Osmoregulation | With Focus on Fluid and Solute Dynamics in Tardigrada

PhD Dissertation KENNETH A. HALBERG

© Kenneth A. Halberg FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN

Osmoregulation│With Focus on Fluid and Solute Dynamics in Tardigrada

PhD Dissertation Kenneth A. Halberg

UNIVERSITAS Dissertation submitted Monday the 14th of May 2012. HAFNIENSIS Supervisor: Associate Professor Nadja Møbjerg, PhD. 2012 Dissertation presented at University of Copenhagen to be publicly examined (provided acceptance in its current form) in Auditorium 1, August Krogh Building, Universitetsparken 13, Thursday, June 28, 2012 at 14:00 for the degree of Doctor of Philosophy. The examination will be conducted in English.

Abstract Halberg, K. A. 2012. Osmoregulation │With Focus on Fluid and Solute Dynamics in Tardigrada.

Osmoregulation is the regulated control of water and solute composition in body fluid compartments. On one hand, the internal composition must be kept within optimal conditions for metabolic processes in the face of external perturbation. On the other hand, the nature of the living state demands a continuous traffic of compounds in and out of the organism. These demands appear to be in fundamental contradiction however cells and animals achieve so-called “steady-state” by means of an array of transport proteins, which provide a stringent control on the exchange of water and solutes across body surfaces. The distinct mechanisms of solute transport have been studied in most animal groups, but there are still large gaps in our understanding of how animals cope with osmotic stress. In the present thesis, osmoregulatory phenomena were studied in vertebrate and invertebrate organism alike, with the main focus being on fluid and solute dynamics in Tardigrada. For example, the inorganic ion composition of several species was investigated, which revealed that tardigrades contain roughly similar relative contributions of inorganic ions to total osmotic concentration, when compared to closely related animal groups. Moreover, it was inferred that cryptobiotic tardigrades (species able to enter a state of latent life) contain a large fraction of organic osmolytes. The mechanisms of organic anion transport in a marine species of tardigrade was investigated pharmacologically, and compared to that of insects. These data showed that organic anion transport is localized to the midgut epithelium and that the transport is both active and transporter mediated with a pharmacological profile similar to that of insects. Tardigrades survive in a variety of osmotic environments (semi-terrestrial, limnic and marine habitats), why the ability to volume and osmoregulate was examined. These studies demonstrated an ability to regulate total body volume during both hypo- and hyperosmotic conditions, and that the ability to hyper-regulate could be a general theme among members of eutardigrades. Thus, the work presented herein, have contributed to establishing tardigrades as an important experimental group in which central physiological questions may be answered, including aspects of osmotic and ionic regulation.

Keywords: osmoregulation, volume regulation, organic anion transport, hyper-regulate, inorganic ions, organic osmolytes, tardigrade, insect,

Kenneth A. Halberg, The August Krogh Centre, Department of Biology, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark

© Kenneth A. Halberg 2012

“Beautiful is what we see,

More beautiful is what we perceive,

Most beautiful is what we do not understand”

- Niels Stensen List of Papers

This thesis is based on the following papers and manuscripts, which are referred to in the text by their Roman numerals.

I. Halberg, K. A., Larsen, K. W., Jørgensen, A., Ramløv, H. & Møbjerg, N. Cryptobiotic tardigrades contain large fraction of unidentified organic solutes: A comparative study on inorganic ion composition in Tardigrada. II. Halberg, K. A., & Møbjerg, N. (2012). First evidence of epithelial transport in tardigrades: A comparative investigation of organic anion transport. Journal of Experimental Biology, 215: 497-507. III. Møbjerg, N. M., Halberg, K. A., Jørgensen, A. Persson., D., Bjørn M, Ramløv H & Kristensen R. M. (2011). Survival in extreme environments – on current knowledge of adaptations in tardigrades. Acta Physiologica, 202: 409-420. IV. Haugen, B.M., Halberg, K.A., Jespersen, Å., Prehn, L.R. & Møbjerg, N. (2010). Functional characterization of the vertebrate primary ureter: Structure and ion transport mechanisms of the pronephric duct of axolotl larvae (Amphibia). BMC developmental Biology, 10: 56. V. Halberg, K. A., Persson, D., Ramløv, H., Westh, P., Kristensen, R. M. & Møbjerg, N. (2009). Cyclomorphosis in Tardigrada: Adaption to environmental constraints. Journal of Experimental Biology, 212: 2803-2811.

Additionally, the following papers and manuscripts were prepared during the course of my PhD studies, but are not included in the thesis:

VI. Halberg K. A., Jørgensen, A. and Møbjerg, N. (in prep.). Surviving without water: Tun formation in tardigrades is an active process mediated by the musculature VII. Halberg K. A., Persson, D., Jørgensen, A. Kristensen, R. M. and Møbjerg, N. (submitted). Population dynamics of a marine tardigrade: Temperature limits geographic distribution of Halobiotus crispae. Marine Biological Research VIII. Persson, D., Halberg K. A., Jørgensen A., Møbjerg N. & Kristensen R. M. (in review). Neuroanatomy of Halobiotus crispae (Eutardigrada: Hypsibiidae): Tardigrade brain structure suggests inclusion into Panarthropoda. Journal of Morphology. IX. Persson, D., Halberg K. A., Jørgensen A., Ricci C., Møbjerg N. & Kristensen R. M. (2010). Extreme stress tolerance in tardigrades: Surviving space conditions in low earth orbit. Journal of Zoological Systematics and Evolutionary Research, 49: 90-97. X. Halberg, K. A., Persson D., Møbjerg N., Wanninger A. & Kristensen R. M. (2009). Myoanatomy of the Marine Tardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae). Journal of Morphology, 270: 996-1013.

Lastly, following paper provides important background knowledge for the work presented herein:

XI. Møbjerg, N., A. Jørgensen, J. Eibye-Jacobsen, K. A. Halberg, D. Persson & R. M. Kristensen (2007). New Records on cyclomorphosis in the marine eutardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae). Journal of Limnology, 66 (suppl. 1): 132-140.

Reprint and publication is made with permission from the respective copyright holders.

Paper II, V © The Company of Biologists. Paper IV, is copyright of the authors. Paper III © Wiley-Blackwell

Statement of authorship

Paper I: KAH was deeply involved in study design. KAH participated in extracting animals and ion chromatography. KAH performed nanoliter osmometry. KAH performed the data analysis, prepared the figures, participated in discussions and interpretation of the data, and drafted the manuscript.

Paper II: KAH performed the major part of the experimental work and data analysis. He participated in planning of experiments, data interpretation, prepared the figures and drafted the manuscript.

Paper III: KAH performed cell counts and provided images of tardigrades. He helped draft parts of the manuscript.

Paper IV: KAH participated in immunostaining experiments, performed CLSM and prepared the 3D images. KAH participated in discussions and interpretation of the data.

Paper V: KAH participated in planning of experiments, sampling, staging, scanning electron microscopy, DSC experiments, experiments on cold hardiness and osmotic stress tolerance, volume measurements, hemolymph sample collections, and nanoliter osmometry. He furthermore participated in discussions and interpretation of data, prepared the figures and drafted the manuscript.

Front cover: Scanning elecron micrographs of the tardigrades Rictersius coronifer (top left), Halobiotus crispae (middle right), and Echiniscus testudo (middle bottom). Contents

Preface...... 9 Introduction ...... 11 Maintaining a stable internal environment ...... 11 Osmoregulators and osmoconformers ...... 12 Osmoregulatory organs...... 12 Filtration-Reabsorption systems...... 13 Secretion-Reabsorption systems...... 14 Phylum Tardigrada...... 17 General morphology ...... 18 Classification ...... 19 Ecology...... 21 Fluid and solute dynamics – an overview ...... 23 Inorganic ion composition ...... 23 Organic anion transport ...... 25 Volume and osmoregulation...... 26 Conclusions and future perspectives ...... 27 Dansk sammenfatning ...... 29 Acknowledgements ...... 30 References ...... 32

8 Preface

The primary aim of this thesis was to address several aspects of the fluid and solute dynamics in tardigrades, and hereby provide new insight into the general stress biology of these enigmatic creatures. This was done by adopting an integrative approach, i.e. applying advanced methods in biology, analytic biochemistry and physical chemistry, which offered functional data from different disciplines to be obtained. The experimental work was carried out mainly at The August Krogh Centre, University of Copenhagen; however, additional experimental work was performed at the Danish Natural History Museum, University of Copenhagen and at the Department of Nature, Systems and Models, Roskilde University. Overall this thesis has contributed to converting tardigrades, from an almost exclusive taxonomic phenomenon into an established and important experimental group, in which central physiological questions can be investigated. This PhD-thesis comprises a short introduction to osmotic and ionic regulation in Metazoa, accompanied by a brief review on the general morphology, classification and ecology of tardigrades. Moreover, an overview of the results presented as well as conclusions and future perspectives are presented. Five papers and manuscripts form the basis of this thesis, of which four are published in peer review journals (Papers II, III, IV and V), and one is prepared for submission (Paper I). I am the first author of three (Paper I, II and V) and second author on two (Papers III and IV) of these papers. Five additional papers and manuscripts were prepared during the course of my PhD studies, but are not included in the thesis. This work was funded by the 2008 Faculty of Science, University of Copenhagen Freja-Programme.

Copenhagen, the 14th of May 2012

Kenneth A. Halberg

9 10 Introduction

Maintaining a stable internal environment “Life is as a thing of macromolecular cohesion in salty water” (Gilles & Delpire, 1997). Albeit a crude statement, it frames the fact that the ability to control salt and water balance is a fundamental prerequisite for both cellular and animal life. Indeed, the internal environment must usually be kept within relatively narrow limits, as substantial deviations in cell composition are incompatible with the optimal function of macromolecules (lipids, proteins, RNA), and may ultimately modify the rate and extent of cellular reactions (Zhao, 2005). The overall mechanism by which animals conserve a proper osmotic balance between cells, extracellular fluid and the environment is termed osmoregulation. The basis for osmoregulation lies in the strict control of the ionic composition and the osmotic pressure of the intra- and extracellular compartments through the regulated accumulation and loss of inorganic ions and organic compounds (Dawson & Liu, 2009). This regulation is achieved through the coordinated activity of an array of transporter proteins (both energy-consuming and passive), which collectively maintain the steady-state condition of cells and animals (Essig, 1968). During steady-state conditions, compositions of the intra- and extracellular compartments are maintained in a non-equilibrium state (Dawson & Liu, 2009). This uneven distribution of solutes is important for keeping an optimal milieu for metabolic processes (Zhao, 2005). Accordingly, the extracellular fraction of the body fluids of animals are typically high in Na+ and Cl- , and relatively low in the other major ions (e.g. K+, Ca2+ and Mg2+), while the + + 3- intracellular environment of most organisms is low in Na but high in K , PO4 and proteins (e.g. Dawson & Lui, 2009; Paper I). As such, the plasma membrane of cells must maintain ionic, but not osmotic, differences, while specialized excretory organs – e.g. antennal glands of crustaceans, Malpighian tubules of insects and tardigrades, rectal glands of sharks and rays, gills and intestine of teleost fishes, salt glands of birds and reptiles, the kidneys of vertebrates etc. – often maintain both ionic and osmotic differences between animals and their environments (Riegel, 1970; Peaker, 1971; Paper IV, Beyenbach & Piermarini, 2011; Reilly et al., 2011; Whittamore, 2012). In general, mechanisms that allowed organisms to respond and adapt to an osmotic challenge over the course of

11 evolution, has been fundamental to the invasion of new osmotic hostile habitats, and such ecological divergence in turn is an important mechanism for the speciation process (Schluter, 2009). Accordingly, if the ability to osmoregulate had not evolved, life on the planet would look quiet different from how we know it.

Osmoregulators and osmoconformers Generally, animals are divided into two broad categories in terms of their responses to osmotic stress: osmoregulators, which maintain an internal osmolarity different from that of the external environment, and osmoconformers, which conform to the external medium in which they are immersed (Fig. 1). Most vertebrates are strict osmoregulators (e.g. Paper IV), as they maintain ionic and osmotic balance within narrow limits; although hagfish, a basal group of vertebrates, are a notable exception (Sardella et al., 2009). Conversely, marine invertebrates are typically categorized as osmoconformers, as many of them appear to be in osmotic balance with sea water over a range of salinities (Fig. 1). However, there are numerous obvious exceptions including the marine tardigrade Halobiotus crispae as well as several members of Crustacea (e.g. Sarver et al., 1994; Normant et al., 2005; Paper V). In fact, the terms ‘strict osmoregulator’ and ‘strict osmoconformer’ must be used with caution, as typical osmoregulators are forced to conform during the most extreme conditions (e.g. Dowd et al., 2010), whereas animals otherwise described as osmoconformers actually maintain slight differences between the internal and external environment (e.g. van Weel, 1957). Although achieved through different mechanisms, both osmoregulating and osmoconforming animals may tolerate wide ranges of external salinities, thus termed euryhaline species, while animals intolerant of large changes are called stenohaline species.

Osmoregulatory organs Osmoregulatory organs are specialized organs involved in maintaining ionic and osmotic homeostasis in the face of osmotic perturbation, as well as in excreting endobiotic and exobiotic waste products (Dawson & Lui, 2009; Papers II, IV). The specific organs mediating these processes may vary between different groups of animals (see above); however, the molecular basis and specific mechanism of solute and water transport often show a highly convergent/homologous pattern across different types of epithelia (e.g. Paper II). In general, two types of systems

12 Fig. 1 Examples of osmotic performance of representative species from various groups exposed to sea/brackish water, expressed as the relation between internal (extracellular) and external (habitat) osmolality. These data show that osmoconformaty (∆osm=0) is present in invertebrate and vertebrate species alike, albeit strong hypo-regulators and the ability to produce a hyperosmotic urine appears restricted to vertebrates. It should be emphasized that the selected species not necessarily represent the osmotic performance of the entire group, as large difference may exist between even closely related species. For original data see: Robertson, 1949a; van Weel, 1957e; Dice, 1968d; Liggins and Grigg, 1985j; Diehl, 1986b; Normant et al., 2005f; Sardella et al., 2009g; Paper Vc ;Reilly et al., 2011h; Whittamore, 2012i).

have evolved by which the initial process of urine formation takes place i) the filtration-reabsorption type and ii) the secretion-reabsorption type.

Filtration-Reabsorption systems The kidneys of vertebrates (fish, amphibians, reptiles, birds and mammals), and the functional analogs of crusteans and molluscs, maintain extracellular fluid homeostasis by producing urine through the filtration of plasma (ultrafiltration), which is subsequently modified by selective reabsorption and secretion of ions, organic molecules and water (Anderson, 1960; Schmidt-Nielsen, 1963; Riegel,

13 1970; Møbjerg et al., 2004; Paper IV; Whittamore et al., 2012). In vertebrates, three temporally and spatially different kidney generations, the pronephroi, mesonephroi and metanephroi, successively maintain fluid and electrolyte homeostasis during morphogenesis, with the pronephroi constituting the functional kidneys of fish and amphibian larvae (Paper IV). The functional unit of the vertebrate kidney is the nephron, which is composed of a filtration unit and a renal tubule (Anderson, 1960; Møbjerg et al., 2004; Paper IV). The filtration process that takes place in the filtration unit (the glomerulus and Bowman’s capsule) is ‘passive’ i.e. entirely driven by the hydrostatic pressure generated by the heart, whereas the reabsorption and secretion processes take place over specialized epithelia of the renal tubule (Schmidt-Nielsen, 1963). The primary membrane transporter for energizing vertebrate tissue is the Na/K-ATPase (e.g. Paper IV). Accordingly, vertebrate kidneys may produce both dilute, iso-osmotic and concentrated urine relative to the body fluids (Paper IV; Whittamore et al., 2012), which has been a dominant factor in allowing vertebrates to penetrate into all types of habitats on Earth. Filtration-reabsorption systems are capable of processing large volumes of fluids, but are energetically costly, as any substance (e.g. glucose) that has been filtered remains in the urine unless subsequently reabsorbed (Schmidt-Nielsen, 1963). The advantage of such a system; however, is that new potentially toxic compounds are eliminated without the need for developing distinct secretory pathways for each new compound, which may be necessary for the secretion-reabsorption type system (Paper II).

Secretion-Reabsorption systems The Malpighian tubules of insects, and possibly tardigrades (Møbjerg & Dahl, 1996; Papers II, III, V), are the functional analogs of the vertebrate kidney, but constitute a secretion-reabsorption system that produces urine in a fundamentally different way than the filtration-reabsorption-systems (Beyenbach & Piermarini, 2011). In the absence of blood vessels (i.e. a closed circulatory system), the hemolymph of insects is circulated at pressures insufficient for filtration, and the Malpighian tubules thus form the (primary) urine entirely by secretion (Beyenbach & Piermarini, 2011). The formation of the primary urine is generally initiated in the distal segments (blind-ended tip) of the Malpighian tubule, and is essentially iso-osmotic (consisting mainly of KCl and NaCl) to the hemolymph (Williams & Beyenbach, 1983). The subsequent reabsorption of water, ions and metabolites in proportions that maintain extracellular homeostasis (a process analogous to that of vertebrates) takes place in downstream structures i.e. proximal tubule, hindgut and rectum (O’Donnell & Maddrell, 1995; Coast, 2007). The final urine composition is

14 adjusted in the rectum (Coast, 2007) and may be either strongly hypo- or hyperosmotic depending on the species and its physiological status (Maddrell & Phillips, 1975; Reynolds & Bellward, 1989). In contrast to vertebrate epithelia, the V-type H+-ATPase is considered ubiquitous in energizing insect epithelia (Beyenbach & Piermarini, 2011); although the Na/K-ATPase is still expressed and functionally relevant for tubular function (Torrie et al., 2004; Paper II). In fact, energized by the H+-ATPase, some of the highest fluid secretion rates per unit area membrane from any tissue have been reported from hematophagous insects (e.g. Aedes aegypti, Rhodnius prolixus) after a blood meal (Williams & Beyenbach, 1983; Maddrell & Phillips, 1975). In addition to playing a key role in osmoregulation, new properties of the Malpighian tubules of insects have emerged in recent years, which suggest that Malpighian tubules are involved in such diverse functions as renal detoxification, metabolism of toxins and immune system responses (Dow & Davies, 2006; Paper II). As holds for insects, ultrastructural studies on the Malpighian tubules of tardigrades indicate that they function as secretion-reabsorption systems involved in fluid and solute transport (Weglarska, 1987; Møbjerg and Dahl, 1996; Peltzer et al., 2007). They are positioned at the transition zone between the midgut and rectum of eutardigrades (Fig. 2), and the positional conformity between insects and eutardigrades has been used as a strong argument for their homology (Greven, 1982; Møbjerg and Dahl, 1996). However, at present no functional data exist relating the Malpighian tubules of tardigrades to an osmoregulatory role. Accordingly, functional studies on the fluid and solute dynamics of tardigrades are greatly needed (Papers I, II, III, V), and due to the close affinity to the euarthropod complex (Aguinaldo et al., 1997), would be highly useful in understanding and reconstructing the evolution of osmoregulation in Insects and other arthropods.

15 Fig. 2 Structure and organization of the Malpighian tubules of the marine eutardigrade Halobiotus crispae. dm, dorsal Malpighian tubule; dp, distal part; is, initial segment; mg, midgut; mv, microvilli,; nu, nucleus; pp, proximal part; re, rectum; From: Møbjerg & Dahl, 1996.

16 Phylum Tardigrada

Tardigrades, also known as water bears, are among the smallest multi-cellular animals on the planet (0.1-1.2 mm). They were discovered in 1773 by the German pastor J. A. E. Goeze, who described them as “kleiner Wasserbär”, or little water bear, due to their strong resemblance to a tiny bear (Ramazzotti & Maucci 1982). Not long after the current name “Tardigrada” was given by the Italian naturalist Spallanzani in 1776 (Lat. tardus – slow, grado – walker). In response to unfavorable environmental conditions, many species of tardigrades have the ability to enter the ametabolic state of suspended animation, also known as cryptobiosis, in which the organism is neither dead nor alive (Møbjerg et al., 2011; Fig. 3). The animal can remain in this state for as much as 20 years (Jørgensen et al., 2007), yet once external conditions again become favorable, the tardigrade resumes activity unaffected. This incredible ability is shared with selected species of nematodes, rotifers and arthropods (Glasheen & Hand, 1988; Crowe & Maddin, 1974; Ricci et al., 2003). In 1962, Tardigrada was recognized as a phylum by Ramazzotti in Il Phylum Tardigrada. Presently, there are more than 1000 described species (Guidetti & Bertolani 2005; Degma & Guidetti, 2007; Degma et al., 2012); however, it has been estimated that several thousand species remain undescribed (Paper III). Tardigrada is included in the invertebrate superclade Ecdysozoa (Aguinaldo et al. 1997); however, their precise phylogenetic position is still being debated. Both molecular and morphological investigations produce conflicting conclusions, and it is currently unclear whether the group is more closely related to the nematodes and nematomorphs or to arthropods and onychophorans (Aguinaldo et al. 1997; Dunn et al. 2008; Zantke et al., 2008; Edgecombe 2010; Rota-Stabelli et al., 2010; Cambell et al., 2011). Regardless, this group is closely related to one of the two most species-rich and economically important groups Nematoda or Arthropoda, and thus maintains a central position in relation to the two major invertebrate model organisms, i.e. Caenorhabditis elegans Maupas, 1900 and Drosophila (Sophophora) melanogaster Meigen, 1830 (Gabriel et al. 2007; Goldstein and Blaxter 2002).

