prevention, 223À226 Genomes (KEGG), 249 terrestrial environment, 182 severity, mechanism limiting, 229 stress, 157À158 innate immune system, 226À229 L xenobiotics, 158 Dscams, 227À228 Labral rejection, 50 Luxury consumption (superfluous GNBP, 226À227 Labrum cell, 214À216 feeding), 49 immune regulation, 228 Lag effect, fecundity, 131 pathogen attack, 228À229 Lathonura M pathogen recognition, 226À228 akinesis, 184 Magnesium, 29 PGRPs, 227 movement, 181 Male physiology, gamogenetic PRR, 226 Lead, 29 reproduction, 143 SR-As, 227 Levers, 156 chemical sensing, 143 TEPs, 227 Life span, 119À120 touch, 143 overview, 219À223 xenobiotics, 127 Male sex hormones, 137À139 Infections Lincomycin, 186 chemical compound exposure, 140 overview, 219À223 Lindane, 105 natural factors influencing prevention, 223À226 Lipids, 19À20, 21t, 192À193 formation, 139À140 barrier defenses, 224À226 D. magna,13À15 Mandible rolling, 45À46 parasites, avoiding, 223À224 D. pulex,20 Manganese, bioaccumulation, 103 severity, mechanism limiting, 229 feeding, 40À43 Marine cladocera, osmotic regulation, Infochemicals, 194 algal, 43À45 110 Ingested chlorophyll, 71À72 Cladocera, 42À43 Mechanical damage, 121 Innate immune system, 226À229 essential fatty acids, 41 Mechanoreception, 176À179 immune regulation, 228 freshwater fish, 192À193 body posture control, 177À179 pathogen attack, 228À229 metabolism, 61À65 buoyancy, 178À179 pathogen recognition, 226À228 Moina macrocopa,16 orientation in space, 177 Dscams, 227À228 Moina rectirostris,19À20 Melanins, 26 GNBP, 226À227 Littoral cladocera Mercury, 195 PGRPs, 227 food consumption quantities, 48 bioaccumulation, 103 PRR, 226 hemoglobin, 79 Messenger RNA (mRNA), 257 SR-As, 227 locomotion, 182 Metabolomics, 255, 257 TEPs, 227 movement trajectories, 153 Metschnikowia bicuspidata, 224 334 SUBJECT INDEX

Metschnikowiella bicuspidata, 149 parthenogenetic eggs, 216À218 hemoglobin, 132À133 Microarray, 257 Optical density (OD). See DNA membrane, 133À134 Microcystins, 251À252 estimation and measurement respiration rates, 134 Microcystis aeruginosa, 251À252 techniques RNA concentration, 134 Microscopy, 10 Organic debris, 38À40 size, 132 modern video, 10 Organic xenobiotics, 196 xenobiotics, 135 SEM, 10 acetylsalicylic acid, 196 environmental factors, 132 Migration. See Vertical migration fipronil, 196 fecundity, 130À132 Model organism, 257 phenol, 196 constraint at low food Modern video microscopy, 10 Orthologue (or orthologous gene), 257 concentrations, 131À132 Moina macrocopa,16 Osmotic regulation lag effect, 131 temperature, 188 anatomical background, 107 resource quality effect, 132 X-ray irradiation, 175 environmental background, 107 resource quantity effect, 131 Moisture content, 13, 14t process, 109À112 xenobiotics, 132 Molting, 125À126 amphiosmotic regulation, 110 sex hormones, 136À140 xenobiotics, 126 freshwater cladocera, 109À110 female, 136À137 Mortality, 120 marine cladocera, 110 male, 137À139 Movement trajectories, 153À155 sodium exchange in hyperosmotic Pasteuria ramosa, 224 littoral cladocerans, 153 cladocerans, 110À112 ecological immunology, 232À233 pelagic cladocera, 153À154 turgor, 112 priming, 233 swimming parameters, 154À155 water, 108À109 Pathogen attack responses, 228À229 Mucopolysaccharide. See Slime anal intake, 108À109 Pathogen recognition, 226À228 Muscle physiology, 155À156 drinking, 108 Dscams, 227À228 joints, 156 integuments’ impermeability, 108 GNBP, 226À227 levers, 156 xenobiotics, 112 PGRPs, 227 Myoglobin, 27 Oxygen consumption, 75À79, 193 PRR, 226 bottom-living cladocera, 77 SR-As, 227 N littoral cladocera, 77 TEPs, 227 Narcotization, 157 pelagic cladocera, 77À79 Pathogen recognition receptors (PRR), Neptunium, 103 226 Neurosecretion, 161À163 P Pelagic cladocera neurosecretory cells, 161À163 Palaeogenomics, 252À254, 257 escape behavior, 185À186 neurosecretory substances, 163 Paralogue (or paralogous gene), food consumption quantities, 48À49 Next-generation sequencing (NGS), 257À258 hemoglobin, 79À81 248À249, 255, 257 Parasites, 149, 219, 220t locomotion, 182 NGS. See Next-generation sequencing avoiding, infection prevention, movement trajectories, 153À154 (NGS) 223À224 oxygen consumption, 77À79 Nickel, 103 evolution and coevolution, 229À232 swarming, 183 Nitric oxide (NO), 228À229 genetic specificity, 230À231, 230f swimming, 155 Nitric oxide synthase (NOS) genes, resistance costs, 231À232 vision, 168 228À229 transmission stages, 224 water clearance rate, 46À48 Nitrogen, 28 Parasitic castration, 149 Peptidoglycan recognition proteins excretion, 100À101 Parthenogenetic reproduction, (PGRP), 227 Nitrogen content, 13 130À140 Perception. See also Vision Nuchal organ, 211À214 abortion of eggs, 135À136 colored light, 171À172 Nucleic acids, 17 eggs and embryos, 132À135 polarized light, 171 carotenoid content, 133 UVR, 173À175 O chemical composition, 132 X-rays, 175 Ocellus. See also Vision color, 132 Peristalsis, 58 À extirpation, 168À169 cultivation, 133 Peritrophic membrane, 53 55 Onychopoda, 208 density, 132 Pesticides, 186, 197 Oogenesis, 216À218 environmental factors, 135 PGRP. See Peptidoglycan recognition amphigonic eggs, 218 hatchability, 133 proteins (PGRP) SUBJECT INDEX 335

Phagocytosis, 96À97 Reproduction Scavenger receptor-A proteins Phenol, 196 anatomical background, 129 (SR-As), 227 Phenotypic plasticity, 258 cyclicity, 129À130 Scrambling, 181À182 Phosphorus, 28À29 gamogenetic, 140À147 Selectivity, feeding metabolism, 61 dormant egg physiology, labral rejection, 50 Phosphorus compounds, excretion, 143À147 morphological background, 50 101À102 male physiology, 143 postabdominal rejection, 50 Phosphorus-containing substances, 17 parthenogenetic, 130À140 regurgitation, 50 Photoperiod, 172À173 abortion of eggs, 135À136 Selenium, 67 Phototaxis, 169À171, 186 eggs and embryos, physiology, SEM. See Scanning electron reversal, 170À171 132À135 microscopy (SEM) Phylogeny, 258 environmental factors, 132 Senescence, 126À127 Pigments, 20À27 fecundity, 130À132 Sense organs, 159À161, 163À165 carotenoids, 22À26 sex hormones, 136À140 chemoreception, 175À176 hemoglobin, 26À27 xenobiotics mechanoreception, 176À179 melanins, 26 inhibitory effects, 147À148 body posture control, 177À179 myoglobin, 27 stimulatory effects, 148 buoyancy, 178À179 Polarized light, perception, 171 transgenerational transfer, orientation in space, 177 Polyacetylglucosamine, 17 148À149 vision. See Vision Polychlorinated biphenyls, 105 Resistance trade-offs, parasites, Sex hormones, 136À140 Polygenic trait (quantitative trait), 258 231À232 female, 136À137 Polyphemus pediculus Respiration male, 137À139 emotional reactions, 184 anatomical background, 75 Sham death. See Akinesis swarming, 183 anoxia, 84À85 Sida crystallina Polysaccharide, 17. See also Glycogen carbon dioxide (CO2), 82 phenol, 196 Postabdominal rejection, 50 energy budget, 82 Signal transducers, 228 Potassium, 195 environmental background, 75 Silica (SiO2), 29 Prophenoloxidase (PO) cascade, 229 hemoglobin, 79À81 Silver, 195 Protein littoral cladocera, 79 bioaccumulation, 104 D. magna,13À15 pelagic cladocera, 79À81 Simocephalus metabolism, 60 hypoxia, 82À84 glycogen, 17 Moina macrocopa,16 iron, 81 pigments, 21À22 Proteomics, 258 oxygen consumption, 75À79 temperatures, 189 PRR. See Pathogen recognition bottom-living cladocera, 77 X-ray, 175 receptors (PRR) littoral cladocera, 77 