17 Fig. 3 Scanning electron micrographs showing the external morphology of Richtersius coronifer (Eutardigrada) A. lateral view of the active, hydrated state B. Dorsal view of the dehydrated, cryptobiotic state C. Ventral view of the dehydrated, cryptobiotic state. The pictures illustrate the morphological changes associated with entry into an ametabolic state (i.e. cryptobiosis), which include the retraction of head and limbs into the body cavity, and the formation of a compact shape – the tun. From: Paper VI.

General morphology Tardigrades are bilaterally symmetric micrometazoans with a body divided into five separate body segments, i.e. a cephalic segment, containing a mouth, eyespots

18 and sensory organs (papillae cephalica or cirri and clavae), and four trunk segments (Nelson, 2002; Fig. 3A). The first three trunk segments each bear a pair of lateroventrally directed legs, while the terminal trunk segment bears a pair of posterioventrally directed legs (Figs. 3, 4). The legs typically terminate in 4 to 13 claws or suction discs (Nelson, 2002). Tardigrades are ventrally flattened with a convex dorsal side, and are covered by a segmented cutinous cuticle, which is periodically shed during molting – formation of the new cuticle is maintained by a single layer of epidermal cells (Nelson, 2002). The digestive system consists of a foregut, midgut and a hindgut with a pair of stylets and stylet glands flanking the buccal tube. Three glands (the Malpighian tubules) are positioned at the transition zone between the midgut and hindgut in eutardigrades (Weglarska, 1987; Møbjerg and Dahl, 1996; Peltzer et al., 2007). Tardigrades posses a hemocoel-type of fluid- filled body cavity, i.e. an open circulatory system as seen in arthropods and nematodes, which likely functions in circulation and respiration. The somatic musculature is composed of structurally independent muscle fibers, which can be divided into a dorsal, ventral, dorsoventral, and a lateral musculature in addition to a distinct leg musculature (Schmidt-Rhaesa & Kulessa, 2007; Fig. 4). Moreover, the buccopharyngeal muscles, intestinal muscles and cloacal muscles comprise the animal’s visceral musculature. Whereas cross striation of the somatic musculature is especially pronounced in Arthrotardigrada, the somatic muscles of Eutardigrada are described as an intermediate between smooth and obliquely striated (Walz, 1974). The nervous system of tardigrades consists of an (at least) three lobed brain (Fig. 5) and a ventral nerve cord with four fused paired ganglia that shows a clear segmental organization.

Classification Originally based on morphological characters, tardigrades are divided into two main evolutionary lines, represented by the extant lineages Eutardigrada and Heterotardigrada. The validity of a third class, Mesotardigrada, is currently considered dubious (Ramazzotti and Maucci, 1983). Heterotardigrada consists of the orders Arthrotardigrada and Echiniscoidea with arthrotardigrades possessing the most plesiomorphic characters. Arthrotardigrada consists exclusively of marine species (Renaud-Mornant 1982; Jørgensen et al. 2010), and are morphologically the most diverse group. They are present in all oceans from intertidal zones to abyssal depths, and inhabit various types of sediment. In contrast, the Echiniscoidea comprises both limno-terrestrial, limnic as well as marine species with the majority of the described species belonging to the family Echiniscidae. Taxonomically, the main characters

19 Fig. 4 Tardigrade musculature as revealed by fluorescently coupled phalloidin in combination with confocal laser scanning microscopy three-dimensional reconstruction A. Lateral view of Halobiotus crispae (Eutardigrada), Paper VIII B. Ventral view of Echiniscoides sigismundi (Heterotardigrada), unpublished data. separating the two groups from Eutardigrada include a separate gonopore, a closed three-lobed anus as well as well-developed cephalic-, trunk-, and leg appendages (Guidetti and Bertolani, 2005). The eutardigrades are divided into the two orders; Apochela and Parachela. Both orders contain mainly limno-terrestrial species, albeit with a few exceptions –

20 Fig. 5 Conceptual drawing based on immunofluorescent and ultrastructural data, showing an interpretation of the brain structure of Halobiotus crispae. A. Lateral view B. Frontal view. clg, claw gland; co, connective; dc, dorsal commissure; ey, eye; g0, sub-pharyngeal ganglion; gI, first ventral trunk ganglion; ic, inner connective; il, inner lobe; lgg, leg ganglion; mg, median ganglion; mo, mouth opening; oc, outer connective; ol, outer lobe; pc, papilla cephalica; pb, pharyngeal bulb; st, stylet; t, temporalia; vll, ventrolateral lobe. From: Paper VIII.

most notably the marine genus Halobiotus (see Paper III). In general, eutardigrades are cylindrically shaped with a more or less distinct segmentation, and exhibit a relatively uniform morphology (Fig. 3A). The key characters of eutardigrades include a cloaca (combined gonopore and anus), the presence of Malpighian tubules and a strong reduction of cephalic sensory structures (Guidetti and Bertolani, 2005). Structures such as the bucco-pharyngeal apparatus and claw shape are important taxonomic characters within Eutardigrada.

Ecology Tardigrades occupy a range of moisture regimes and often constitute a major component of meiofaunal communities in terrestrial, limnic and marine ecosystems throughout the globe. However, they are distinctly aquatic organisms, requiring a film of water to be active. Tardigrades are predominantly egg-laying, with both sexual and parthenogenetic modes of reproduction described (Bertolani, 2001). Molting occurs continuously throughout their lifecycle, which may be between 3-30 months (Nelson, 2002). Populations of tardigrades have been studied in a variety of habitats; including mosses, lichens, leaf litter and soil, and

21 Fig. 6 Population dynamics of the marine tardigrade Halobiotus crispae showing a unimodal pattern of maximal frequency. Graphic representation of sampling data (2006-2012) comparing the temporal pattern in abundance of H. crispae to abiotic parameters (temperature, (─ ─); salinity, (- - -); pH, (- ─ -), and the seasonal appearance of the different cyclomorphic stages (shown on top), from the locality of Vellerup Vig, Isefjord, Denmark. Light grey area is the period in which exuvia containing eggs were found, and thus indicates the period of sexual reproduction. From: Paper VII. the life history and population dynamics have received some attention (Martinez, 1975; Morgan 1977; Guidetti et al. 1999; Uhía and Briones 2002; Jönson 2003; Suzuki 2003). Tardigrade population dynamics may show both unimodal and bimodal patterns of annual variation (Martinez, 1975; Morgan, 1977; Fig. 6), albeit the specific pattern appears to be both species and habitat specific. Factors such as temperature, moisture and food availability have been suggested to be correlated with population density (Hallas & Yeates, 1972; Morgan, 1977). However, other factors including competition, predation and parasitism may play a role in controlling population density and diversity (Nelson, 2002). Tardigrades possess an amazing reproductive capacity, as indicated by the large changes in animal density on short temporal scales (Morgan, 1977).

22 Fluid and solute dynamics – an overview

Inorganic ion composition Knowledge of the composition as well as concentrations of dissolved particles in internal fluids of an organism, and how these change during various exposures, is fundamental to the understanding of its basic physiology. However, practically nothing is known about these aspects in tardigrades, which has been a major obstacle to the study of the fluid and solute dynamics in these animals. In Paper I, the inorganic ion content of five different species (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) covering both a large phylogenetic and habitat spectrum was analyzed (Fig. 7A). These data demonstrated that Na+ and Cl- are the principle inorganic + + 2+ ions in tardigrade fluids, albeit substantial concentrations of K , NH4 , Ca , 2+ - 2- 3- Mg , F , SO4 and PO4 were also detected. Moreover, tardigrades appear to contain roughly similar relative contributions of the respective inorganic ions to total osmotic concentration, when compared to selected species of arthropods, nematodes and onychophorans (Hobson et al., 1952; Sutcliffe, 1962; Campiglia, 1975; Wilder et al., 1998; Normant et al., 2005), albeit a large relative contribution of calcium appears characteristic of tardigrade fluids. Inorganic ions destabilize macromolecules and affect the rate and extent of metabolic reactions at high concentrations, which invariably leads to impairment of cellular function (Zhao, 2005; Yancey, 2005). However, apparently R. coronifer does not exclude inorganic ions during dehydration (Fig. 7B), which suggests a concomitant accumulation of organic solutes. Moreover, as evidenced by the differences between the calculated osmotic concentrations of the known ions and the measured total osmolarity in the different species (Fig. 7A), it was inferred that cryptobiotic tardigrades (in steady-state) contain a large fraction of unidentified organic osmolytes. Organic osmolytes can be divided into a few major categories (sugars, polyols, amino acids and various derivatives), and several of these groups possess known protective functions in relation to osmotic stress (Hincha and Hagemann, 2004; Yancey, 2005). Accordingly, the future detection and analysis of such compounds are likely to provide new insight into the biochemistry and physiology of superior tardigrade adaptations.

23 Fig. 7 Ionic contributions to total osmotic concentration A. Concentrations (mM) of the respective cations and anions measured in each investigated species, as well as the corresponding total osmotic concentration (mOsm/kg). The blank area represents the osmotic deficit (OD). B. Concentrations (mg/l) of the respective cations and anions measured in hydrated, active specimens compared to dehydrated cryptobiotic animals of Richtersius coronifer. Data are expressed as mean ± s.d. From: Paper I.

24 Organic anion transport The ability to excrete endogenous waste products as well as environ- mental toxins is an essential step to avoid these compounds reach toxic levels and to keep metabolic reactions within optimal conditions. One of the better known systems involved in such an excretion is the organic anion transport system, the function of which has been studied in several vertebrate (fish, amphibians, reptiles, birds and mammals) and invertebrate (nematodes and insects) model organisms (George et al., 1999; Dantzler, 2002; Dow and Davies, 2006). In Paper II, these data were expanded as the sites, characteristics Fig. 8 Tentative cellular model for the and pharmacological profile of the transepithelial transport of organic anions in transepithelial transport of chloro- tardigrades. From: Paper II. phenol red (CPR), a prototypical substrate of the classic organic anion secretion pathway, was investigated in the tardigrade Halobiotus crispae Kristensen, 1982 and compared to corresponding data from the desert locust Schistocerca gregaria Forskål, 1775. This was done by introducing a new method for quantifying non-fluorescent dyes. Our study revealed i) that tardigrades posses an organic anion transport system, ii) that it was localized to the midgut epithelium, and iii) that organic anion secretion was both active and transporter mediated, with possible members of the SLC21/SLCO transport families mediating the basolateral entry step in tardigrade midgut cells. Transport by insect Malpighian tubules showed a similar pharmacological profile, but higher concentrations of CPR were achieved. Based on the observed transport characteristics in the presence and absence of transport inhibitors, a tentative cellular model for the transepithelial transport of CPR in tardigrades was suggested (Fig. 8). Specifically, a large lumen positive potential generated by the H+-ATPase could provide the driving force for accumulation of anions in the lumen, although the exact coupling between electrochemical gradients generated by the pumps and transport of ions is unknown. This study was the first to provide evidence for epithelial transport in tardigrades.

25 Volume and osmoregulation Tardigrades survive in a variety of osmotic environments and must protect the internal tissues from the vagaries and extremes of the external environment. The secondary marine species Halobiotus crispae colonizes habitats characterized by especially large fluctuations in external salinity, why the ability to respond to an osmotic challenge was investigated in this species (Paper V). Animals were exposed to both hypo- and hyperosmotic media and the subsequent changes in total body volume and internal osmotic pressure were recorded. These data revealed that H. crispae is able to regulate total body volume back to control values when immersed in hypotonic solutions, yet was unable to do so in concentrated media. Instead a new steady-state was achieved significantly below control conditions. Animal activity was only markedly affected in very dilute media, suggesting a possible effect on neuro-muscular function at low salt concentrations. Conversely, when analyzing the concomitant changes in hemolymph concentrations, it appeared that H. crispae is a euryhaline osmoconformer, in which the hemolymph osmotic pressure is largely governed by the external environment. However, expressing the hemolymph osmolality measured during exposure to dilute as well as concentrated media as a function of external salinity revealed that H. crispae is in fact a strong hyper-regulator (Fig. 9A). Expanding these studies to include the limno-terrestrial species Richtersius coronifer Richters, 1903 (Paper III) showed that this is could be a general feature of all eutardigrades (Fig. 9B), which was further supported by data presented in Paper I. The ability to hyper-regulate indicates the excretion of dilute urine. The three glands positioned at the transition zone between the midgut and rectum of eutardigrades, are generally considered to have an excretory function. This assumption is based on the probable homology of the Malpighian tubules of eutardigrades and insects (Greven, 1982; Møbjerg and Dahl, 1996), and on several ultrastructural studies that shows that the epithelium is likely involved in fluid and solute transport (Weglarska, 1987; Møbjerg and Dahl, 1996; Peltzer et al., 2007). However, data in support of an osmoregulatory function of both the rectum (Dewel and Dewel, 1979) and midgut (Paper II) also exists, which emphasizes the need for functional studies on these organs at both the molecular and cellular level.

26 Fig. 9 Osmotic performance of A. Halobiotus crispae and B. Richtersius coronifer during exposure to media of varying osmotic strength. From: Paper III.

27 Conclusions and future perspectives

This dissertation has provided new insight into the fluid and solute dynamics of metazoans, particularly relating to one of the most enigmatic groups on the planet – the tardigrades. Using a multi-disciplinary approach, crucial information was provided on both organs and systems of several species, representing vertebrates, arthropods and tardigrades, and general patterns in especially tardigrade physiology have emerged (e.g. Paper III). By comparing our data on tardigrades to several evolutionary related groups, including nematodes, onychophorans and arthropods, basic physiological principles have been discovered (e.g. Paper II), which emphasizes the importance of comparative physiology. In this respect, future work on tardigrade physiology could encompass an extension of the work presented herein. Additional species from different habitats and evolutionary lineages should be investigated, in order to further explore the diversity as well as common trends in tardigrade biology. In particular, studies on marine cryptobionts (e.g. Echiniscoides sigismundi) and additional non- cryptobiotic species would help clarify whether the osmotic deficits observed in cryptobiotic animals (Fig. 5; Paper I) in fact are related to cryptobiotic ability or alternatively to habitat preference. Regardless, large scale analyses and characterization of the organic solutes of cryptobiotic tardigrades should be performed, which surely would provide an enhanced resolution of several aspects relating to tardigrade stress responses. Other studies could include a characterization of the volume and osmoregulatory capacity of heterotardigrade species. Our results on eutardigrades show a capacity to hyper-regulate over a broad range of external salinities (Fig. 7); however, do heterotardigrades without Malpighian tubules posses the same ability? True limnic species (e.g. Bertolanius nebulosus) should also be investigated. In addition to the suggested whole animal experiments, studies on the molecular and cellular level are needed to fully understand how fluid and electrolyte homeostasis is achieved in these animals. For example, electrophysiological investigations using single cell glass microelectrode impalements on dissected native tissue would help characterize the cellular transporters involved in e.g. urine formation. Collectively, these studies would be important to elucidate how tardigrades functionally have solved colonizing every major type of habitat on the planet, and perhaps be useful in reconstructing how osmoregulation has evolved in metazoans.

28 Dansk sammenfatning

Osmoregulering er kontrollen af kropsvæskernes sammensætning af vand og opløste stoffer. Foruden at opfylde de optimale betingelser for metaboliske processer under indflydelse af eksterne påvirkninger, skal denne sammensætning samtidig imødekomme den kontinuerlige transport af stoffer ind og ud af den levende organisme. Disse krav forekommer umiddelbart fundamentalt uforenelige, men celler og dyr opnår et såkaldt ”steady-state” ved hjælp af et spektrum af transportproteiner, der udøver streng kontrol over udvekslingen af vand og opløste stoffer over forskellige kropsoverflader. De forskellige transportmekanismer ansvarlige for denne kontrol er blevet undersøgt i de fleste dyregrupper, men vores viden om hvorledes dyr håndterer osmotisk stress, er stadig ufuldstændig. I dette arbejde er osmoregulatoriske fænomener blevet undersøgt i såvel vertebrater som invertebrater, omend der hovedsageligt er blevet fokuseret på dynamikkerne af vand og opløste stoffer i Tardigrada. Eksempelvis blev sammensætningen af uorganiske ioner undersøgt i flere forskellige arter af bjørnedyr, hvilket afslørede, at de relative bidrag af uorganiske ioner til den totale osmotiske koncentration overordnet set er ens i bjørnedyr og nært beslægtede dyregrupper. Desuden blev det udledt, at kryptobiotiske bjørnedyr (arter, der er i stand til indtræde i et stadie af latent liv) indeholder en stor andel af organiske osmolytter. Mekanismerne for aniontransport blev undersøgt farmakologisk i en marin art af bjørnedyr, og sammenlignet med de tilsvarende mekanismer i insekter. I bjørnedyret blev den organiske aniontransport lokaliseret til epitelet i midttarmen, og viste sig at være en aktiv transport, med en farmakologisk profil svarende til den i insekter. Bjørnedyr kan overleve et bredt spektrum af osmotiske miljøer (semiterrestrisk, limnisk og marine habitater), hvorfor evnen til at volumen- og osmoregulere blev undersøgt. Disse studier demonstrerede at bjørnedyr kan regulere den samlede kropsvolumen under såvel hypo- som hyperosmotiske forhold, samt indikerede at hyperregulering kan være en gennemgående tendens hos eutardigrader. Indeværende arbejder har altså bidraget til at fastslå bjørnedyr som en vigtig eksperimentel gruppe, hvori centrale fysiologiske spørgsmål kan besvares, heriblandt aspekter af ionregulering og osmoregulering.

29 Acknowledgements

During the course of my PhD studies I was fortunate to have the generous help of many great people, which has made this work possible. First and foremost, I thank my supervisor Nadja Møbjerg for being an unwavering source of support, encouragement and guidance during past years, but also for constantly challenging me to develop intellectually as well as a researcher. I truly appreciate all you have done for me, and hope to be able to continue our collaboration in the future. I also greatly appreciate the support of Reinhardt Møbjerg Kristensen (close to being my second supervisor) who initially introduced me to strange world of tardigrades, and who took me under his wing, offering me guidance and support (and equally important coffee). You have had enormous impact on my education as a young researcher, and I have sincerely enjoyed working with you. I have had the great fortune of working with many wonderful colleagues at University of Copenhagen, both at The August Krogh Center and the Natural History Museum who all are warmly thanked. Thanks to you it has been a pleasure coming to work every day. I am especially grateful to Erik Hviid Larsen with whom I shared many interesting discussions, and who was an endless source of inspiration. Aslak Jørgensen is thanked for great collaborations and discussions over the years as well as for critical review of my thesis. Dennis Krogh Persson is thanked for being a good colleague, but more importantly, a good friend. Since we started this journey in research together, I have come to depend on our collaborations and camaraderie, which has been an invaluable help along the way (by the way, it is your turn to buy cake). I thank Jette Lyby Michelsen for technical assistance over the years. During my visits to Roskilde University I was fortunate to collaborate with several great people. I especially thank Hans Ramløv for allowing me to come work in his laboratory (on several occasions), which scientifically turned out to be quite fruitful. I have particularly enjoyed our conversations and discussions, and I thank you for both your encouragement and support. I also thank Kristine Wulff Larsen for unparalleled project teamwork as well as our many conversations on everything and anything. It was an absolute pleasure,

30 which I hope to repeat some time in the future. Peter Westh is thanked for allowing me to work in his laboratory and for his enormous expertise and help. Perhaps most importantly I thank the help and support of family and friends. I especially thank my parents and parent in-laws for their invaluable support during difficult times, and for helping me and my family in more ways than I care to mention. You’re the best! Lastly I thank my girlfriend Iben Rønn Veland for helping me, supporting me, tolerating me and loving me, but mostly for taking care of our son when I was working – none of this was possible without your help! I also thank my little son Hannibal for tolerating that I had to work sometimes, and couldn’t be home when you wanted me to. I am looking forward to making up for lost time. I love you both more than you know. Thank you. This work was funded by the 2008 Faculty of Science, University of Copenhagen Freja-Programme.

31 References

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35

UNIVERSITAS HAFNIENSIS 2012

36 Paper I

Inorganic ion composition in Tardigrada: cryptobionts contain large fraction of unidentified organic solutes

1,* 2 3 2 Kenneth Agerlin Halberg , Kristine Wulff Larsen , Aslak Jørgensen , Hans Ramløv and Nadja Møbjerg1

1 Department of Biology, the August Krogh Centre, University of Copenhagen, Universitetsparken 13, DK- 2100 Copenhagen Ø, Denmark, 2 Department of Nature, Systems and Models, University of Roskilde, Universitetsvej 1, DK-4000 Roskilde, Denmark and 3 Laboratory of Molecular Systematics, Natural History Museum of Denmark, University of Copenhagen, Sølvgade 83, DK-1307 Copenhagen K, Denmark *Author for correspondence ([email protected])

Submitted June 2012

SUMMARY Tardigrades are a group of micrometazoans known to tolerate extreme environmental stress. Significant efforts have been devoted to the field, however; mechanisms explaining the extreme adaptations found among tardigrades is still lacking. Here we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (E. testudo, M. tardigradum, R. coronifer, M. cf. hufelandi and H. crispae) using high-performance anion-exchange chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na+ and Cl- are the principle inorganic ions + + 2+ 2+ - 2- 3- in tardigrade fluids, albeit substantial concentrations of K , NH4 , Ca , Mg , F , SO4 and PO4 also were detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared to that of the external medium (Na+, ×70-800; K+, ×20-90; Ca2+ and Mg2+, ×30-200; Cl-, ×20-50; 2- + - 2- SO4 , ×30-150), whereas in the marine species H. crispae Na , Cl and SO4 are almost in ionic equilibrium with (brackish) salt water, while K+, Ca2+ and Mg2+ are slightly concentrated (×2-10). However, there is an anion deficit of ~120 mEq/l in M. tardigradum and H. crispae, indicating that there are ionic components that remain unidentified in these species. Body fluid osmolality ranged from 361±49 in R. coronifer to 961±43 mOsm/kg in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not exclude large amounts of ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicates that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.

Key words: tardigrades, inorganic ions, ion chromatography, nanoliter osmometry, organic osmolytes, cryptobiosis

INTRODUCTION intensive research efforts have been devoted to the Tardigrades are a group of minute multi-cellular field, as the translational output associated with a animals that are known to tolerate extreme detailed understanding of their complex stress environmental stress (Guidetti et al., 2010; biology is expected to include new methods for Møbjerg et al., 2011). This capacity derives preserving and stabilizing biological materials mainly from their ability to enter a state latent of (Wełnicz et al., 2010). Several advances have been life, i.e. cryptobiosis, in which their resistances to made in our understanding of tardigrade stress adverse environmental conditions are greatly responses, especially regarding i) the role of increased (Møbjerg et al., 2011). In recent years, selective carbohydrates (trehalose), ii) the K. A. Halberg and others differential expression of stress proteins (heat 1834 and Milnesium tardigradum Doyère, 1840 shock proteins and late embryogenesis abundant were extracted from moss collected at Öland, proteins), and iii) the identification of anti- Sweden, while Echiniscus testudo Doyère, 1840 oxidant defenses and DNA repair mechanisms was found in moss collected at Nivå, Denmark. (see reviews by Guidetti et al., 2010; Wełnicz et These species were extracted by washing the al., 2010; Møbjerg et al., 2011). However, a respective moss samples with tap water through general mechanism explaining the extreme six different sieves of progressively smaller mesh adaptations found among tardigrades is still size, so as to concentrate the tardigrades and to lacking. Consequently, new approaches that may remove large debris. Specimens of Halobiotus provide increased insight into the superior stress crispae Kristensen, 1982 were isolated from adaptations of tardigrades are greatly needed. marine algae and sediment collected from Knowledge of the composition as well as Vellerup Vig, Denmark according to the method concentrations of dissolved particles in internal of Halberg and Møbjerg (2012). The total number fluids is fundamental to the understanding of of animals used in all experiments was 2220 R. basic physiological processes, such as fluid and coronifer, 326 M. tardigradum, 630 M. cf. electrolyte homeostasis, signal transduction and hufelandi, 426 E. testudo and 268 H. crispae. solute transport. Accordingly, such data have been provided for most major groups of animals Inorganic cation and anion analysis (cnidarians, echinoderms, annelids, molluscs, The dominant cations and anions present in the crustaceans, insects, chelicerates, tunicates, fish, different tardigrade species were determined by amphibians and mammals) more than half a HPAEC using a Metrohm chromatography system century ago (Macallum, 1910; Robertson, 1949, (830 IC interface, 818 IC pump, 819 IC 1954; Sutcliffe, 1962; Hronoski & Armstrong, conductivity detector, columns C4 - 150/4.0 1977). However, practically nothing is known (cations) and A supp 5 150/4.0 (anions); Metrohm, about the chemical composition of tardigrades, Herisau, Switzerland). The eluents (mobile which has been a major obstacle to the phases) were made according to manufacturer’ understanding of the fluid and solute dynamics in instructions: For cations, the eluent consisted of these animals (Halberg et al., 2009b; Møbjerg et 0.7 mM C7H5NO4 + 1.7 mM (65%) HNO3. For al., 2011; Halberg & Møbjerg, 2012). Questions anions, it consisted of 3.2 mM Na2CO3 + 1.0 mM relating to this area of research are especially NaHCO3. The eluents were filtered (mesh size: 45 important to address, if we wish to unravel the µm) prior to use. The analysis settings employed biological mechanisms mediating the unique were a flow-rate of 0.9 ml/min (cations) and 0.7 tolerance to extreme desiccation (anhydrobiosis) ml/min (anions) with a pressure of ~6.4 MPa. – the most widespread form of cryptobiosis in Cation analyses were performed non-suppressed, Tardigrada. whereas anion detection was conducted using In the present study, we use a combination of chemical suppression. Fluka multi-element cation high-performance anion-exchange chromato- and anion standards (Sigma-Aldrich, St. Louis, graphy (HPAEC) and nanoliter osmometry, to MO, USA) were used to construct calibration identify and quantify inorganic cations and anions curves for the respective ions bracketing the present in tardigrade homogenates, and to concentration range of interest. Based on these measure the total osmotic concentrations of five calibration curves, the ion chromatography (IC) different species of tardigrades, covering both a software (IC Net 2.3, Metrohm, Herisau, broad phylogenetic and habitat spectrum. Our Switzerland) calculated the ion concentrations of study indicates that tardigrades possess powerful all subsequent samples (mg/l), which were ion-retentive and osmoregulatory capacities, and recalculated to a different unit of concentration that (only) cryptobiotic species contain a large (mM) and adjusted according to the appropriate fraction of organic solutes. dilution factor (see below). Representative chromatograms of both the cationic and anionic MATERIALS AND METHODS fractions are shown for all investigated species Tardigrade sampling (Figs. 1, 2). The empirically determined elution Specimens of Richtersius coronifer Richters, order and retention times of the investigated ions + + 1908, Macrobiotus cf. hufelandi C.A.S. Schultze, were Na (tR = 5.37 min), NH4 (tR = 6.03 min), Inorganic ion composition in Tardigrada

Fig. 1. Representational chromatograms revealing the principal inorganic cations present in each investigated species: 1, sodium; 2, ammonium; 3, potassium; 4, calcium; 5, magnesium; nOAp, negative organic acid peak. Stitched square indicates an unidentified compound (tR = 10.36 min) that increases app. two-fold in absolute concentration in dehydrated animals of Richtersius coronifer (data not shown). Column, Metrohm C4-150/4.0; mobile phase, 0.7 mM dipicolinic acid + 1.7 mM (65%) nitric acid; flow-rate, 0.9 ml/min; Conductivity detector without suppression. Injection volume, 60 µl.

+ 2+ 2+ K (tR = 7.73 min), Ca (tR = 18.12 min), Mg performed as fast and uniform as possible. A total - - (tR = 23.17 min), F (tR = 4.05 min), Cl (tR = 6.01 of 40-225 animals were transferred to each test 3- 2- min), PO4 (tR = 14.74 min) and SO4 (tR = 16.58 tube and the sample was subsequently min). homogenized using a sterile plastic pestle; great care was taken to ensure complete homogenization Sample preparation (visually confirmed at 50× magnification), and the Following extraction, specimens were washed pestle was subsequently rinsed with a small repeatedly with ddH2O (Halobiotus crispae was volume of cation eluent to ensure total transfer of washed in filtered salt water; SW, 20 ‰), and ions to the test tube. The number of animals per subsequently transferred, using an Irwin loop, to sample (N) varied according to species size and sample tubes containing cation eluent (75-100 availability (Tables 1, 2). The entire sample was µl); samples dissolved in cation eluent allows for then centrifuged (10 min at 5600 rpm) to remove a more precise quantification of cations due to solid particles (e.g. cuticle fragments), and the increased signal to noise ratio (pers. comm.; supernatant was filtered (mesh size: 0.20 µm) Metrohm Nordic, Denmark). Prior to transfer, using a single use syringe filter (Sartorius AG, surface water was removed by blotting the Göttingen, Germany). The samples were animals with tissue paper in an attempt to avoid subsequently frozen at -20 ºC, if not quantified unwanted dilution of the samples. Pilot immediately. A total of five to seven samples were experiments revealed that this process was critical prepared for each species (Table 2). to acquire reproducible data, and was accordingly K. A. Halberg and others

Fig. 2. Representational chromatograms revealing the principal inorganic anions present in each investigated species: 6, fluoride; 7, chloride; 8, phosphate; 9, sulfate; W, negative water peak. Stitched square indicates an unidentified compound (tR = 7.24 min) that increases app. two-fold in absolute concentration in dehydrated animals of Richtersius coronifer (data not shown). Column, Metrohm A supp 5 150/4.0; mobile phase, 3.2 mM sodium carbonate + 1.0 mM sodium hydrogen carbonate; flow-rate, 0.7 ml/min; Conductivity detector with chemical suppression. Injection volume, 60 µl.

The ionic concentration and composition of dehydration, tissue paper saturated with ddH2O the external media from the different habitats i.e. was used to rinse the surface of the animals, and a moss water (MW) and SW, were additionally dry tissue paper was used to remove excess determined. Moss samples were rehydrated in moisture. This was done in order to remove ddH2O for several hours, and MW samples were potential solutes extruded on the surface of the subsequently collected from between the leaf- animals – the surface of some animals was covered stems. SW samples were prepared by inaccessible due to animal clumping, and therefore diluting SW (1:200) collected at the locality. could not be rinsed. A volume of 90 µl of cation Samples were quantified in triplets using both eluent was added, and the animals were vapor pressure osmometry (Vapro 5520, Wescor immediately homogenized. The samples were then inc., UT, USA) and HPAEC (Table 3). prepared as described above with six samples In order to document whether changes in prepared in total (Table 2). Data (mg/l) from this inorganic ion content occur during dehydration experiment was directly compared to that of from an active to a cryptobiotic state, samples of hydrated animals (Table 4; Fig. 3B), as both sets dehydrated Richtersius coronifer were of samples contained near identical number of additionally prepared. Groups of 75 animals were animals per unit volume (i.e. 0.81 and 0.83 transferred to each sample tube, and excess water animals/µl eluent respectively), which was removed by blotting the animals with tissue circumvented the need for recalculations (see paper. The animals were subsequently allowed to below). In order to test whether R. coronifer dehydrate over the ensuing 24 h at ambient actually produced viable tuns during the temperature and humidity. Following complete abovementioned conditions, post-cryptobiotic Inorganic ion composition in Tardigrada survival was assessed. Using four groups of 50 tardigrade, r is the radius and h the length of the specimens, a survival rate of 94 ± 4% (mean ± trunk and hind legs respectively, while W (0.72 i.e. s.d.) was observed, which is comparable to the mean fractional water content of R. coronifer and maximally reported survival rate of R. coronifer H. crispae) is the gravimetrically measured dehydrated on Whatman filters (Persson et al., fractional water content. Using these data (Table 2010). 1), the total tardigrade test volume was calculated by multiplying the volume of an individual with Calculation of ion concentrations the number of animals included in the sample The IC software expressed the integration of according to equation (2). peaks as a concentration (mg/l), which was recalculated using the respective molecular (Eq 2) Vtotal = Vindividual × N weights of each compound to a different concentration (mM) prior to adjusting for the Vtotal is the total tardigrade test volume, and N the dilution factors. number of animals included in the sample. Lastly, The volume of each investigated species was the concentrations of the dominant cations and calculated according to an adjusted method of anions in the investigated species of tardigrades Halberg et al. (2009b). In brief, micrographs were were calculated by multiplying the measured ion taken of N = 20 animals of each species, using a concentrations with the dilution factor, which was digital camera (C-5050, Olympus, Japan) calculated according to equation (3). mounted on an Olympus BX 51microscope (Olympus, Japan), and median length (h) and (Eq 3) D = F / Vtotal width (2r) of the trunk and legs were measured. Approximating the geometric shape of the trunk Where D is the dilution factor and F is the final and legs as a cylinder, and adjusting the volume volume (i.e. volume of cation eluent the of liquid according to the gravimetrically tardigrades were transferred to + Vtotal). Sample measured water content (based on Westh and information for the respective species is listed in Kristensen, 1992; Halberg et al., 2009b), the fluid Table 2. volume of an individual tardigrade of each species was calculated using equation (1). Nanoliter osmometry The total osmotic concentration of tardigrades 2 2 (Eq 1) Vindividual = π(r trunkhtrunk + 8r leghleg) × W from each investigated species was estimated using nanoliter osmometry. This was done in order Where Vindividual is the volume of an individual to determine the fraction that the identified

Table 1. Volume estimations. Mean values of length (h) and width (2r) of the trunk and legs (N = 20 animals), as well as the calculated volume (Eq 1), of each investigated species. W is the average of the gravimetrically determined water content (72%) of Richtersius coronifer (Westh and Kristensen, 1992) and Halobiotus crispae (Halberg et al., 2009). Data are expressed as mean ± s.d. K. A. Halberg and others inorganic ions constitute of the total osmotic respect to the external medium; hyper-regulation concentration in each species, and to provide an has previously been suggested to be a general independent verification of our HPAEC data, i.e. theme among members of Eutardigrada (Halberg total osmotic concentration should be higher than et al., 2009b; Møbjerg et al., 2011). Comparing the accumulated concentration of the respective the measured total osmotic concentrations with the inorganic ions (Table 3). Using the same calculated total ionic concentrations reveals that procedure for removing excess water as described there is a large ‘osmotic deficit’, especially in the above, individual specimens were transferred into limno-terrestrial cryptobiotic species E. testudo, sample oil wells (loading oil type B; cST=1250 ± M. tardigradum, R. coronifer and M. cf. hufelandi 10%; Cargille laboratories, Cedar grove, NJ (Fig. 3A; Table 3). Furthermore, in most species 07009, USA) of a calibrated nanoliter osmometer the measured positive and negative charges do not (Clifton Technical Physics, Hartford, NY, USA), entirely maintain electro-neutrality (Table 3). This and the osmolality (mOsm/kg) was determined by charge deficit indicates that there are ionic freezing point depression (FPD = 1.858 components that remain unidentified, especially in °C/Osmol). Six to ten animals of each species M. tardigradum and H. crispae, and that these were used in this experiment (Table 3). unidentified ions contribute to the observed osmotic deficits. Bicarbonate for example, is Statistics important for pH-regulation of the extracellular Significant changes in the individual inorganic fluid of most animals, and could contribute ion concentrations between active and significantly to the unidentified anionic fraction. cryptobiotic animals of Richtersius coronifer The charge deficit appears to account for the entire were tested using an unpaired, two-sample t-test osmotic deficit in H. crispae (Table 3), however; with significance levels of P>0.05 (not even when accounting for the charge deficits, a significant, NS), P<0.05 (significant, *) and large fraction of unidentified osmolytes remain P<0.01 (very significant, **). unaccounted for in the limno-terrestrial, cryptobiotic species (Fig. 3A). In fact, this RESULTS difference is likely even greater than indicated in Ionic composition and total osmotic Figure 3A, as our ionic data was not corrected concentration in tardigrades according to osmotic coefficients. Total osmotic concentration range 361±49 in R. Na+ and Cl- are the principal inorganic ions of coronifer to 961±43 mOsm/kg in H. crispae from tardigrade fluids, accounting for 11-56% of the (Table 3), indicating that both limno-terrestrial total osmotic concentration in all investigated and marine species are hyper-osmotic with species; with Macrobiotus cf. hufelandi and

Table 2. Sample data. Number of samples (n), number of animals per sample (N), total number of animals used (n × N), total tardigrade test volume (Vtotal, Eq 2), final volume (F), as well as the resulting dilution factor (D, Eq 3) is listed. Dehydrated animals of R. coronifer have no measurable water content, why Vtotal, F and D cannot be listed. Inorganic ion composition in Tardigrada

Figure 3. Graphical representation of the respective ionic contributions to total osmotic concentration. A. Concentrations (mM) of the respective cations and anions measured in each investigated species, as well as the corresponding total osmotic concentration (mOsm/kg), as measured by nanoliter osmometry (see also Table 3). The blank area represents the osmotic deficit (OD), i.e. other solutes. The phylogenetic position and habitat preference of each species is listed. The light micrographs of the animals are shown to scale (see Table 1 for average species size). B. Concentrations (mg/l) of the respective cations and anions measured in hydrated, active specimens compared to dehydrated cryptobiotic animals of Richtersius coronifer (see also Table 4). Data are expressed as mean ± s.d. K. A. Halberg and others

Halobiotus crispae containing the lowest and bone of vertebrates and exoskeletons of highest concentrations respectively (Fig. 3A; invertebrates, and is for example found in Table 3). There are notable differences in the Na+/ extremely high levels in the cuticle of marine Cl- ratio between the animals, i.e., the ratio is less crustaceans (Adelung et al., 1987; Sands et al., than unity in the limno-terrestrial herbivores E. 1998). In contrast, the [F-] in blood/hemolymph testudo (0.61), R. coronifer (0.60) and M. cf. and soft tissue is negligible (Adelung et al., 1987; hufelandi (0.33), higher than unity in the limno- Sands et al., 1998). Therefore, the [F-] of our terrestrial predator M. tardigradum (1.19) and samples could reflect ions bound to cuticular close to unity in the marine herbivore H. crispae structures that do not contribute to the dissolved (0.92). anionic fraction. Indeed, high levels of dissolved Compared with Na+ and Cl-, generally, the F- are deleterious to enzymatic function (Eagers, [K+] is relatively low in all species, ranging from 1969). In line with this suggestion, the observed 19-73 mM (Tab. 3). Thus, the Na+/ K+ ratio is differences in [F-] could relate to differences in higher than unity in E. testudo (2.39), M. cuticle composition, with a lower F- content in tardigradum (2.05), R. coronifer (1.32) and H. members of Macrobiotiidae (R. coronifer and M. crispae (6.12), however; lower than unity in M. cf. hufelandi) compared to other species (Table 3). 3- 2- cf. hufelandi (0.77). Interestingly, the relative Conversely, the detected [PO4 ] and [SO4 ] both contribution of K+ to total osmotic concentration contribute to the anionic fraction in all tardigrade is low constituting <10 % in all investigated species, accounting for approximately 1-6% and species (Table 3), regardless of dietary and 1-4% of the total osmotic concentration, habitat preferences – an observation that is respectively, with the highest concentrations paralleled by insects, in which K+ usually detected in the marine H. crispae (Fig. 3A; Table contributes 2-10% of total hemolymph 3). concentration (Sutcliffe, 1963). The highest absolute [K+] is found in the predator M. Changes in ionic composition during tardigradum, and is app. 2-3 times the dehydration concentration of the herbivorous species (Table Surprisingly, the concentrations of the examined 3). ions showed little variation from the active to the In general, high [Ca2+] seems characteristic of cryptobiotic state in specimens of Richtersius tardigrade fluids, just as reported for aquatic coronifer (Fig. 3B; Table 4). Excluding small yet insects (Sutcliffe, 1962). In contrast, [Mg2+] is significant changes in the K+ and Cl- contents, only + comparatively low in all limno-terrestrial species, NH4 was very significantly reduced from the + and ranges from 2-13 mM. Conversely, in the hydrated to the dehydrated state (Table 4). NH4 is marine tardigrade H. crispae the [Mg2+] remains a waste product of protein metabolism, so the relatively high (Table 3; Fig. 3), which is in observed reduction could reflect the general shut- agreement with the high [Mg2+] in SW (Table 3). down of metabolic processes during dehydration. 2+ + Comparable high [Mg ] have also been reported NH4 is a weak acid that easily converts to NH3 from marine crustaceans (Tentori and Lockwood, depending on the pH of the solution, so the 1990). However, it should be taken into account measured final biological concentration is no more that the measured cation pools include ions that than tentative. typically form complexes with proteins and other macromolecules. For example, the stylet and DISCUSSION stylet supports of tardigrades are known to be Ionic composition in tardigrades composed of CaCO3 (Bird and McClure, 1997), Our analyses of osmotic and ionic concentrations why the concentrations of e.g. Ca2+ in tardigrade have been performed on homogenates of the + fluids could be lower than measured. NH4 also investigated species with no distinction between appears to contribute to the total osmotic intra- and extracellular compartmentalization. concentrations measured in all species, with Nevertheless, our data provide a much needed values of 6-31 mM detected. indication of osmolyte composition in Tardigrada, Substantial [F-] was detected in all tardigrade which is fundamental to our understanding of samples, ranging from 7-52 mM (Table 3). This tardigrade physiology. ion is typically localized to hard tissue, such as Inorganic ion composition in Tardigrada charges) are listed; the polarity polarity the are listed; charges) cations and anions detected in each investig detected anions and cations as mean ± s.d. difference in ionic concentration, and total or osmometry nanoliter by measured as (mOsm/kg), concentration composition and Table 3. Ionic of the charge deficits is indicated in parent of the charge deficits total osmotic concentration of the investigated osmotic concentration), as well theosmotic obse ated species, as moss water (MS) as well vapor pressure osmometry, respect hesis. Numbers noted Numbers noted in hesis. species of tardigrades and the corresponding ext and 20 ‰ salt water (SW) samples, in addit rved charge deficits (calculated as the (calculated deficits rved charge brackets indicates the n brackets indicates ively. In addition, the osmotic umber of samples tested difference between and negativepositive ion to the correspo the to ion ernal media. Concen deficits (calculated as the . Data are expressed nding total osmotic trations (mM) of K. A. Halberg and others