Simocephalus, immobilization of, 10 Pseudochydorus globosus pelagic cladocera, 77À79 Size, 6À7 vertical migration, 183 xenobiotics, 85À86 Slime, 17 Respiration rates, parthenogenetic salivary gland, 17 Q eggs, 134 thoracic limb glands, 17 QPCR. See Quantitative polymerase Resurrection ecology, 258 Sodium, 29 chain reaction (qPCR) Ribosomal DNA (RNA), 17 Sodium exchange, in hyperosmotic QTL. See Quantitative trait loci (QTL) RNA interference (RNAi), 255, 258 cladocerans, 110À112 mapping RNA sequencing (RNA-Seq), 258 Somersaulting, 185À186 Quantitative genetics, 258 Spectrophotometry, 11 Quantitative polymerase chain S Spirobacillus cienkowskii, 224 reaction (qPCR), 248, 258 Salivary gland, 17, 18f SR-As. See Scavenger receptor-A Quantitative trait loci (QTL) mapping, intravital staining, 10, 17 proteins (SR-As) 258 Saponin, 148 Starvation, 72À74 Scanning electron microscopy (SEM), Sterilization, 9 R 10 Stress, 157À158 Regeneration, body form Scanning stage densitometry, 201 Strontium, 29 modification, 121 Scapholeberis metabolism, 67 Regurgitation, 50 swarming, 183 Sulfated mucopolysaccharide, 17 336 SUBJECT INDEX

Surface film, 182 Turbulence, body form modification, X Surgical methods, 11 121 Xenobiotics, 11 Suspended minerals, feeding, 51 Turgor, 112 behavioral impact, 186 Swarming, 183 chemical composition, Swimming, 154À155 U 29À32 littoral species, 154À155 Ultraviolet radiation (UVR), 194 Cladoceran body, pelagic species, 155 perception, 173À175 29À32 trajectories, 155 Ultraviolet (UV)-B irradiation, 189 cytology and metabolic factors, Synteny, 258 Unbalanced diet, 70À71 114À117 Uranium, 195À196 enzyme activity, 115À117 T UVR. See Ultraviolet radiation (UVR) digestion, 74 Tandem gene cluster, 258 excretion Tandemly repeated genes, 258 V elimination route, 105À106 TAR. See Transcriptionally active Vertical migration, 182À184 transformation, 105 regions (TAR) Vision fecundity, 132 Temperature antomical background, 166À175 feeding, 51 ecophysiology, 188À189 bottom-living cladocera, 168 growth, 123À124 low temperatures, 189À190 pelagic cladocera, 168 heart rate, 97 impact, 194 environmental background, 168 inorganic, 194À196 TEP. See Thioester proteins (TEPs) perception aluminum, 195 Terrestrial environment, 182 colored light, 171À172 arsenic, 195 Testosterone, 137À139. See also Male polarized light, 171 cadmium, 195 sex hormones UVR, 173À175 copper, 195 metabolism, 134f, 137 X-rays, 175 mercury, 195 Thioester proteins (TEPs), 227 photoperiod, 172À173 potassium, 195 Through the Looking Glass (Carroll), phototaxis, 169À171 silver, 195 230 reversal, 170À171 uranium, 195À196 Tissues, metabolism, 113À114 surgical methods, 168À169 locomotion, 158 acetylcholinesterase (AChE), extirpation of eye and ocellus, molting, 126 113À114 168À169 organic, 196 acid phosphatase, 114 Vitamins, 20 acetylsalicylic acid, 196 alkaline phosphatase, 114 Vitellogenesis, 217À218 fipronil, 196 choline esterase, 114 phenol, 196 enzymes, 113 W osmotic regulation, 112 phenoloxidase activity, Water parthenogenetic eggs, 135 114 excretion, 102 reproduction proteins, 113 osmotic regulation, 108À109 inhibitory effects, 147À148 Toll receptors, 228 anal intake, 108À109 stimulatory effects, 148 Toxicity. See also Xenobiotics drinking, 108 transgenerational transfer, nutrition, 74 integuments’ impermeability, 148À149 testing, 196À197 108 respiration, 85À86 Transcriptionally active regions Water clearance rate, 46À48 vision, 179 (TAR), 242, 258 littoral cladocera, 46 X-rays, perception, 175 Transcriptomics, 248À249, 258 pelagic cladocera, 46À48 Transgenerational transfer, of Water quality testing, 196À197 Z xenobiotics, 148À149 Weight, 6À7 Zinc, 104