In the present study we provide data on the ionic tardigradum resembles the heterotardigrades more composition of five different species of than the other eutardigrades (Table 5) tardigrades covering a large phylogenetic Sutcliffe (1962, 1963) argued that definitive spectrum. Our study is represented by members types of hemolymph are related to phylogenetic of Heterotardigrada (Echiniscoidea) and position within Insecta. Apart from small Eutardigrada (Apochela and Parachela), four differences in absolute concentrations of Na+ and 3- evolutionary distant families (Echiniscidae, PO4 , the ionic compositions and relative Milnesiidae, Macrobiotidae and Hypsibiidae), as contributions of the different components in well as both limno-terrestrial and marine habitats. Richtersius coronifer and Macrobiotus cf. Accordingly, we will discuss the ionic hufelandi (Eutardigrada: Macrobiotidae) are compositions of the respective species in relation similar (Tables. 3 and 5). As a testable hypothesis, to systematic position and habitat preference, as these similarities would suggest that the relative well as make comments on our data in relation to ion composition among the species relates to hemolymph composition in representatives of phylogeny and systematic position in Tardigrada. phylogenetically related groups (i.e. Arthropoda In contrast to the other groups of limno-terrestrial and Onychophora). tardigrades, the inorganic content of R. coronifer Echiniscus testudo (Heterotardigrada: and M. cf. hufelandi is characterized by a Echiniscidae) belongs to another evolutionary relatively small contribution of Na+ (~2-7 times lineage than the other tardigrades in the present lower) and Cl- (~2-3 times lower), and conversely, 2- study. Compared to limno-terrestrial members of a relatively large contribution of SO4 (~4-10 Eutardigrada, the ionic composition of this times higher). The physiological significance of heterotardigrade is characterized by a large these variations is unknown. contribution of Na+ and Cl- (~45%), and a very Halobiotus crispae (Eutardigrada: 2+ 2- low contribution of Mg (0.4%), SO4 (0.6%) Hypsibiidae) is a truly marine species, and is the 3- and PO4 (1.4%), respectively (Tab. 5). The large species with the highest total concentration of both contribution of Na+ and Cl- to total osmotic ions and total solutes measured. Na+ and Cl- concentration, which is comparable to that seen in account for more than 50% of its total osmotic the marine species H. crispae (Table 5), could concentration. The divalent cations, Ca2+ and reflect the supposed marine origin of tardigrades Mg2+, are also detected in high concentrations, (Jørgensen et al., 2010). This hypothesis can be both absolute and relative. In contrast to the tested by data on members of the ‘ancient’ and limno-terrestrial species, the total osmotic exclusively marine Arthrotardigrada. concentration of H. crispae is almost exclusively The family Milnesiidae, represented by the accounted for by the measured ionic predator Milnesium tardigradum, is currently concentrations, which becomes evident when considered the sister-group of all other considering the charge deficit indicated in Table 3. eutardigrades (Guidetti et al., 2009). M. The contribution of the total diffusible ions to tardigradum contains the highest total osmotic- as the total osmotic concentration of tardigrades is well as ionic concentration among the limno- roughly similar to that of the hemolymph of terrestrial species, with conspicuously high levels arthropods, nematodes and onychophorans (Table of both K+ and Ca2+ (Table 3). The high [K+] in 5). In fact, as Na+ predominantly is an M. tardigradum compared to the phytophagous extracellular ion, whereas K+ and Ca2+ mainly are species is somewhat surprising, as e.g. intracellular ions, the ionic composition of carnivorous insects typically contain low levels of tardigrade hemolymph is expected to resemble K+ (Sutcliffe, 1962). Conversely, phytophagous that of closely related animal groups, e.g. other tardigrades are known to feed on bryophytes high members of Panarthropoda (see Table 5). in K+ and low in Na+ (Smith, 1978), and were, It is relevant to compare concentrations of ions analogous to phytophagous insects (Sutcliffe, in tardigrade body fluids with those of the 1962), expected to reflect this relative ion respective external media (Table 3). The strongest composition in their extracellular body fluids. ability to concentrate ions is seen in limno- Interestingly, the relative ion contributions to terrestrial species, which hyper-regulate by as total osmotic concentration suggests that M. much as ~350-750 mOsm/kg (Fig. 3A). The marine H. crispae maintains hemolymph osmotic Inorganic ion composition in Tardigrada

Table 4. Changes in ionic composition during dehydra- tion. Concentrations (mg/l) of the respective cations and anions measured in hydrated, active specimens compared to dehydrated cryptobiotic animals of Richtersius coronifer. Both sets of samples contained near identical number of animals per unit volume (i.e. 0.8 animals/µl eluent), and were accordingly directly comparable. Numbers noted in brackets indicates the number of samples tested. Data are expressed as mean ± s.d. Significant difference in the concentration of the respective inorganic ion concentrations were tested using an unpaired, two-sample t-test with significance levels of P>0.05 (not significant, NS), P<0.05 (significant, *) and P<0.01 (very significant, **).

pressure ~300 mOsm/kg above that of the (Halberg et al., 2009b; Halberg and Møbjerg, environment, and does so over much larger range 2012). of external salinities compared to the limno- terrestrial species R. coronifer (Halberg et al., Changes in ionic composition during 2009b; Møbjerg et al., 2011). In this context it dehydration should be emphasized that the MW samples were Desiccation is the most severe form of osmotic acquired by rehydrating moss samples with stress, and tardigrades are among the most ddH2O, and not precipitation from the locality, desiccation tolerant animals on Earth (Møbjerg et however; as atmospheric precipitation generally al., 2011). As cryptobiotic tardigrades dehydrate, contains few dissolved particles, with a liquid water is slowly reduced to immeasurable concentration of less than 400 µM (Granat, 1972), levels (Westh and Ramløv, 1991), which leads to a this is likely to be of little consequence. gradual increase in dissolved particles and osmotic Accordingly, Na+ is concentrated by a factor of pressure of the body fluids. In general, the basis ×70-800, K+ by ×20-90, Ca2+ and Mg2+ by ×30- for osmotic stress tolerance includes the active 200, whereas Cl- is concentrated by ×20-50, and extrusion of inorganic ions combined with the 2- SO4 by ×30-150 in limno-terrestrial tardigrades. accumulation of organic ‘compatible’ osmolytes + - 2- In contrast, Na , Cl and SO4 are basically in (Yancey, 2005) – at very high concentrations, ionic equilibrium with respect to the external SW inorganic ions bind to and destabilize proteins and in H. crispae, while K+, Ca2+ and Mg2+ are nucleic acids, which invariably damages cellular concentrated by a factor of ×2-10. The large function (Yancey, 2005). In this regard it was osmotic and ionic gradients maintained by both surprising to learn that R. coronifer apparently do limno-terrestrial and marine species are indicative not exclude inorganic ions during dehydration of powerful ion retentive mechanisms in (Fig. 3B; Table 4), which indicates a concomitant Tardigrada – functions that presumably are accumulation of organic osmolytes. Organic maintained by such organ systems as the osmolytes can accumulated in large amounts Malpighian tubules (only found in eutardigrades, without perturbing cellular function, hence the see. e.g. Møbjerg and Dahl, 1996) and gut system term ‘compatible’, and hereby increase K. A. Halberg and others intracellular osmotic potential so that osmotic makes tardigrades especially interesting for equilibrium between intra- and extracellular identification of osmolytes with protective fluids is maintained. Moreover, organic functions, as well as for the study of their physio- osmolytes are known to stabilize macromolecular chemical mechanisms. The non-reducing sugar, structures by direct interaction with proteins and trehalose, has already received much attention membrane lipids (Crowe et al., 1987; Hincha and (Westh and Ramløv, 1991; Hengherr et al., 2007; Hagemann, 2004; Yancey, 2005). As evidenced Jönsson and Persson, 2010). Interestingly, by the large osmotic deficits, which appears however, trehalose is present only in minute restricted to cryptobiotic tardigrades (Fig. 3A; quantities in active tardigrades (e.g. 0.1% dry Table 3), it is tempting to suggest that a large weight in R. coronifer), and is accumulated to no quantity of organic osmolytes are synthesized in more than 2.9% of the dry weight in Macrobiotus these species, thus enabling the animals to islandicus (Jönsson and Persson, 2010). respond quickly to decreases in external water Obviously, it cannot account for the observed potential. Such a strategy seems favorable in light osmotic deficits demonstrated in the present study, of the continuous dehydration-rehydration cycles although it appears sufficient to confer protection that may occur in limno-terrestrial habitats, against dehydration in the investigated members additionally supported by the short time span of Macrobiotidae. In fact, cell concentrations of (<20 min) with which tardigrades can enter the trehalose for maximal cell survival during tun stage successfully. De novo synthesis of dehydration of murine fibroblasts are about 5.3 × osmolytes during dehydration of cryptobiotic 1010 molecules per cell (Chen et al., 2001). tardigrades has also been reported (Westh and Assuming that this applies to tardigrade cells also Ramløv, 1991; Hengherr et al., 2007; Jönsson and and that the cell number of Richtersius coronifer is Persson, 2010), and indirectly confirmed in our comparable to that of Halobiotus crispae (1058 ± study by the observed doubling of two 53 cells/animal excluding gametes; Møbjerg et al., (unidentified) osmolytes following dehydration of 2011), the estimated number of trehalose required R. coronifer (stitched square in Figs. 1, 2. would be about 5.6 × 1013 molecules per animal. Chromatograms of dehydrated animals are not Trehalose is accumulated to 2.3% of the dry shown). weight of R. coronifer (Westh and Ramløv, 1991), and using the measured dry weight of 2.9 µg per Cryptobiotic species contain large fraction of animal (Westh and Ramløv, 1991) a single unidentified solutes individual accumulates on average 6.67 × 10-8 g As evidenced by the differences between the trehalose. With a molecular weight of 342.3 g/mol calculated ionic concentrations of the known ions this corresponds to 1.96 × 10-10 mol per animal, and the measured total osmolalities in the and by multiplying with Avogadro’s constant different species (Fig. 3A, Table 3), it is inferred (6.023 × 1023 molecules per mol) this equals 1.18 that cryptobiotic tardigrades contain a large × 1014 trehalose molecules per animal, which is fraction of unidentified organic osmolytes. twice the estimated number of molecules required Because the osmotic deficits were restricted to for protection against dehydration. In fact, the cryptobiotic species, it is reasonable to assume calculated number of trehalose molecules is that the fraction of organic solutes includes probably several times higher than that required, elements associated with cryptobiotic survival. as tardigrade cells are much smaller than murine Organic osmolytes fall into a few major fibroblasts, and therefore have a smaller total area categories (sugars, polyols, amino acids and of membranes to be protected. However, trehalose various derivatives) and appear universally is only accumulated in significant amounts in exploited by both plants and animals (Yancey, selected species (Hengherr et al., 2007; Jönsson 2005). Several chemical compounds of the above and Persson, 2010), which suggests that a diverse groups have been shown to have protective pattern of dehydration mechanisms have evolved function during osmotic stress (Hincha and in Tardigrada. Accordingly, these as yet Hagemann, 2004; Yancey, 2005). The unidentified biochemical pathways, would have to demonstration of survival during extreme osmotic be identified and characterized in order to obtain a conditions – in both dehydrated and active states profound insight into the metabolic and chemical (Halberg et al., 2009b; Møbjerg et al., 2011) – mechanisms of cryptobiosis. Inorganic ion composition in Tardigrada nematodes, crustaceans, in inves the concentration in of internal each Table 5.

Total osmotic concentration (mOsm/ concentration Total osmotic sects and onychophorans is included fo is included and onychophorans sects tigated species of tardigrades. Correspondi species of tardigrades. tigated kg) of the external medium and internal body body and internal the external medium kg) of r comparative purposes ng data on hemolymph concentration and co and concentration hemolymph on ng data . TR, terrestrial; fluids, as well the ─ , not measured. , not measured. osmotic contribution (%) contribution osmotic mposition of selected species of selected species of mposition of the respective ions to the K. A. Halberg and others

We would like to thank Anne Lise Maarup for technical Halberg, K. A. & Møbjerg, N. (2012). First evidence of assistance and Reinhardt Møbjerg Kristensen (Natural History epithelial transport in tardigrades: Comparative Museum of Denmark) for loan of the Olympus BX 51 investigation of organic anion transport. J. Exp. Biol., microscope. Station Linné (Porten til Alvaret), Ölands Skogsby, Sweden is warmly thanked for accommodation during sampling 215:497-507. of tardigrades. Funding came from the Carlsberg Foundation Halberg K. A., Persson, D., Jørgensen, A. Kristensen, and the Freja-Programme (Faculty of Science, University of R. M. and Møbjerg, N. submitted. Population Copenhagen). dynamics of a marine tardigrade: Temperature limits geographical distribution of Halobiotus crispae. Mar. Biol. Res. Hengherr, S., Heyer, A. G., Köhler, H. R. & Schill, R. LIST OF ABBREVIATIONS (2009). Trehalose and anhydrobiosis in tardigrades – FPD freezing point depression evidence for divergence in response to dehydration. HPAEC high-performance anion-exchange chromatography IC ion chromatography FEBS. J., 275:281-288. MW moss water Hincha, D. K. & Hagemann, M. (2004). Stabilization SW salt water of model membranes during drying by compatible solutes involved in the stress tolerance of plants and microorganisms. Biochem. J. 383:277-283. Hobsen, A. D., Stephenson, W. and Eden A. (1952). REFERENCES Studies on the physiology of Ascaris lumbricoides: Campiglia, S. (1976). The blood of Peripatus acacioi II. The inorganic composition of the body fluid in Marcus & Marcus (Onychophora) – III. The ionic relation to that of the environment. J. Exp. Biol., composition of the hemolymph. Comp. Biochem. 29:22-29. Physiol., 54(A):129-133. Hronowski, L. & Armstrong, J. B. (1977). Ion Chen, T., Acker, J. P., Eroglu, A. flere authors composition of the plasma of Ambystoma (2001). Beneficial effect of intracellular trehalose on mexicanum. Comp. Biochem. Physiol. A, 58:181-183. the membrane integrity of dried mammalian cells. Jönsson, K. I. & Persson, O. (2010). Trehalose in three Cryobiol. 43:168-181. species of desiccation tolerant tardigrades. Open Crowe, J. H., Crowe, L. M., Carpenter, J. F. and Zool. J., 3:1-5. Wistrom, C. A. (1987). Stabilization of dry Jørgensen, A., Faurby, S., Hansen, J. G., Møbjerg, N. phospholipid bilayers and proteins by sugars. and Kristensen, R. M. (2010). Molecular phylogeny Biochem. J., 242:1-10. of Arthrotardigrada (Tardigrada). Mol. Phylogen. Bird, A. F., McClure, S. G. (1997). Composition of the Evol., 54:1006-1015. stylets of the tardigrade, Macrobiotus cf. Normant, M., Kubicka, M., Lapucki, T., Czarnowski, pseudohufelandi. Trans R Soc. S Aust., 121:43-50. W. and Michalowska M. (2005). Osmotic and ionic Eagers, R. Y. (1969). Toxic properties of inorganic haemolymph concentration in the Baltic Sea fluoride compounds. Elsevier, Amsterdam London amphipod Gammarus oceanicus in relation to water New York, 382 pp. salinity. Comp. Biochem. Physiol. A, 141:94-99. Granat, L. (1972). On the relation between pH and the Macallum, A. B. (1910). The inorganic composition of chemical composition in atmospheric precipitation. the blood in vertebrates and invertebrates, and its Tellus XXIV, 6. origin. Proc. R. Soc. Lond. B, 82(559):602-624. Guidetti, R., Altiero, T. & Rebbecchi, L. (2010). On Møbjerg N, Dahl C. (1996). Studies on the morphology dormancy strategies in tardigrades. J. Insec. and ultrastructure of the Malpighian tubules of Physiol., 57(5):567-576. Halobiotus crispae Kristensen, 1982 (Eutardigrada). Guidette, R., Schill, R. O., Bertolani, R., Dandekar Zool. J. Linn. Soc., 116:85-99 T. & Wolf, M. (2009). New molecular data for Møbjerg, N., A. Jørgensen, J. Eibye-Jacobsen, K. A. tardigrade phylogeny, with the erection of Halberg, D. Persson & R. M. Kristensen (2007). Paramacrobiotus gen. nov. J. Zool. Syst. Ecol. Res., New Records on cyclomorphosis in the marine 47(4):315-321. eutardigrade Halobiotus crispae (Eutardigrada: Halberg, K. A., Persson D., Møbjerg N., Wanninger Hypsibiidae). J. Limnol., 66 (suppl. 1): 132-140. A. & Kristensen R. M. (2009a). Myoanatomy of Møbjerg, N. M., Halberg, K. A., Persson., D., the Marine Tardigrade Halobiotus crispae Jørgensen, A. & Kristensen R. M. (2011). Survival (Eutardigrada: Hypsibiidae). J. Morphol., 270:996- in extreme environments – on current knowledge of 1013. adaptations in tardigrades. Acta Physiol., 202: 409- Halberg, K. A., Persson, D., Ramløv, H., Westh, P., 420. Kristensen, R. M. & Møbjerg, N. (2009b). Persson, D., Halberg K. A., Jørgensen A., Ricci C., Cyclomorphosis in Tardigrada: Adaption to Møbjerg N. & Kristensen R. M. (2010). Extreme environmental constraints. Journal of Experimental stress tolerance in tardigrades: Surviving space Biology, 212:2803-2811. Inorganic ion composition in Tardigrada

conditions in low earth orbit. J. Zool. Syst. Evol. Res., 49: 90-97. Persson, D., Halberg K. A., Jørgensen A., Møbjerg N. & Kristensen R. M. (2012). Neuroanatomy of Halobiotus crispae: Tardigrade brain structure suggests inclusion into Panarthropoda. J. Morphol. (in review). Robertson, J. D. (1949). Ionic regulation in some marine invertebrates. J. Exp. Biol., 26:182-200. Robertson, J. D. (1954). The chemical composition of the blood of some aquatic chordates, including members of the Tunicata, Cyclostomata and Osteichthyes. J. Exp. Biol., 31:424-442. Sands, M, Nicol, S. & McMinn, A. (1998). Fluoride in Antarctic marine crustaceans. Mar. Biol., 132:591- 598. Smith R. I. L. (1978). Summer and winter concentrations of sodium, potassium and calcium in some maritime Antarctic cryptogams. J. Ecol., 66 (3):891-909. Sutcliffe, D. W. (1962). The composition of haemolymph in aquatic insects. J. Exp. Biol., 39:325-343. Sutcliffe, D. W. (1963). The chemical composition of haemolymph in insects and some other arthropods, in relation to their phylogeny. Comp. Biochem. Physiol., 9(2):121-135. Tentori E. and Lockwood, A. P. M. (1990). Haemolymph magnesium levels in some oceanic crustaceans. Comp. Biochem. Physiol. A, 4:545-548. Wilder, M. N., Ikuta, K., Atmomarsono M., Hatta, T. and Komuro, K. (1998). Changes in osmotic and ionic concentrations in the hemolymph of Macrobrachium rosenbergii exposed to varying salinities and correlation to ionic and crystalline composition of the cuticle. Comp. Biochem. Physiol. A, 119:941-950. Wyatt, G. R. (1961). The biochemistry of insect hemolymph. Ann. Rev. Entomol., 6:75-102. Westh, P. and Kristensen, R. M. (1992). Ice formation in the freeze-tolerant eutardigrades Adorybiotus coronifer and Amphibolus nebulosus studied by differential scanning calorimetry. Polar Biol. 12:693-699. Westh, P. and Ramløv, H. (1991). Trehalose accumulation in the tardigrade Adorybiotus coronifer during anhydrobiosis. J. Exp. Zool. 258:303-311. Yancey, P. H. (2005). Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol., 208:2819-2830.

Paper II

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The Journal of Experimental Biology 215, 497-507 © 2012. Published by The Company of Biologists Ltd doi:10.1242/jeb.065987

RESEARCH ARTICLE First evidence of epithelial transport in tardigrades: a comparative investigation of organic anion transport

Kenneth Agerlin Halberg* and Nadja Møbjerg Department of Biology, The August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark *Author for correspondence ([email protected])

Accepted 31 October 2011

SUMMARY We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP (1mmoll–1; 81% reduction), the Na+/K+-ATPase inhibitor ouabain (10mmoll–1; 21% reduction) and the vacuolar H+-ATPase inhibitor bafilomycin (5mmoll–1; 21% reduction), and by the organic anions PAH (10mmoll–1; 44% reduction) and probenecid (10mmoll–1; 61% reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile, albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalization of the Na+/K+-ATPase -subunit in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalization data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our results, we cautiously suggest that apical H+ and possibly basal Na+/K+ pumps provide the driving force for the transport; the exact coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades. Key words: organic anion transport, Chlorophenol Red, 2,4-dinitrophenol, ouabain, bafilomycin, probenecid, para-aminohippuric acid, tardigrade, insect, V-type H+-ATPase, Na+/K+-ATPase, Malpighian tubule.

INTRODUCTION specialized reproductive and excretory organs (Dewel and Dewel, The ability to excrete metabolic waste products as well as 1979; Rebecchi and Bertolani, 1994; Møbjerg and Dahl, 1996; environmental toxins (xenobiotics) is a fundamental prerequisite Schmidt-Raesa and Kulessa, 2007; Pelzer et al., 2007; Zantke et al., for animal life. One of the earliest identified systems involved in 2008; Halberg et al., 2009a; Møbjerg et al., 2011; Rost-Roszkowska such an excretion is the classic organic anion transport system et al., 2011). The alimentary canal can be divided into several (Marshall and Vickers, 1923). In vertebrates, this system is morphologically distinct regions i.e. bucco-pharyngeal apparatus, (especially) well known in the proximal tubule where it rids the oesophagus, midgut and hindgut, with the Malpighian tubules (MTs) organism of various xenobiotic and endobiotic compounds, of eutardigrades positioned at the junction between these last two through an ATP-dependent, net transepithelial secretory pathway sections. Interestingly, the same basic organizational pattern is found (reviewed by Dantzler, 2002; Burckardt and Burckardt, 2003; in insects, and has been used as a strong argument for the homology Russel, 2010). The importance and understanding of similar of these two organ systems (Greven, 1982; Møbjerg and Dahl, 1996). transport activities in various invertebrate phyla, however, is Among multi-cellular animals, tardigrades exhibit an extraordinary extremely limited. In this study, we expand on the current ability to resist environmental extremes, and are known to survive knowledge by investigating organic anion transport in the conditions greatly exceeding those encountered in their natural habitat tardigrade Halobiotus crispae (Tardigrada), as compared with the – even in space (Jönsson et al., 2008; Rebecchi et al., 2008; Persson desert locust Schistocerca gregaria (Arthropoda). et al., 2011). The biochemical and physiological mechanisms The phylum Tardigrada consists of a group of minute, eight-legged, mediating this unique tolerance, however, remain largely unidentified. multicellular animals that, like arthropods and nematodes, belong to Previously, we have shown that the marine eutardigrade H. crispae the invertebrate superclade Ecdyzosoa (Aguinaldo et al., 1997). They tolerates large changes in external salinity surviving periods of osmotic are considered essential to our understanding of early metazoan stress by maintaining haemolymph osmotic pressure above that of evolution, yet fundamental questions concerning their basic biology the external medium (Halberg et al., 2009b); an adaptive mechanism remain unanswered (Møbjerg et al., 2011). In spite of their small size likely present in all eutardigrades (Møbjerg et al., 2011). Here, we (~50–1200mm), tardigrades are relatively complex animals; they are identify organs involved in transepithelial transport of organic anions composed of >1000 cells, and possess a well-developed musculature and investigate the transport characteristics with the aim of providing and nervous system, as well as a complex alimentary canal and a better understanding of the unique biology in these animals.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 498 K. A. Halberg and N. Møbjerg

Organic anion transport has previously been described in other groups of invertebrates (George et al., 1999; Torrie et al., 2004; 6000 Faria et al., 2010). Notably, the alimentary canal and MTs of insects, pH 11 r2=0.996 which collectively form the functional analogue of the vertebrate 5000 kidney, have been studied (reviewed by Phillips, 1981; O’Donnell pH 7–10 r2=0.987 et al., 2003; Dow and Davies, 2006). As in vertebrates, the excretory 4000 organs of insects transport a wide range of organic solutes and pH 6 r2 exogenous toxins through organic anion transporters (Oats), organic 3000 =0.994 anion-transporting peptides (Oatps), P-glycoproteins (Mdr/P-gp) as 2000 well as multidrug resistance-associated proteins (Mrps) (Maddrell relationship (%) et al., 1974; Bresler et al., 1990; Lanning et al., 1996; Linton and O’Donnell, 2000; Torrie et al., 2004; Neufeld et al., 2005; Leader 1000 and O’Donnell, 2005; O’Donnell and Leader, 2006; Chahine and O’Donnell, 2009; Chahine and O’Donnell, 2010). Organic anions 0

Difference in red spectrum/green spectrum Difference 0 1 2 3 4 5 are divided into type I and type II organic anions, on the basis of –1 structural and chemical properties (e.g. Wright and Dantzler, 2004). [CPR] (mmol l ) The different groups of transporters vary in their transport Fig.1. Standard curves showing the percentage difference in the red mechanisms, but overlap in their substrate specificity, as they spectrum/green spectrum relationship as a function of Chlorophenol Red (CPR) concentration at different pH values (pH6, 7–10 and 11). The curves transport carboxylates and sulphonates interchangeably (Neufeld et were fitted by regression analysis using OriginPro 7.5 with a third order al., 2005; Chahine and O’Donnell, 2009). Oats and Oatps (solute polynomial providing the best fit with r2 values close to unity. carriers belonging to the SLC22 and SLC21/SLCO family) transport both small (<400–500Da) hydrophilic type I organic anions (Oats) and large (>450Da) hydrophobic type II organic anions (Oatps), changes in spectral light to that of dye concentration (Fig.1). As whereas Mdr/P-gp and Mrps (ABC transporters, ABCB and ABCC CPR exhibits a pH-dependent shift in colour (from yellow to violet) subfamilies) generally transport large (>500Da) polyvalent type II between pH4.6 and 7.0, and a secondary shift (to purple) at pH>10, organic anions (Wright and Dantzler, 2004; Russel, 2010). From a standard curve was constructed at three discrete colours relevant the insect’s perspective, the clearance of exogenous toxins is of to our investigation (red, violet and purple corresponding to particular interest, as insects often live in environments with high pH6–11). This was done in order to correct for potential pH xenobiotic exposure, potentially at harmful or lethal concentrations. dependent effects on CPR quantification caused by the tissue. The Accordingly, they are forced to process naturally occurring plant standard curves were constructed from optical analysis of toxins (Torrie et al., 2004; Neufeld et al., 2005), as well as micrographs taken of samples (40ml) with known dye concentrations anthropogenic contaminants, such as insecticides (Lanning et al., (ranging from 0.1 to 5mmoll–1) at each respective pH value. The 1996; Neufeld et al., 2005; Buss and Callaghan, 2008). The optical measurements provided relative, arbitrary values for the light physiological importance of this detoxification system – and intensities of the red, green and blue spectrum of spectral light from implicitly the MTs – is emphasized by the fact that transcripts for each sample, and were acquired using the image analysis and these transporters are enriched in the transcriptome of the MTs visualization software Imaris 6.4 (Bitplane, Zurich, Switzerland). (Wang et al., 2004), and because the expression of several Oatp and The standard curves are expressed as percentage difference in the Mdr transporters is significantly upregulated upon dietary exposure red spectrum/green spectrum relationship as a function of CPR to Oatp and Mdr substrates (Mulenga et al., 2008; Chahine and concentration (Fig.1). The curves were fitted by regression analysis O’Donnell, 2009; Chahine and O’Donnell, 2010). Indeed, it has been using OriginPro 7.5 (OriginLab, Northampton, MA, USA) with a suggested that organic solute excretion is the most significant third order polynomial providing the best fit with r2 values close to function of the insect MT (Dow and Davies, 2006). Nevertheless, unity. The CPR concentration in a given tissue was subsequently in spite of significant efforts in the past decades, the mechanisms calculated by employing the appropriate standard curve. The underlying transepithelial transport of organic anions is far from standard curve was chosen by visually comparing the colour of the understood, and our knowledge is limited to relatively few taxa. accumulated CPR with that of the respective standard curves. In this study we examined epithelial transport in tardigrades. Contrary to our expectations, the midgut of H. crispae was the Using a comparative approach, we investigated the sites, only organ in which CPR accumulation was clearly visualized. characteristics and pharmacological profile of the net transepithelial No accumulation was observed in the tardigrade MTs. As such, transport of Chlorophenol Red (CPR; 3Ј,3Љ-dichlorophenol- quantification of dye accumulation was only performed from the sulphone-phthalein), a pH indicator and a prototypic substrate of tardigrade midgut, and compared with the accumulation in the the classic organic anion secretory pathway, in the tardigrade H. MTs of S. gregaria. Specifically, 6–12 regions from areas of the crispae and the desert locust S. gregaria. Our results show that the tardigrade midgut containing the highest dye intensity were tardigrade midgut is the principal site of CPR transport, and that selected arbitrarily, in addition to a similar number from the this transport is active and transporter mediated. Additionally, our adjacent haemolymph, while four different regions were selected data show that the pharmacological profiles of CPR transport in the from each insect MT. Each region selected represented a circle tardigrade midgut and locust MT are surprisingly similar. with a diameter of 5mm for H. crispae and 25mm for S. gregaria. The average light intensity within these circles was measured by MATERIALS AND METHODS the Imaris program. Averaging the measured intensity of all Quantification of CPR accumulation selected regions provided an overall average intensity for the red, The quantification of CPR accumulation was performed by green and blue colour spectrum within each investigated organ introducing a new method for quantifying non-fluorescent dyes. The following each exposure. As CPR predominantly appears red (to method exploits the optical properties of CPR by relating relative violet) in the midgut of H. crispae and in the MTs of S. gregaria,

THE JOURNAL OF EXPERIMENTAL BIOLOGY Epithelial transport in tardigrades 499 the red spectrum/green spectrum relationship offered an estimate A 1 μmol l–1 10 μmol l–1 100 μmol l–1 1 mmol l–1 5 mmol l–1 of dye accumulation, as calculated from the appropriate standard curve. The final CPR concentration was normalized according to tc tc the background light intensity, i.e. the tardigrade haemolymph or tc lu lu lulu lu untreated (control) insect MTs. For tardigrades, only regions lu tc without gut content were selected for quantification, in order to avoid the influence of gut content on the wavelength of captured B 2.2 light. Deviations in animal depth did not influence CPR –1 Km=81.8 μmol l 2.0 –1 quantification within the variation encountered in this study. The Vmax=1.58 mmol l MTs of S. gregaria consist of three structurally distinct regions, 1.8 i.e. proximal, middle and distal relative to the gut (Garret et al., ) 1.6 1988). Dye accumulation was estimated from the proximal region –1 1.4 and parts of the middle region within 5mm of the junction with 1.2 200 the gut, as dye accumulation was highest here. (mmol l 1.0 150

lumen Test solutions 0.8 The haemolymph osmolality of H. crispae, kept at a salinity of 100

[CPR] 0.6 20p.p.t., was previously measured by nanolitre osmometry to 50 ~950mOsmkg–1 (Halberg et al., 2009b). At present we do not know 0.4 Lumen/bath ratio 0 the composition of tardigrade extracellular fluids. As such, the 0.2 0 0.5 1 5 experimental solution (control solution) was prepared from 0 evaporative reduction of seawater (SW; salinity 20p.p.t., pH8) 0 1 2 3 4 5 collected at the locality, and 35mmoll–1 glucose was added to [CPR] (mmol l–1) alleviate potential variation in experiments caused by differences bath in nutrient availability. This yielded a final measured osmolality of 950±3mOsmkg–1 (N3). For S. gregaria an insect saline (control Fig.2. Accumulation of CPR as a function of external CPR concentration in solution) was prepared containing (in mmoll–1): 130 NaCl, 10 KCl, Malpighian tubules (MTs) of Schistocerca gregaria. (A)Representative light micrographs of the MTs following exposure to different concentrations 4 NaHCO3, 2 MgSO4, 2 CaCl2, 6 NaH2PO4, 35 glucose and 5 Hepes, (1 mol l–1, 10 mol l–1, 100 mol l–1, 1 mmol l–1 and 5 mmol l–1) of CPR. –1 m  m  m      titrated to pH7.2, with a measured osmolality of 336±2mOsmkg Scale bars, 100mm. lu, lumen; tc, trachea. (B)Luminal CPR concentration (N3). as a function of bath CPR concentration, revealing the kinetic parameters In order to explore the kinetics of CPR transport by MTs of S. Km and Vmax for CPR transport. Each point shows the mean ± s.d. for gregaria, a concentration–response curve was constructed over a N4–6 animals with 3–5 MTs providing the estimate for each animal. The 5000-fold range of CPR concentrations (1mmoll–1 to 5mmoll–1) solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis (using error as weight). Insert shows the lumen/bath (Fig.2). These data revealed that CPR transport is saturated at an –1 –1 ratio of CPR as a function of external CPR concentration. external CPR concentration of ~1.6mmoll (Vmax1.58mmoll , –1 Km81.8mmoll ; Fig.2). However, the MTs are unable to concentrate the dye at high concentrations of CPR (>1mmoll–1; Fig.2, inset). Experiments, on both animals, were performed at a Specimens of S. gregaria Forskål 1775 were acquired from a concentration of 1mmoll–1 CPR. specialized animal shop (www.exopark.dk), and kept at room Test solutions were prepared from the two control solutions temperature (RT) with light:dark periods of 16h:8h and fed annual containing 1mmoll–1 CPR, and one of the following inhibitors: 2,4- meadow grass (Poa annua) ad libitum. dinitrophenol (DNP, 1mmoll–1), ouabain (10mmoll–1), bafilomycin –1 –1 A1 (5mmoll ), para-aminohippuric acid (PAH; 10mmoll ) or Exposure to test solutions probenecid (0.1–10mmoll–1). The final osmolality of the solutions Halobiotus crispae was measured on a Vapro 5520 vapour pressure osmometer (Wescor, Evaluation of CPR transport by tardigrade epithelia was performed Logan, UT, USA). All solutions were titrated with NaOH to pH8 on whole animals immersed in the dye solution. Data obtained from for H. crispae and pH7.2 for S. gregaria. 82 animals were used for the study – no distinction was made between male and female specimens. Initial observations revealed Experimental animals that the animals did not take up dye through the mouth or cuticle Specimens of H. crispae Kristensen 1982 were collected on 11 (CPR non-punctured, Fig.3) and test solutions were therefore February 2008 and 17 January 2010 at Vellerup Vig, Isefjord, introduced to the haemolymph of single specimens (length Denmark (55°44.206ЈN, 11°51.258ЈE) (see Fig.3A). Bottom samples 300–500mm) through a small hole made in the cuticle in the anterior were collected with a mini van Veen grab at a depth of 1–2m (salinity part of the animal. The animal was incubated for a period of 60min ~20p.p.t., pH8). Rocks, algae and sediment collected with the grab at RT in the respective test solution, and quickly washed in SW were freshwater shocked. The debris was decanted into a conical net prior to photography. Light micrographs of the specimens were taken (mesh size 63mm) and subsequently transferred to SW from the in bright-field at a ϫ40 magnification, using an Olympus DP20 locality and kept at 4°C. Animals in the active stage (see Kristensen, camera mounted on an Olympus BX50 microscope (Olympus, 1982; Møbjerg et al., 2007; Halberg et al., 2009b) were identified Hamburg, Germany). Additionally, in order to investigate whether using a dissection microscope. The tardigrades were kept for a period the test solutions were ingested during the experimental period, intact of up to 6months at 4°C in SW (salinity 20p.p.t., pH8) and regularly non-punctured animals were incubated in the CPR test solution for supplied with fresh substrate, consisting mainly of sediment, organic a corresponding period. The animals were washed and photographed debris, filamentous algae and diatoms. as described above.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 500 K. A. Halberg and N. Møbjerg

+Bafilomycin Fig. 3. CPR accumulation in the gut lumen of Halobiotus crispae, SW CPR non- +DNP +Ouabain +DMSO +PAH +Probenecid  control punctured CPR –81% –21% –21% –44% –61% and the effect of inhibitors on dye accumulation. (A)Light B nn nn micrograph of H. crispae from a ventral view showing the animalʼs basic morphology. mg, midgut. Asterisks indicate MTs. Scale bar, mg mg mg mg mg mg mg mg 100mm. (B)Representative light micrographs of the midgut (mg) following exposure to the various test solutions. Seawater control shows the midgut of a punctured animal following immersion in 1.6 C A seawater, while CPR non-punctured shows the midgut of an intact animal after immersion in CPR solutions. Estimations of luminal 1.4 Leg 1 CPR concentration were performed solely from areas devoid of gut content, i.e. brown material in the gut. The percentage change in CPR concentration, compared with experiments on CPR alone, is ) 1.2 mg Leg 2

–1 noted. Scale bars, 50mm. (C)Corresponding luminal concentrations Leg 3 of CPR. Data are depicted as means ± s.d. Asterisks refer to a 1.0 * * * significant difference from CPR alone (*P<0.05, significant;

(mmol l 0.8 (13) **P<0.01, highly significant). Numbers in parentheses indicate the Leg 4 number of animals tested.

luminal * 0.6 (6)* (8) **

[CPR] (11) 0.4 ** ** (10) 0.2 (5) (12) (6) 0

DNP PAH –1 CPR –1 –1 –1 ouabain SW control bafilomycin –1 –1 probenecid 1 mmol l +1 mmol l +10 mmol l CPR non-punctured +10 mmol l +5 μmol l +10 mmol l A separate experiment was performed to evaluate whole-organism MTs of S. gregaria were fixed for 60min at RT in an aldehyde activity of H. crispae following puncture of the cuticle and exposure fixative containing: 1.2% glutaraldehyde, 1% paraformaldehyde, to a pure SW control and the various test solutions, following the 0.05moll–1 sucrose and 0.05moll–1 sodium cacodylate buffer procedure described above. The animals were scored with a (pH7.4) and subsequently rinsed and stored in 0.05moll–1 sodium numerical value according to their activity, which was defined as: cacodylate buffer with 0.05moll–1 sucrose. Following 1h post- –1 0, no movement discerned; +1, small movement of leg or claw; +2, fixation in 2% OsO4 with 0.1moll sodium cacodylate, tubules clear movement of body and extremities. Thirty specimens were were dehydrated through a graded series of ethanol and used for each test solution, and a cumulative score was given for propylenoxide and embedded in Araldite®. Semi-thin sections each group (see Fig.4). were cut on a Leica ultramicrotome EM UC6 (Leica,

Schistocerca gregaria 60 The entire alimentary canal including ~233 MTs (Garret el al., 1988) * Clear was dissected from adult specimens (see Fig. 5A) (imago females movement and males; 2–10 days post-fifth instar nymph) under insect saline; 94 45 animals were used in total. Prior to transfer into the test solutions, Small 30 several MTs were randomly chosen and removed, and micrographs * movement were taken in order to acquire an estimate of the background light intensity for each animal. The background light intensity was later Activity score 15 used for normalizing the estimate of the CPR concentration (see * * No movement above). The gut systems were incubated in the respective test 0 solutions for 60min at RT. Following a quick rinse in insect saline, CPR DNP –1 –1 –1 PAH individual MTs representative of overall dye intensity were –1 ouabain SW control bafilomycin –1 –1 probenecid subsequently isolated under microscope (Zeiss Stemi 200-CS, Carl Not punctured 1 mmol l +1 mmol l Zeiss International, Oberkochen, Germany), and prepared for +10 mmol l +10 mmol l photography. MTs obviously damaged during dissection (i.e. that did +5 μmol l not accumulate CPR) were avoided. Details pertaining to the method +10 mmol l of photography were as described above. Five to 11 locusts with 3–5 Fig.4. Animal activity of H. crispae (with punctured cuticle) following MTs per specimen were used in estimations of the CPR concentration exposure to test solutions. Thirty animals were tested for each exposure. for each exposure; a minimum of 20 and a maximum of 40 MTs Each animal was scored with a numerical value according to their activity, were analysed in total for each test solution. defined as: 0, no movement discerned; +1, small movement of leg or claw; +2, clear movement of body and extremities. A cumulative score was given for each group. The statistical significance of differences in activity score Midgut and MT structure and immunostaining was tested using Mann–Whitneyʼs U-test. Asterisks refer to significant Whole animals of H. crispae and MTs dissected from S. gregaria difference from 1mmoll–1 CPR (no asterisk, P>0.05, not significant; were prepared for structural investigation and immunostaining. *P<0.05, significant).

THE JOURNAL OF EXPERIMENTAL BIOLOGY Epithelial transport in tardigrades 501

Microsystems, Wetzlar, Germany) with glass knives and Saline +DNP +Ouabain +Bafilomycin +PAH +Probenecid control CPR –80% –20% –26% –40% –77% subsequently stained with Toluidine Blue. B For immunocytochemistry, whole animals of H. crispae and MTs tc lu lu tc lu of S. gregaria were fixed in 3% paraformaldehyde in 0.1moll–1 lu lu tc tc tc tc lu lu sodium cacodylate buffer (pH7.4) for 60min and subsequently –1 transferred to 0.1moll sodium cacodylate buffer. The tissue was A then dehydrated through a graded series of ethanol and xylene, 1.6 C embedded in paraffin and sectioned into ~10mm sections, or (11) 1.4 transferred to PBS and used as whole mounts for immunostaining. (10)* Paraffin sections were deparaffinized through a graded series of xylene 1.2 (10)* Leg 3 Leg 2 Leg 1 and alcohol, washed in saline (control solution; see ‘Test solutions’ ) –1 ** above) and blocked with 10% normal goat serum (Invitrogen, 1.0 (9) Carlsbad, CA, USA) for 30min, prior to incubation with primary antibody. Paraffin sections, as well as whole mounts, were incubated (mmol l 0.8

overnight at 4°C in insect saline (MTs) or PBS (tardigrades) containing luminal 10% normal goat serum, 0.1% Triton-X and primary antibody. The 0.6

+ + [CPR] ** (8)** Na /K -ATPase -subunit monoclonal mouse primary antibody 5- 0.4 (8) IgG (10mgml–1) was developed by D. M. Famborough, and obtained from the Developmental Studies Hybridoma Bank (University of 0.2 Iowa, Iowa City, IA, USA). This antibody has been used to identify the Na+/K+-ATPase in numerous excretory tissues, including MTs of 0.0 Drosophila melanogaster (Lebovitz et al., 1989; Torrie et al., 2004), CPR DNP PAH the gills of the blue crab Callinectes sapidus (Towle et al., 2001) and –1 –1 –1 –1 ouabain bafilomycin the pronephros of Ambystoma mexicanum (Haugen et al., 2010). –1 probenecid Saline control –1 1 mmol l +1 mmol l Following an extensive wash in saline, the tissue was incubated with +10 mmol l +10 mmol l (anti-mouse) Alexa Fluor 594 (1:100) secondary antibody (Invitrogen) +5 μmol l overnight at 4°C. The tissue was rinsed with saline then counterstained +10 mmol l with Alexa Fluor 488-conjugated phalloidin (luminal marker; 1:40; –1 Fig.5. CPR accumulation in MTs of S. gregaria, and the effect of inhibitors Invitrogen) and DAPI (50mgml ; Invitrogen) for 2h, washed and on dye accumulation. (A)Photo of S. gregaria. Scale bar, 1cm. mounted on glass coverslips in Vectashield (Vector Laboratories Inc., (B)Representative light micrographs of the MTs from exposure to the Burlingame, CA, USA). Images were acquired using a Leica DM various test solutions. Control saline shows an MT prior to immersion in RXE 6 TL microscope equipped with a Leica TCS SP2 AOBS CPR solution. The percentage change in CPR concentration, compared confocal laser scanning unit (Leica Microsystems, Wetzlar, Germany). with experiments on CPR alone, is noted. Scale bars, 100mm. The tissue was scanned employing sequential scanning (setting: (C)Corresponding luminal concentration of CPR in the MTs. Asterisks refer to significant difference from 1mmoll–1 CPR alone (*P<0.05, significant; between frames) using the 488nm line of an argon/krypton laser and **P<0.01, highly significant). Numbers in parentheses indicate the number the 594nm line of a helium laser, in addition to the 405nm UV laser of animals tested. For S. gregaria, measurements on 3–5 MTs provided a line. The image series was processed and edited using Imaris software. mean for each animal. Confocal images are based on 240 optical sections of a Z-series performed at intervals of 0.7mm. Experiments were conducted multiple times with corresponding results. All control preparations performed solely on this structure (slight residual coloration was without primary antibody were negative for immunostaining. occasionally observed in the haemolymph). These results were compared with the accumulation in S. gregaria MTs. The luminal Chemicals concentration of CPR in the gut of H. crispae and in the MT of S. All chemicals were obtained from Sigma-Aldrich (St Louis, MO, gregaria when exposed to a 1mmoll–1 CPR solution, in the presence USA). Bafilomycin was dissolved and stored in dimethyl sulphoxide or absence of inhibitors, is shown in Figs3 and 5. Activity of H. crispae (DMSO). Inhibitors were allowed to dissolve in the CPR solution during the corresponding exposures is depicted in Fig.4. overnight prior to use. In H. crispae, areas void of gut contents appear transparent, whereas those with gut contents are a light to dark brown colour Statistics (Fig.3B, SW control). Only areas devoid of gut content were used Data are expressed as means ± s.d. unless otherwise stated. The for quantification of dye accumulation. In order to expose internal statistical significance of differences between the various exposures epithelia to the test solutions a small hole was made in the cuticle was tested using one-way ANOVA followed by a Tukey multiple in the anterior part of the animal. Although clearly affected by the comparisons of means. The statistical tests were performed using punctured cuticle compared with untreated specimens, animal the data analysis program OriginPro 7.5 (OriginLab). Significance activity was surprisingly high under control conditions, even 60min levels were P>0.05 (not significant), P<0.05 (significant, *) and after the small hole in the cuticle was made (Fig.4). This implies P<0.01 (highly significant, **). that the osmolality as well as the ionic composition of the used SW are within tolerable limits of extracellular fluid conditions. RESULTS When intact, non-punctured animals were exposed to the CPR accumulation 1mmoll–1 CPR solution, no internal CPR accumulation was evident, During our initial observations on CPR transport in punctured H. showing that the surrounding medium is not ingested by the animals crispae, dye accumulation was almost exclusively observed in the within the experimental period (Fig.3B). However, following tardigrade midgut, and for this reason subsequent analyses were exposure of punctured animals to the same 1mmoll–1 CPR solution,

THE JOURNAL OF EXPERIMENTAL BIOLOGY 502 K. A. Halberg and N. Møbjerg

+Bafilomycin +Bafilomycin Fig. 6. The effect of bafilomycin and dimethylsulphoxide (DMSO) on +DMSO +DMSO +DMSO +DMSO  CPR –14% –21% CPR +5% –26% CPR accumulation. (A)Representative light micrographs of the A C tardigrade midgut (mg) following exposure to the test solutions. The percentage change in CPR concentration, compared with experiments with CPR alone, is noted. Scale bars, 50 m. mg mg gm lu tc m tc lu tc lu (B)Corresponding luminal concentration of CPR in midgut lumen. (C)Representative light micrographs of locust MTs following exposure to the test solutions. The percentage change in CPR ** concentration, compared with experiments on CPR alone, is noted. 1.6 B 1.6 D ** Scale bars, 100mm. (D)Corresponding luminal concentration of (11) CPR in the MTs. Asterisks refer to significant difference from (10) –1 1.4 1.4 1mmoll CPR alone (*P<0.05, significant; **P<0.01, highly significant). Numbers in parentheses indicate the number of animals tested. For S. gregaria, measurements on 3–5 MTs 1.2 1.2 (8) ) provided a mean for each animal. –1 ** 1.0 * 1.0 (13) (mmol l 0.8 * 0.8

lumen 0.6 (5) (8) 0.6

[CPR] 0.4 0.4

0.2 0.2

0 0

CPR –1 –1 CPR

bafilomycin –1 –1 bafilomycin 1 mmol l +DMSO (0.5%) 1 mmol l +DMSO (0.5%) μmol l +DMSO (0.5%) +DMSO (0.5%) +5 μmol l +5 dye accumulation was readily visible in the midgut lumen, experiment showed that the observed inhibition is partly due to suggesting that organic anion transport is mediated by the midgut DMSO – see below). Although the mean CPR concentration in the epithelium; CPR concentration in the midgut lumen reached a mean presence of bafiolmycin A1 was higher in S. gregaria, i.e. of 0.68±0.1mmoll–1 (Fig.3B,C). Similarly, dye accumulation was 0.97±0.18mmoll–1 (Fig.5), a corresponding 26% reduction was evident in the lumen of the insect MTs, reaching a mean CPR observed. The activity of H. crispae was markedly affected by –1 concentration of 1.3±0.13mmoll (Fig.5B,C). bafilomycin A1, with most animals showing movements only DNP is a mitochondrial un-coupler that inhibits mitochondrial following tactile stimuli (Fig.4). Notably, visual colour changes to ATP production. For H. crispae, ~81% reduction in the mean CPR CPR were observed (i.e. the dye turned purple) in some specimens concentration was detected in experiments using DNP, resulting in immediately after transfer to SW, and in all animals 10min post- a midgut CPR concentration of only 0.1±0.07mmoll–1 (Fig.3). The exposure, suggesting an alkalization of the midgut content, which tardigrades almost all became passive (Fig.4), indicating that a range is consistent with the specific inhibition of an apical H+-ATPase. of tissues was affected by the DNP application. In comparison, a Because bafilomycin was dissolved in DMSO, we tested whether similar reduction of ~80% was observed in the MTs of S. gregaria, the used concentrations of DMSO (0.5%) had an effect on CPR with CPR concentrations of 0.26±0.12mmoll–1 (Fig.5). This finding accumulation in both animals. In S. gregaria, no significant change shows that CPR accumulation is ATP dependent. The Na+/K+- in CPR accumulation was detected (Fig.6C,D), but in H. crispae, ATPase and vacuolar H+-ATPase are likely ATP-consuming a significant change was observed in the presence of 0.5% DMSO candidates, which could energize transepithelial dye transport. We (Fig.6A,B). A significant difference between 0.5% DMSO-treated therefore investigated CPR transport during applications of known animals and 0.5% DMSO + bafilomycin-treated specimens, pump inhibitors. however, was also detected (Fig.6A,B), revealing that the observed Solutions containing ouabain, a plant alkaloid that binds to and inhibition is only partly caused by bafilomycin in H. crispae. The inhibits the Na+/K+-ATPase, revealed a significant effect on CPR observation that DMSO has an effect on CPR transport in tardigrades transport in both H. crispae and S. gregaria. The mean CPR is in contrast to previous reports from insects, stating that a concentration was 0.44±0.11mmoll–1 (Fig.3) in H. crispae, which concentration of DMSO below 1% has no effect on the fluid is a 21% reduction in luminal CPR concentration, and secretion rate in the MTs of Drosophila (Linton and O’Donnell, 1.04±0.19mmoll–1 (Fig.5) in S. gregaria, corresponding to ~20% 2000). reduction. Activity of H. crispae was not affected by this test solution Furthermore, we investigated the impact of two well-known (Fig.4). substrates of organic anion transporters, PAH and probenecid, on + The vacuolar H -ATPase inhibitor bafilomycin A1 similarly the ability to accumulate CPR. Both PAH and probenecid are type affected CPR transport significantly, showing a mean CPR I organic anions (carboxylates), which appeared to reduce the CPR concentration of 0.44±0.13mmoll–1 (Fig.3) in H. crispae, which is concentration in our pilot experiments on both H. crispae and S. a 21% reduction compared with animals treated with CPR alone gregaria. Addition of 10mmoll–1 PAH caused a reduction of ~44% (note, bafilomycin was dissolved in DMSO, and a control in luminal CPR concentration in H. crispae and ~40% in S.

THE JOURNAL OF EXPERIMENTAL BIOLOGY Epithelial transport in tardigrades 503

+0.1 mmol l–1 +1 mmol l–1 +10 mmol l–1 +0.1 mmol l–1 +1 mmol l–1 +10 mmol l–1 Fig.7. Titration of the inhibitory effect of CPR –26% –56% –61% CPR –9% –47% –77% probenecid. (A)Representative light A CPR C micrographs of the tardigrade midgut (mg) lu following exposure to the different mg –1 mg mg mg lu lu lu tc concentrations (0.1, 1 and 10mmoll ) of tc probenecid. The percentage change in CPR concentration, compared with experiments with CPR alone, is noted. Scale bars, 50mm. ** (B)Corresponding luminal concentration of 1.6 B 1.6 D ** CPR in midgut lumen. (C)Representative (11) ** light micrographs of the insect MTs following 1.4 1.4 (5) exposure to the different concentrations (0.1, ** 1 and 10mmoll–1) of probenecid. The 1.2 1.2 percentage change in CPR concentration,

)

–1 ** compared with experiments on CPR alone, is 1.0 ** 1.0 ** noted. Scale bars, 100mm. (D)Corresponding ** (5) luminal concentration of CPR in the MTs. (mmol l 0.8 (13) 0.8 Asterisks refer to significant difference from * 1mmoll–1 CPR alone (*P<0.05, significant;

lumen 0.6 0.6 **P<0.01, highly significant). The numbers in (7) parentheses indicate the number of animals

[CPR] (9) 0.4 0.4 tested. For S. gregaria, measurements on (4) (10) 3–5 MTs provided a mean for each animal. 0.2 0.2

0 0

CPR –1 –1 CPR

probenecid probenecid probenecid probenecid –1 –1 –1 –1 –1 probenecid–1 probenecid 1 mmol l 1 mmol l

+1 mmol l +1 mmol l +0.1 mmol l +10 mmol l +0.1 mmol l +10 mmol l gregaria, with a mean concentration of 0.31±0.07mmoll–1 (Fig.3) noticeably passive during probenecid exposure (Fig.4), indicating and 0.78±0.19mmoll–1 (Fig.5), respectively. In solutions containing that, at the high concentration given, probenecid affects processes 10mmoll–1 probenecid, dye accumulation was drastically reduced in addition to organic anion transport. Indeed, there is evidence that in H. crispae by ~61% compared with solutions containing CPR probenecid inhibits cellular oxidative metabolism at concentrations only, averaging merely 0.21±0.05mmoll–1 in luminal concentration of >1mmoll–1 (Masereeuw et al., 2000). Consequently, we titrated (Fig.3). Dye accumulation was even more reduced in S. gregaria the effect of probenecid through successive dilution of the inhibitor, (by ~77%), averaging 0.31±0.1mmoll–1 in luminal concentration using 0.1 and 1mmoll–1 concentrations, in addition to 10mmoll–1 of probenecid-exposed tubules (Fig.5). Halobiotus crispae became (Fig.7). These results show an inverse relationship between

mu. f. α Fig.8. MT structure and subcellular localization of the - A D G -subunit + + F-actin subunit of the Na /K -ATPase in S. gregaria. (A–C) Sections DAPI (2mm Araldite®) stained with Toluidine Blue. A prominent brushborder is present, as well as several transverse muscle mv fibers; contraction creates peristalsis, which aids in luminal lu clearance (visual observation). Mononucleated principal-like cells and double-nucleated cells (dashed circles) were tc observed. (D)Imaris reconstruction (shadow projection mode) of a partial confocal laser scanning microscopy stack B displaying a median, longitudinal cross-section. (E)Imaris reconstruction (shadow projection mode) displaying the mu. f. E tubular lumen. Basal localization of the -subunit is evident. (F) Immunohistochemistry on paraffin sections confirms the mu. f. mv  basal localization of the pump. (G)Imaris three-dimensional lu mv lu reconstruction of an entire stack, again showing that the Na+/K+-ATPase localizes to the basal plasma cell membranes. A longitudinal muscle fiber (mu. f.) runs the mu. f. length of the MT. lu, lumen; mv, microvilli. Scale bar, 75mm. C mu. f. F

lu lu tc mv

THE JOURNAL OF EXPERIMENTAL BIOLOGY 504 K. A. Halberg and N. Møbjerg probenecid concentration and CPR accumulation (and activity; data ATPase (bafilomycin and ouabain reduce CPR accumulation), and not shown). At a concentration of 0.1mmoll–1 probenecid, CPR possibly transporter mediated (inhibited by the prototypic organic concentration was reduced by 26% with a luminal concentration of anions PAH and probenecid). The latter suggests that CPR transport 0.42±0.07mmoll–1 in H. crispae, and by 9% corresponding to a is (at least partly) transcellular. Comparing the transport characteristics concentration of 1.2±0.14mmoll–1 in S. gregaria. When applied at of CPR transport between the tardigrade midgut and the insect MT a concentration of 1mmoll–1, probenecid reduced CPR concentration reveals a surprisingly similar overall pharmacological profile of the by 56% corresponding to a luminal concentration of investigated tissues, albeit with markedly higher concentrations of 0.25±0.05mmoll–1 in H. crispae, and by 47% equivalent to CPR observed in the insect MT. In addition to transcellular transport 0.7±0.16mmoll–1 in S. gregaria (Fig.7). of the dye, fluid secretion may augment transport of organic anions by convective secretion through the paracellular pathway, and/or Midgut and MT structure and immunostaining reduce diffusive back-flux of organic anions from the tubule lumen Our transport studies indicate that CPR transport is active and to the haemolymph (O’Donnell and Leader, 2006; Chahine and transporter mediated in the midgut of H. crispae as well as the MTs O’Donnell, 2010). Consequently, without a measure of fluid secretion of S. gregaria. It is well established that the vacuolar H+-ATPase rates, and thereby a measure of net CPR secreted, we cannot conclude is important for energizing transepithelial transport in insects, whether the difference in relative concentration of CPR in the gut whereas (and in contrast to vertebrate literature) the role of the lumen of H. crispae, compared with the MTs of S. gregaria, is a Na+/K+-ATPase is controversial (e.g. Torrie et al., 2004; Beyenbach consequence of a lower dye transport capacity, or whether it reflects et al., 2010). The data obtained in the present study using ouabain differences in fluid transport rates. Also, we cannot be certain that would suggest that the Na+/K+-ATPase is important for CPR the concentration of CPR in the haemolymph of H. crispae is exactly transport in both animals. Conversely, ouabain could act as a the same as that of the surrounding bath – although, the relatively competitive inhibitor, being transported by the same transporters as high dye concentration used along with the small diffusion distances CPR. We therefore investigated whether the Na+/K+ pump is involved, the relatively long exposure time (60min) and the animal expressed in the epithelia. movements that facilitate fluid exchange, would make potential Unfortunately, our attempt to localize Na+/K+-ATPase in concentration differences negligible. tardigrades was unsuccessful, both on whole mounts and on paraffin A vast number of papers have investigated various aspects of sections, and also in attempts at antigen retrieval. organic anion transport in insect MTs. In the present study we Our observations on the MTs of S. gregaria generally correspond observed a mean CPR concentration of 1.3±0.13mmoll–1 when no with those previously described (Garret et al., 1988), when focusing inhibitors were added. This concentration is elevated above the bath on the proximal region and parts of the middle region used in the concentration (1mmoll–1), albeit by a much smaller factor than that present study. In general, we observed a prominent brushborder reported from other insects (Maddrell et al., 1974; Bresler et al., (Fig.8B,C) and at least two cell types. One type, a principal-like cell, 1990; Linton and O’Donnell, 2000; Leader and O’Donnell, 2005). has a single nucleus, while the other is a very large double-nucleated However, when exposed to external CPR concentrations of 1, 10 cell (dashed circles in Fig.8). Cross-striated muscle fibers are present and 100mmoll–1, the mean luminal CPR concentrations were in both the transverse (Fig.8A,B) and longitudinal direction 0.16±0.03, 0.73±0.09 and 0.89±0.07mmoll–1, respectively, which (Fig.8E,G). The transverse muscle fibers were only observed in the is a factor of ~160, 70 and 9 above bath concentrations (Fig.2). proximal-most region of the MTs, whereas the longitudinal muscle When exposed to a bath concentration of 5mmoll–1 CPR, the mean extended the entire length of the tubule. Visual observations revealed luminal concentration was 1.79±0.33mmoll–1, a factor of 2.8 below that the transverse muscles aid the clearance of luminal content into external concentrations. Consequently, the luminal CPR the midgut through peristaltic contractions. Our immunocytochemical concentration is maximally elevated (~160-fold) above that in the analysis shows that the Na+/K+-ATPase is expressed in the MTs of bathing medium when the latter contains CPR at a concentration of S. gregaria, and is localized to the basal plasma membranes 1mmoll–1. This concentrative ability is among the highest measured (Fig.8D–G), although to a lesser extent in the double-nucleated cells for insects, and is an additional confirmation of active transport of (Fig.8D,G). The localization was confirmed by counterstaining of CPR by the insect MTs. nuclei by DAPI and staining apical microvilli with phalloidin. This Ouabain, a well-characterized, potent inhibitor of the Na+/K+- is, to our knowledge, the first study showing immunolocalization of ATPase, reduced the CPR concentration in both the tardigrade the Na+/K+-ATPase in S. gregaria MTs. Interestingly, the localization midgut and the insect MTs by ~20–21%. A similar 23% reduction of the transporter substantiates previously published data on isolated in PAH secretion in the presence of 1mmoll–1 ouabain was reported basal membrane fractions from MTs and hindgut from S. gregaria, from MTs of D. melanogaster (Linton and O’Donnell, 2000), while which show a prominent Na+/K+-ATPase activity (Al-Fifi, 2007). fluorescein uptake was reduced in MTs of Blaberus giganteus by 30% and in MTs of Locusta migratoria by 20%, when ouabain was DISCUSSION used at 1 and 0.1mmoll–1, respectively (Bresler et al., 1990). Our Our study is significant in two main aspects: (i) we are the first to immunocytochemical investigation on the MTs of S. gregaria provide evidence for active epithelial transport in tardigrades, and revealed expression of the Na+/K+-ATPase in the basal cell (ii) our data show that tardigrades possess an organic anion transport membranes, although to a lesser extent in the double-nucleated cells. system. Consequently, our study is important for understanding the A basal localization of the Na+/K+-ATPase in principal cells of MTs evolution of transport systems. of D. melanogaster has previously been reported (Torrie et al., 2004). CPR is categorized as a sulphonate, but is known to compete with The fact that there is expression of the Na+/K+-ATPase in the basal substrates for both solute carriers (SLC22 and SLC21/SLCO) and membranes, and that CPR transport is ouabain sensitive, suggests ABC transporters in a number of organisms (Pritchard et al., 1999; that the pump is important for CPR transport. Alternatively, it could Linton and O’Donnell, 2000; Chahine and O’Donnell, 2009). In H. be argued that the inhibitory effects of ouabain are due to competitive crispae, CPR transport is ATP dependent (strong inhibition by DNP), inhibition, rather than to non-competitive inhibition, given the fact probably energized by both the Na+/K+-ATPase and the V-type H+- that ouabain is actively transported by members of the SLC21/SLCO

THE JOURNAL OF EXPERIMENTAL BIOLOGY Epithelial transport in tardigrades 505 subfamily (Oatps) in D. melanogaster (Torrie et al., 2004). Whether Haemolymph this is ubiquitous in insects is at present not known; however, several Solute carrier (SLC21/SLCO) members of the Oatp family are known to transport ouabain in Na+/K+-ATPase human tissue (see Hagenbuch and Gui, 2008). Competition studies Basal lamina using fluorescently labeled ouabain would help in clarifying this K+ OA– matter. Considering the striking similarities between the pharmacological profiles of the tardigrade midgut and insect MT, ATP + + + it seems reasonable to assume that the Na /K -ATPase is similarly Pi+ADP Na present in the basal cell membranes of the tardigrade midgut (in spite of failed attempts to localize this transporter). In support of Tardigrade Nucleus midgut cell this interpretation is the fact that transcripts for the -subunit of the + + Na /K -ATPase were found in the expressed sequence tags (EST) H+ OA– library from Hybsibius dujardini Doyère, 1840 (TardiBASE cluster Pi+ADP ATP ID: HDC01733 TardiBASE; http://xyala.cap.ed.ac.uk/research/ tardigrades/tardibase.shtml). Bafilomycin is a specific inhibitor of the V-type H+-ATPase and was found to reduce CPR accumulation in both H. crispae and S. V-type H+-ATPase gregaria. This observation is consistent with the fact that the V-type Apical junction H+-ATPase is viewed as being central to the transport activities of Lumen complex the MT in insects (Weng et al., 2003; Beyenbach et al., 2010) and perhaps also for transport in tardigrades; the B-, C-, D-, E-, G- and Fig.9. Tentative model of the tardigrade midgut cell derived from the H-subunits of the V-type H+-ATPase were found in the EST library current study on H. crispae. Based on the pharmacological profile of the of H. dujardini (TardiBASE). Indeed, visual changes to CPR colour tardigrade midgut epithelium, both the Na+/K+-ATPase and the V-type H+- were observed in the midgut of H. crispae (CPR became purple; data ATPase are potential candidates for providing an electrochemical driving force for the transepithelial movement of organic anions. Transport not shown) 10min post-incubation, suggesting an alkalization of the characteristics and the presence in tardigrade EST libraries suggest that a midgut content. This observation is in accordance with specific member of the SLC21/SLCO transporter family may mediate the + inhibition of an apical V-type H -ATPase in the tardigrade midgut. basolateral entry of organic anions in tardigrades. The exact coupling PAH is a prototypical substrate of the classic organic anion transport between electrochemical gradients generated by the pumps and transport system (i.e. the SLC22 subfamily) and was shown to be an effective of ions, as well as the nature of the apical exit step, are not known. inhibitor of CPR transport in both H. crispae and S. gregaria (~40% reduction in both animals). This finding is a strong indication that PAH (carboxylate) and CPR (sulphonate) transport are mediated by oxidative phosphorylation (Masereeuw et al., 2000). It is therefore a common transporter, or alternatively through two separate transport possible that the reduction in CPR transport is non-specific when systems overlapping in affinity, in both investigated epithelia. Whether the drug is applied in concentrations ≥1mmoll–1. At lower carboxylates (e.g. PAH and probenecid) and sulphonates (e.g. CPR) concentrations of probenecid (<1mmoll–1), however, animal motility are handled by a common transporter (Bresler et al., 1990), or by two was regained (in a concentration-dependent manner), and was not separate transport systems (Maddrell et al., 1974; Linton and significantly different from that in solutions containing CPR only O’Donnell, 2000; Chahine and O’Donnell, 2009), appears to be highly (data not shown). Noticeably, the relative inhibition of CPR transport variable and/or species specific. Indeed, transport of PAH and by probenecid was of a greater magnitude in the midgut of H. crispae probenecid (carboxylates) and CPR and methotrexate (MTX; than in the MT of S. gregaria at the lower (specific) concentrations sulphonates) are all mediated by one tranporter (OAT1) in humans, of probenecid. This could indicate a higher specificity for probenecid but not in rats (Burckardt and Burckardt, 2003), whereas MTX uptake by the tardigrade transporter(s) compared with that of S. gregaria. is competitively inhibited by CPR and probenecid, but not PAH in D. melongaster (Cahine and O’Donnell, 2009). The tardigrade midgut cell transports organic anions Probenecid is implicitly regarded as a competitive inhibitor of The observed CPR transport by the tardigrade midgut and insect MT organic anion transport, and has been shown to compete with both presumably takes place because CPR is structurally and chemically solute carrier and ABC transporter substrates (Bresler et al., 1990; similar to compounds encountered in the natural habitat of these Linton and O’Donnell, 2000; Neufeld et al., 2005; Leader and animals. In terrestrial insects, organic anion transport seems especially O’Donnell, 2005). It is typically applied in a concentration of important for excreting xenobiotics such as plant toxins and pesticides 1mmoll–1; here, we examined the effects of probenecid on CPR (Lanning et al., 1996; Torrie et al., 2004; Neufeld et al., 2005; Buss transport in the range 0.1–10mmoll–1. In H. crispae, probenecid and Callaghan, 2008); however, a similar physiological requirement reduced CPR accumulation by 26%, 56% and 61% at a concentration is not as readily apparent in tardigrades. The handling of endogenous of 0.1, 1 and 10mmoll–1, respectively, whereas the corresponding toxins derived from metabolism, in contrast, is a ubiquitous reduction in CPR concentration was 9%, 47% and 77% in S. prerequisite for all organisms, and it is likely that this is the primary gregaria. In the presence of very high probenecid concentrations function of the organic anion transport system in tardigrades. (10mmoll–1), we observed an almost total loss of motility in H. Because of the broad overlap in substrate specificity between the crispae. Indeed, the level of animal movement was comparable to different groups of organic anion transporters, we cannot definitively that in treatments containing DNP. This observation suggests that identify, on the basis of the data presented here, the type of the drug affected processes in addition to CPR transport at the given transporter(s) responsible for the observed CPR transport. However, concentration. In fact, when applied at concentrations of 1mmoll–1 searching the EST library of H. dujardini (TardiBASE) revealed or above, probenecid is reported to induce a range of non-specific that putative genes of Oatp (cluster ID: HDC00927) (SLC21/SLCO effects presumably initiated by the uncoupling of mitochondrial protein family), P-gp (cluster ID: HDC01698) and Mrps (cluster

THE JOURNAL OF EXPERIMENTAL BIOLOGY 506 K. A. Halberg and N. Møbjerg

ID: HDC00004, HDC02687 and HDC03352) (ABCB and ABCC Burckardt, B. C. and Burckardt, G. (2003). Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev. Physiol. Biochem. Pharmacol. 146, protein families) are present and expressed in tardigrades – just as 95-158. they are in insects (Maddrell et al., 1974; Lanning et al., 1996; Buss, D. S. and Callaghan, A. (2008). Interaction of pesticides with p-glycoprotein and other ABC proteins: A survey of the possible importance to insecticides, herbicide and Bresler et al., 1990; Linton and O’Donnell, 2000; Torrie et al., 2004; fungicide resistance. Pestic. Biochem. Physiol. 90, 141-153. Neufeld et al., 2005; Leader and O’Donnell, 2005; O’Donnell and Chahine, S. and OʼDonnell, M. J. (2009). Physiological and molecular characterization of methotrexate transport by Malpighian tubules of adult Drosophila melanogaster. J. Insect Leader, 2006; Chahine and O’Donnell, 2009). Comparing the Physiol. 55, 927-935. transport characteristics of the different groups of transporters reveals Chahine, S. and OʼDonnell, M. J. (2010). Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules of that it is unlikely that members of the ABC transporter family (i.e. Drosophila melanogaster larvae. Arch. Insect Biochem. Physiol. 73, 128-147. P-gps and Mrps) mediate the basolateral entry of CPR in H. crispae Dantzler, W. H. (2002). Renal organic anion transport: a comparative and cellular perspective. Biochim. Biophys. Acta 1566, 169-181. and S. gregaria. This assumption is based on the fact that P-gp Dewel, R. A. and Dewel, W. C. (1979). Studies on the tardigrades. J. Morphol. 161, 79- transporters predominantly recognize large cationic species (Russel, 110. 2010), and because Mrp transporters mainly transport large Dow, J. A. T. and Davies, S. A. (2006). The Malpighian tubule: rapid insights from post- genomic biology. J. Insect. Physiol. 52, 365-378. (>500Da) polyvalent type II organic anions (Wright and Dantzler, Faria, M., Navarro, A., Luckenbach, T., Piña, B. and Barata, C. (2010). Characterization 2004; Russel, 2010) – the apical transporter MRP2, however, was of the multixenobiotic resistance (mxr) mechanism in embryos and larvae of the zebra mussel (Dreissena polymorpha) and studies on its role in tolerance to single and mixture shown to transport PAH in humans (see below). In addition, PAH combinations of toxicants. Aqua. Toxicol. 101, 78-87. does not compete with MTX (an Mrp substrate) in D. melongaster, Garret, M. A., Bradley, T. J., Meredith, J. E. and Phillips, J. E. (1988). Ultrastructure of the Malpighian tubules of Schistocerca gregaria. J. Morphol. 195, 313-325. suggesting distinct transport systems for these compounds in insects George, R. L., Wu, X., Huang, W., Fei, Y. J., Leibach, F. H. and Ganapathy, V. (1999). (Chahine and O’Donnell, 2009). In contrast, both members of the Molecular cloning and functional characterization of a polyspecific organic anion transporter from Caenorhabditis elegans. J. Pharmacol. Exp. Ther. 291, 596-603. SLC22 and SLC21/SLCO protein families (Oats and Oatps) have Greven, H. (1982). Homologues or analogues? A survey of some structural patterns in been reported to transport all the investigated organic anions, Tardigrada. In Proceedings of the Third International Symposium on the Tardigrada (ed. D. R. Nelson), pp. 55-76. Johnson City, Tennessee: East Tennessee State University including probenecid, PAH and CPR (Pritchard et al., 1999; Lee Press. and Kim, 2004; Torrie et al., 2004). Therefore, it seems probable Hagenbuch, B. and Gui, C. (2008). Xenobiotic transporters of human organic anion transporting polypeptides (OATP) family. Xenobiotica 38, 778-801. that an Oat or Oatp homologue mediates the basolateral entry of Halberg, K. A., Persson, D., Møbjerg, N., Wanninger, A. and Kristensen, R. M. CPR in the tardigrade midgut cell as well as the insect MTs (Fig.9). (2009a). Myoanatomy of the marine Tardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae). J. Morphol. 270, 996-1013. At present, our data do not allow us to make assumptions on the Halberg, K. A., Persson, D., Ramløv, H., Westh, P., Kristensen, R. M. and Møbjerg, N. nature of the luminal exit. Evidence suggests that MRP2 is involved (2009b). Cyclomorphosis in Tardigrada: adaption to environmental constraints. J. Exp. Biol. 212, 2803-2811. in the efflux of organic anions across the brush-border membrane Haugen, B. M., Halberg, K. A., Jespersen, Å., Prehn, L. R. and Møbjerg, N. (2010). in the human kidney proximal tubule (Leier et al., 2000), and a Functional characterization of the vertebrate primary ureter: structure and ion transport mechanisms of the pronephric duct of axolotl larvae (Amphibia). BMC Develop. Biol. 10, similar situation could exist in tardigrades and insects. Our data 56. tentatively suggest that the V-type H+-ATPase, and perhaps also Jönsson, K. I., Rabbow, E., Schill, R. O., Ringdahl, M. H. and Rettberg, P. (2008). + + Tardigrades survive exposure to space in low Earth orbit. Curr. Biol. 18, R729-R731. the Na /K -ATPase, provide the driving force for the transepithelial Kristensen, R. M. (1982). The first record of cyclomorphosis in Tardigrada based on a transport of organic anions in both H. crispae and S. gregaria new genus and species from Arctic meiobenthos. Z. zool. Syst. Evolut.-forsch. 20, 249- 270. (Fig.9). It is likely that a large lumen-positive transepithelial Lanning, C. L., Fine, R. L., Corcoran, J. J., Ayad, H. M., Rose, R. L. and Abou-Donia, potential, generated by an apical H+-ATPase provides a significant M. B. (1996). Tobacco budworm P-glycoprotein: biochemical characterization and its involvement in pesticide resistance. Biochim. Biophys. Acta. 1291, 155-162. driving force for the accumulation of anions in the lumen. However, Leader, J. P. and OʼDonnell, M. J. (2005). Transepithelial transport of fluorescent p- the exact coupling between electrochemical gradients generated by glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets. J. Exp. Biol. 208, 4363-4376. the pumps and transport of the ions is not known. Lebovitz, R. M., Takeyasu, K. and Fambrough, D. M. (1989). Molecular characterization In future studies, it would be of interest to investigate whether and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J. 8, 193-202. substrates of Mrps (e.g. Texas Red and MTX) also accumulate in Lee, W. and Kim, R. B. (2004). Transport and renal drug elimination. Annu. Rev. the midgut of tardigrades, and whether transport of these anions Pharmacol. Toxicol. 44, 137-166. occurs via a separate or a common transporter to CPR. 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Acta Physiol 2011, 202, 409–420

REVIEW Survival in extreme environments – on the current knowledge of adaptations in tardigrades

N. Møbjerg,1 K. A. Halberg,1 A. Jørgensen,2 D. Persson,1,2 M. Bjørn,3 H. Ramløv3 and R. M. Kristensen2 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark 2 Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark 3 Department of Science, Systems and Models, University of Roskilde, Roskilde, Denmark

Received 17 October 2010, Abstract revision requested 13 November Tardigrades are microscopic animals found worldwide in aquatic as well as 2010, terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa, revision received 6 January 2011, accepted 10 January 2011 as do the two major invertebrate model organisms: Caenorhabditis elegans Correspondence: N. Møbjerg, and Drosophila melanogaster. We present a brief description of the tardi- Department of Biology, August grades and highlight species that are currently used as models for physio- Krogh Building, Universitetsparken logical and molecular investigations. Tardigrades are uniquely adapted to a 13, DK-2100 Copenhagen Ø, range of environmental extremes. Cryptobiosis, currently referred to as a Denmark. reversible ametabolic state induced by e.g. desiccation, is common especially E-mail: [email protected] among limno-terrestrial species. It has been shown that the entry and exit of cryptobiosis may involve synthesis of bioprotectants in the form of selective carbohydrates and proteins as well as high levels of antioxidant enzymes and other free radical scavengers. However, at present a general scheme of mechanisms explaining this phenomenon is lacking. Importantly, recent research has shown that tardigrades even in their active states may be ex- tremely tolerant to environmental stress, handling extreme levels of ionizing radiation, large fluctuation in external salinity and avoiding freezing by supercooling to below )20 C, presumably relying on efficient DNA repair mechanisms and osmoregulation. This review summarizes the current knowledge on adaptations found among tardigrades, and presents new data on tardigrade cell numbers and osmoregulation. Keywords cell numbers, cryptobiosis, evolution, osmoreglation, supercool- ing, tardigrade.

Tardigrades, also known as water bears, are micro- 2007). Tardigrades are exceptional among metazoans in scopic metazoans (approx. 0.1–1.2 mm). They were their adaptations to the most extreme environments. As discovered in the 18th Century with the development of is also known from selected species of arthropods, early microscopes and were first described by the nematodes and rotifers, many species have the ability to German zoologist Goeze in 1773, who named them enter cryptobiosis; a state of suspended animation ‘kleiner Wasserba¨r’ or little water bear because of their believed to be ametabolic (Keilin 1959, Clegg 2001). strong resemblance to a little bear (see e.g. Ramazzotti Corti already noted these adaptations in tardigrades in & Maucci 1983, Nelson 2001, Schill 2010). Shortly 1774, when he observed that these animals could be after in 1776, the current name, Tardigrada (from Latin revived after desiccation (Kinchin 1994). In 1962, tardigradus, slow-moving), was given by the Italian Tardigrada was recognized as a phylum by Ramazzotti natural scientist Spallanzani (see e.g. Rebecchi et al. in Il Phylum Tardigrada (Ramazzotti 1962). There are

2011 The Authors Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 409 Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420 currently approx. 1000 described species (Guidetti & between the two major invertebrate model organisms – Bertolani 2005, Degma et al. 2010), but this is likely far the nematode Caenorhabditis elegans Maupas, 1900 from the real number of tardigrade species, as especially and the arthropod Drosophila (Sophophora) melanog- the marine arthrotardigrades remain relatively unex- aster Meigen, 1830 (Goldstein & Blaxter 2002, Gabriel plored. & Goldstein 2007). Tardigrada belongs to the invertebrate superclade Little is known about the physiological mechanisms Ecdysozoa, however; their precise phylogenetic position underlying adaptations to extreme environmental con- is still debated and it is presently not clear whether the ditions in tardigrades. Past centuries of tardigrade group is more closely related to arthropods and research have mainly focused on species descriptions onychophorans or to the nematodes and nematomorphs and morphological investigations related to phyloge- (Fig. 1) (Aguinaldo et al. 1997, Dunn et al. 2008, netic analysis. In recent years, however, research in the Edgecombe 2010). In any case, as noticed in recent field has taken advantage of new molecular tools and an papers, this group has a central position placed in increasing number of scientists find the group fasci-

Loricifera

Kinorhyncha

Priapulida

Nematomorpha ECDYSOZOA

Nematoda* ? Tardigrada*

Arthropoda*

Onychophora

PROTOSTOMIA

Brachiopoda

Annelida

LOPHOTROCHOZOA Mollusca

Rotifera*

Platyhelminthes

Chordata DEUTEROSTOMIA Echinodermata

Cnidaria

Porifera

Figure 1 Evolutionary position of tardigrades in the Animal Kingdom. Phylogeny of the Metazoa (animals) based on Aguinaldo et al. (1997) and Dunn et al. (2008) showing selected phyla with emphasis on the position of the Tardigrada. The inferred position of the tardigrades is based on EST sequences from Richtersius coronifer and Hypsibius dujardini. Ecdysozoa includes all molting animals and is one of the two protostome superclades. The marked phyla have cryptobiotic species.

2011 The Authors 410 Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x Acta Physiol 2011, 202, 409–420 N. Møbjerg et al. Æ Adaptation to extreme environments in tardigrades nating. In particular, the mechanisms underlying the Dewel 1996, Møbjerg & Dahl 1996, Eibye-Jacobsen ability to enter cryptobiosis have attracted considerable 1997, Jørgensen et al. 1999, Greven 2007, Halberg scientific interest. This interest is no doubt associated et al. 2009a). They are found worldwide in aquatic as with the suspected translational output related to a well as terrestrial environments, but depend on free detailed understanding of the complex stress physiology water to be in their active, reproducing state. It has been of tardigrades, i.e. in connection with cryopreservation suggested that tardigrades, like e.g. nematodes, have and dehydration of biological material. Noticeably, the eutely, but detailed studies on the subject are still phenomenon cryptobiosis touches upon our conception lacking. Cell counts based on nuclear staining with of life and death; one of the largest enigmas being how DAPI (4¢,6-diamidino-2-phenylindole) in four active metabolism is restarted after years of suspension. This stage adults of the marine eutardigrade Halobiotus review puts focus on physiological and molecular crispae Kristensen, 1982 revealed a total cell number of adaptations to extreme conditions found among tardi- around 1060 cells, when excluding gametes (Fig. 2). grades. We additionally present a brief description of This number could represent a slight underestimate of the phylum and highlight species that are currently used the total somatic cell number as body cavity cells (also as models for these investigations. known as storage cells) may have escaped two of the specimens, which were punctured prior to the staining (Fig. 2). We did not observe cell divisions (mitosis) Phylum Tardigrada during the counts. Mitosis has, however, previously been reported in post-embryonic eutardigrades (Berto- Tardigrade phylogeny and evolution lani 1970a,b, 1982). About 35 extant animal groups have body plans and There are two main evolutionary lines within the genes that are distinct enough to warrant elevation to tardigrades, represented by the classes Eutardigrada and phylum status (Nielsen 2001). The tardigrades, com- Heterotardigrada (Fig. 3) (see e.g. Jørgensen & Kris- prising the phylum Tardigrada, are one of these groups tensen 2004). The validity of a third class, Mesotardi- (Fig. 1). Tardigrades are microscopic invertebrates with grada, is currently uncertain. Mesotardigrada only a well developed organization including brain and contains a single species, Thermozodium esakii Rahm, sensory organs, muscles, a complex feeding apparatus 1937 originally found in a hot spring in Japan. The type and alimentary tract, reproductive and osmoregulatory specimens of T. esakii no longer exist and the type organs (see e.g. Rebecchi & Bertolani 1994, Dewel & locality was apparently destroyed in an earthquake

AP

br pb eo go mg gI gII c.gl. gIII c.gl. c.gl. gIV Leg 1 Leg 4 Leg 2 c.gl. Leg 3

Figure 2 Cell numbers in Halobiotus crispae. 3-D reconstruction of cell arrangement in the eutardigrade H. crispae based on confocal laser scanning microscopy of a DAPI stained specimen. In order to obtain an estimate of somatic cell numbers in this species, specimens were relaxed in CO2-enriched water, fixed in 4% paraformaldehyde and ultrasonicated. Two of the four specimens were additionally delicately punctured with a fine needle. The specimens were subsequently incubated with DAPI and thoroughly rinsed before mounting. Image acquisition was performed using a Leica DM RXE 6 TL microscope equipped with a Leica TCS SP2 AOBS confocal laser scanning unit. Cell counts and image processing were performed using the software program Imaris (Bitplane, Zurich, Switzerland). The total number of somatic cells in H. crispae was estimated at approx. 1060, based on stainings of four male specimens (mean Æ SD: 1058 Æ 53). This number could represent a slight underestimate of the total somatic cell number as so-called body cavity cells may have escaped the two specimens that were punctured prior to staining (cell counts for the punctured specimens: 998 and 1088; counts for the non-punctured specimens: 1036 and 1112). The largest number of cells is clearly present in the anterior part of the animal containing the brain and buccopharyngeal apparatus. A, anterior; P, posterior; br, brain; c.gl., claw gland; eo, esophagus; gI–IV, ventral ganglia I–IV; mg, midgut; go, gonad; pb, pharyngeal bulb. Scale bar: 50 lm.

2011 The Authors Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 411 Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420

Richtersius Hypsibius* Echiniscoides Echiniscus Milnesium* Halobiotus Paramacrobiotus Ramazzottius* Echiniscoidae Echiniscidae Milnesiidae Isohypsibioidea Macrobiotoidea Hypsibioidea

Arthrotardigrada Echiniscoidea Apochela Parachela marine limno-terrestrial limno-terrestrial limno-terrestrial non-cryptobionts Intertidal Cryptobionts cryptobionts Cryptobionts non-cryptobionts

Heterotardigrada Mesotardigrada (?) Eutardigrada Figure 3 Tardigrade phylogeny. Phylog- eny of tardigrades showing major clades and position of model/discussed species. The phylogeny is based on Sands et al. Tardigrada *Genome projects (2008).

(Nelson 2002). However, a thorough re-sampling for Tardigrade genomes this species has to our knowledge not been performed. Tardigrades most likely evolved within the marine There is a huge variation in the genome size of environment, and marine species are especially numer- tardigrades ranging from about 75–100 Mb in Hypsi- ous within the heterotardigrade order Arthrotardigrada bius and Ramazzottius to 800 Mb in Bertolanius (Renaud-Mornant 1982, Maas & Waloszek 2001, (Gregory 2010, C-values converted from picograms to Jørgensen et al. 2010). Arthrotardigrades are present base pairs using the conversion 1 pg = 978 Mb accord- in all oceans from intertidal zones to abyssal depths, ing to Dolezel et al. (2003), Bertolanius was previously inhabiting different sediment types. In addition, marine named Amphibolus). For comparison, the genome sizes species are found within the other main heterotardi- of Caenorhabditis elegans and Drosophila melanogas- grade order, Echiniscoidea, represented by the intertidal ter are about 100 Mb and 175 Mb respectively (Greg- Echiniscoides sigismundi (M. Schultze, 1865). This ory 2010). The general diploid chromosome numbers of species may very well be the toughest creature on the eutardigrades are 10–12 and 14 for the heterotar- Earth, having to endure periods of desiccation and low digrade Echiniscus (Bertolani 1982). Polyploidy with oxygen tension as well as large perturbations in salinity up to 24 chromosomes is common in eutardigrades and freezing (Kristensen & Hallas 1980). Nevertheless, (Bertolani 1982). Three major sequencing projects are the exact range of this tardigrade’s tolerances remains currently ongoing within Tardigrada. However, all to be investigated. It may be hypothesized that an three projects are investigating eutardigrade species; Echiniscoides-like tardigrade invaded the freshwater/ no data are presently available for the other main terrestrial environment and gave rise to the almost tardigrade group – the heterotardigrades. The interna- exclusively limno-terrestrial eutardigrades (Kinchin tional collaborative Ecdysozoan Sequencing Project is 1994). This is however currently not supported by assembling the genome of Hypsibius dujardini (Doye`re, molecular data (Sands et al. 2008, Jørgensen et al. 1840) as part of an investigation into the ancestral 2010; Jørgensen et al. 2011). genome of the Ecdysozoa lineage. Prior to this project, The eutardigrades are divided into two orders; the Edinburgh based TardiBASE project, generated Apochela and Parachela. Two genera within the latter more than 5000 EST sequences for H. dujardini order, Ramajendas represented by Ramajendas renaudi (GenBank 2010). The German based FUNCRYPTA (Ramazzotti 1972) in the Southern Hemisphere and project was focused on investigating cryptobiosis in Halobiotus in the Northern Hemisphere, have second- Milnesium tardigradum Doye`re, 1840 through studies arily invaded the marine environment (Ramazzotti of gene and protein expression (Fo¨ rster et al. 2009, 1972, Kristensen 1982, Møbjerg et al. 2007). Although Mali et al. 2010, Schokraie et al. 2010). The project cryptobiosis is common in most eutardigrades, our had in 2010 generated approx. 7000 quality EST recent findings in H. crispae suggest that among these sequences and aimed at advancing the basic understand- secondary marine species, adaptations are present that ing of protein expression in tardigrades through tran- are quite extraordinary (Halberg et al. 2009b). The scriptomic and proteomic studies (Mali et al. 2010). The tardigrades stay active while experiencing large fluctu- Japanese based Kumamushi Genome Project is assem- ations in abiotic factors, fluctuations that in other bling the genome of Ramazzottius varieornatus Berto- tardigrades would induce cryptobiosis. lani and Kinchin, 1993 and has preliminarily predicted

2011 The Authors 412 Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x Acta Physiol 2011, 202, 409–420 N. Møbjerg et al. Æ Adaptation to extreme environments in tardigrades about 20 000 candidate genes in this species (Horikawa level, anatomically as well as physiologically. In the tun et al. 2008, Katayama et al. 2009). The great challenge state tardigrades may not only stand long periods of in the years to come will be to correlate the description of desiccation and exposure to toxic chemicals but also tardigrade genes with studies on the function of these very low subzero temperatures, vacuum, high pressure, genes as nicely illustrated in a recent study on a radiation, extreme pH, anoxia and to some extent high purinergic P2X receptor from H. dujardini (Bavan et al. temperature (see e.g. Wright 2001, Rebecchi et al. 2009). 2007). It has been suggested and to some degree shown that adaptations to these extremes may involve synthe- sis of bioprotectants in the form of selective carbohy- Adaptation to extreme environments drates and proteins, high levels of antioxidant enzymes Tardigrades are extraordinary in their tolerance to and other free radical scavengers, biological membranes extremes, including limno-terrestrial habitats that fre- containing specific phospholipids as well as powerful quently dry out, habitats that freeze and habitats that DNA repair mechanisms (Westh & Ramløv 1991, Schill experience large fluctuations in e.g. osmotic pressure et al. 2004, Jo¨ nsson et al. 2005, Rizzo et al. 2010). and oxygen tension. Cryptobiosis, referred to as a In addition to cryptobiotic tardigrade species, we also reversible ametabolic state induced by unfavorable find species that form cysts and enter diapause (see e.g. environmental conditions, is a common adaptation Møbjerg et al. 2007, Guidetti et al. 2008). Importantly, especially among limno-terrestrial tardigrade species it has been shown that tardigrades even in their active (see e.g. Wright 2001). Four cryptobiosis inducing states may be extremely tolerant to environmental stress physical extremes are traditionally recognized: dehy- (May et al. 1964, Jo¨ nsson et al. 2005, Horikawa et al. dration (anhydrobiosis), extremely low temperatures 2006, Halberg et al. 2009b). Virtually nothing is known (cryobiosis), lack of oxygen (anoxybiosis) and high salt about the normal physiology of tardigrades, and concentration (osmobiosis) (Keilin 1959), with desicca- answers to how they tolerate these extremes may tion induced anhydrobiosis and freezing induced cryo- actually be found here. We have recently shown that biosis being the most extensively studied states. the littoral eutardigrade Halobiotus crispae handles Anhydrobiosis and cryobiosis are not equivalent phe- large fluctuation in external salinity and avoids freezing nomena and likely involve different mechanisms for by supercooling to around )20 C in its active stage protection of cells and tissues (Crowe et al. 1990, (Halberg et al. 2009b). This species is characterized by 1992). Little is known of cryobiosis in tardigrades seasonal cyclic changes in morphology and physiology (Westh et al. 1991, Ramløv & Westh 1992, Westh & known as cyclomorphosis, one of the cyclomorphic Kristensen 1992, Halberg et al. 2009b, Hengherr et al. stages being freeze tolerant (Kristensen 1982, Møbjerg 2009, 2010), whereas a great deal of attention has been et al. 2007, Halberg et al. 2009b). The morphological paid to anhydrobiosis (Wright et al. 1992, Wright changes occurring during cyclomorphosis in H. crispae 2001, Rebecchi et al. 2007, Schill 2010). The anhydro- in some respects resemble the formation of dauer larvae biotic state is characterized by the formation of a so- in C. elegans (see e.g. Cassada & Russell 1975, Burnell called tun with withdrawn legs and a longitudinally et al. 2005). contracted body (see e.g. Bertolani et al. 2004). Tun formation is also seen in bdelloid rotifers, whereas Cryptobiosis in tardigrades desiccation tolerant nematodes coil into a tight spiral. Obviously, the ability to pack internal organs during Many experiments on tardigrade cryptobiosis have been tun formation is an important adaptation to desicca- performed on the eutardigrade Richtersius coronifer. tion. Importantly, tun formation is an active, regulated R. coronifer, also known as the giant yellow water bear, event and not merely an effect of water removal (Crowe has a body length of up to 1 mm (Fig. 4a–c). Both males 1972). Along this line, our unpublished data show that and females are present in some populations, but, as when we expose the active state of the cryptobiotic commonly found among eutardigrades, several popula- tardigrade Richtersius coronifer (Richters, 1903) to tions reproduce by parthenogenesis. R. coronifer lives in water containing high levels of chemical substances, the moss in alpine and arctic environments and is further- tardigrades will respond by contracting their bodies more numerous in moss on carbonated bedrock in dry initiating tun formation thus undergoing chemobiosis–a Swedish lowland areas known as Alvar (see e.g. Westh cryptobiotic response to environmental toxins. The & Kristensen 1992, Jo¨nsson et al. 2001). R. coronifer is formation of the tun is a critical and necessary event a true cryptobiont, tolerating extreme desiccation as for tardigrades entering anhydrobiosis. Much more imposed by e.g. space vacuum conditions (Jo¨ nsson et al. work needs to be done in order to understand the 2008, Persson et al. 2011) and it additionally survives processes undertaken during transformation to this exposures to very low temperatures as encountered by ametabolic state, from the molecular to whole organism transfers into liquid nitrogen (approx. )196 C) in the

2011 The Authors Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 413 Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420

(a) A (b) A (c) A st pb eo P P P

st mg pb mg

* * *

(d) (e) (f) st st

pb st pb pb eo eo

mg mg mg

go * * * * * * * * *

Figure 4 Cryptobiotic and non-cryptobiotic survival in extreme environments. (a–c): Microscopy of Richtersius coronifer from Stora Alvar on the Swedish island O¨ land in the Baltic Sea. Richtersius coronifer has been a model tardigrade for investigations on cryptobiotic survival. (a) Active state; (b) light microscopy and (c) scanning electron microscopy of the cryptobiotic tun state induced by desiccation. In this state R. coronifer e.g. tolerates complete desiccation as experienced by vacuum condition and freezing in liquid nitrogen. (d–f): Microscopy of Halobiotus crispae from the Danish population at Vellerup Vig, Denmark. This tardigrade is one of a few species of eutardigrades that have secondarily invaded the marine environment. Halobiotus crispae in the active stage (d) uniquely adapted to cope with profound changes in ambient salinity occurring in tidal and subtidal habitats. Upon transfers to dilute salt water solutions, active stage H. crispae swell (e) and subsequently regulate their body volume to near control conditions. Halobiotus crispae is characterized by appearing in different cyclomorphic stages. The pseudosimplex 1 stage (f) is freeze tolerant. eo, esophagus; mg, midgut; pb, pharyngeal bulb; st, stylet. Asterisks mark the position of three Malpighian tubules presumably involved in osmoregulation. Scale bars: 50 lm. tun as well as active hydrated state, however,with the infections. A recent investigation in Paramacrobiotus tuns tolerating considerable longer time of exposure richtersi (Murray, 1911) has shown that regulation of (Ramløv & Westh 1992, Persson et al. 2011). The antioxidant metabolism likely plays an important role anhydrobiotic state survives temperatures of up to in defense against the potential oxidative damage approx. 70 C for 1 h, but survival rapidly decreases associated with dehydration (Rizzo et al. 2010). It has when the temperature exceeds 70 C, and no specimens long been known that there is a positive correlation survives exposure to 100 C (Ramløv & Westh 2001). between the time spent in the anhydrobiotic state and The increase in life span offered by anhydrobiosis in this the time required for recovering active life after rehy- species seems to be restricted to approx. 5 years (Westh dration (Crowe & Higgins 1967). Recent investigations & Kristensen 1992). This is less than the cryptobiotic indicate that the longer the time spent in anhydrobiosis, life expansion observed for the heterotardigrade Echi- the more damage is inflicted to DNA (Neumann et al. niscus testudo (Doye`re, 1840), for which we have 2009, Rebecchi et al. 2009b), which would explain the revived specimens from moss cushions dried for approx. prolonged recovery time, indicating that considerable 20 years (Jørgensen et al. 2007). What then sets the repair of DNA (and other molecules) occurs in the post- limits for cryptobiotic survival? Probably accumulated anhydrobiotic state. damage to DNA and other molecules as well as to tissues and organs obtained during the ametabolic state. Trehalose accumulation. Current knowledge suggests This includes damage obtained through oxidative pro- that specific carbohydrates and proteins are important cesses as well as predation, bacterial and fungal in protecting the cells from damage encountered during

2011 The Authors 414 Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x Acta Physiol 2011, 202, 409–420 N. Møbjerg et al. Æ Adaptation to extreme environments in tardigrades entry and exit of cryptobiosis. Bioprotectants may response to desiccation, ionizing radiation and heating. interact directly with macromolecular structures such They reported elevated levels of Hsp70 following both as membranes, DNA and proteins, or they may act as heat and radiation treatment as well as in tardigrades osmolytes during osmotic or dehydration stress. As has rehydrated after a period of desiccation. Noticeably, been shown for other cryptobiotic invertebrates, notice- the authors found that tardigrades in the desiccated ably in the cysts of the brine shrimp Artemia francis- state had reduced Hsp70 levels as compared to the non- cana Kellogg, 1906, some, but not all, cryptobiotic treated control group and accordingly suggested that tardigrades accumulate the dissacharide trehalose dur- Hsp70 may be involved in the repair processes after ing desiccation (Clegg 1965, Westh & Ramløv 1991, desiccation rather than acting as a biochemical stabi- Hengherr et al. 2008, Jo¨ nsson & Persson 2010). Tre- lizer in the dry state. Based on the M. tardigradum EST halose has been proposed to act as a molecular stabilizer library, several additional heat-shock proteins have replacing water and to further stabilize cellular struc- been identified, including two a-crystalline heat-shock ture through the formation of amorphous glasses, a proteins, and the relative abundance of the transcripts process known as vitrification (Clegg 2001). The highest coding for these stress proteins have been investigated trehalose concentrations measured in anhydrobiotic during phases of dehydration and rehydration (Reuner tardigrades ranges from 2.3% d.w. in R. coronifer et al. 2010). The results obtained suggested a limited and Macrobiotus krynauwi Dastych and Harris, 1995 role for heat-shock proteins in the desiccation tolerance to 2.9% d.w. in Macrobiotus islandicus Richters, 1904 of M. tardigradum. The authors found a variable (Westh & Ramløv 1991, Jo¨nsson & Persson 2010). pattern of expression with most of the candidate genes These trehalose values are, however, relatively low being down regulated, and only one of the genes (Mt- when compared with the above mentioned crustacean, hsp90), being significantly upregulated in the dehy- which accumulates the disaccharide in concentrations of drated state (Reuner et al. 2010). Comparable studies around 15% d.w. Moreover, trehalose levels are barely in Paramacrobiotus richtersi did not show evidence for measurable and do not increase during dehydration in an increased expression of either Hsp70 or Hsp90 the cryptobiotic Milnesium tardigradum (see Hengherr between hydrated and dehydrated animals (Rizzo et al. et al. 2008, Jo¨ nsson & Persson 2010). The latter 2010). Additionally, specimens of P. richtersi sent into investigations, analysing trehalose contents in eutar- space (Foton-M3 mission) revealed no significant digrades as well as heterotardigrades, show that change as compared to ground controls in the expres- trehalose accumulation does not represent a universal sion of these heat-shock proteins (Rebecchi et al. protective mechanism enabling tardigrades to undergo 2009a). These contrasting results indicate that Hsp cryptobiosis. expression is species specific. A general role in the cryptobiotic survival of tardigrades can at present not Expression of heat-shock proteins. Cryptobiosis has be attributed to these stress proteins. Nevertheless, a been suggested to rely on the synthesis of molecular synergistic effect between HSPs and other bioprotec- chaperones such as heat-shock-proteins, which may tants such as trehalose or LEA (late-embryogenesis assist folding of newly synthesized proteins, control abundant) proteins might exist (see e.g. Goyal et al. their final intracellular location, as well as protect them 2005). Notably, LEA proteins have been described in from stress-associated denaturation and aid in renatur- many other desiccation tolerant organisms from plants ation (Clegg 2001). During entrance into anhydrobiosis to arthropods and might analogously be functionally Ramløv & Westh (2001) described the upregulation of important in tardigrades (see e.g. Schill 2010, Warner a protein with a molecular weight of approx. 71 kDa in et al. 2010). Indeed, a putative LEA protein has R. coronifer and proposed that this protein might recently been detected in M. tardigradum (Schokraie belong to the heat-shock-protein (Hsp70) family. Schill et al. 2010). Consequently, future studies on these and et al. (2004) subsequently investigated RNA expression other bioprotectants might offer additional clues in our patterns of three Hsp70 isoforms in active and anhyd- search to understand the phenomenon cryptobiosis. robiotic states of M. tardigradum as well as during Obviously, cryptobiosis is far from understood and entrance into and exit out of anhydrobiosis. Only one much more research is needed in order to understand of these isoforms (isoform 2) was significantly induced the mechanisms underlying this state. Interestingly, a by the transition from the active to cryptobiotic state preliminary study from the Kumamushi Genome and interestingly, it showed a considerable increase in Project on R. varieornatus suggests that comparative expression during the post-cryptobiotic phase, while metabolome profiling of active and anhydrobiotic the other isoforms were down regulated during cryp- states may provide a novel insight into anhydrobiosis tobiosis as well as in transitional states. Jo¨ nsson & (Arakawa et al. 2009). Additionally, clues to cryptobi- Schill (2007) used an immuno-westernblot method to otic survival may be found in the extreme tolerance quantify the induction of Hsp70 in R. coronifer in seen in active state tardigrades.

2011 The Authors Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 415 Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420

tolerance to perturbations in body volume and osmotic Non-cryptobiotic survival in tardigrades pressure (Halberg et al. 2009b). The tidal habitat in While tardigrades are well known for their abilities to which this tardigrade lives is characterized by large cope with extreme environmental conditions by enter- fluctuations in abiotic factors; most noticeably, altera- ing cryptobiosis, little focus has been on their ability to tions are seen in salinity and temperature (Møbjerg et al. sustain metabolism and remain active during fluctuating 2007). One way of coping with these extremes would be external circumstances. In this context it is interesting to to enter cryptobiosis. However, H. crispae is not a note that tardigrades are genuine aquatic animals – they cryptobiont. In the active stage the tardigrade handles are dependent on free water to be in their active feeding extremes by expending energy on active regulatory and reproducing states. Truly terrestrial conditions are mechanisms. In addition, the so-called pseudosimplex 1 only survived following entry into the cryptobiotic (P1) stage of this animal is freeze tolerant (Halberg et al. state. Furthermore, limno-terrestrial tardigrades that 2009b); it is not, however, a cryobiont as the P1 stage does have the ability to enter the tun state may do so in not tolerate gradual freezing to )80 C. Cryobiosis is response to environmental challenges (e.g. exposure to defined by the apparent absence of a lower lethal chemical substances) that in marine species would not temperature (Wright 2001). The P1 stage is distinctly force the animals into cryptobiosis. In the following we characterized by a double cuticle and closed mouth and discuss what is currently known about the physiology cloaca (Kristensen 1982, Møbjerg et al. 2007). and stress tolerance of tardigrades in their active states. Halobiotus crispae kept at a salinity of 20 ppt have an extensive ability to supercool (avoid freezing) down to Radiation tolerance. Investigations on radiation toler- around )20 C, enabling active stage animals to with- ance in Richtersius coronifer have revealed that exposure stand subzero temperatures without freezing (Halberg to c-radiation at doses up to 1 kGy does not affect et al. 2009b). Similar supercooling points have been survival of desiccated nor hydrated animals, with reported from limno-terrestrial eutardigrades (Macro- hydrated animals tolerating doses of up to 5 kGy (Jo¨ ns- biotus, Paramacrobiotus and Milnesium), whereas het- son et al. 2005). Horikawa and co-workers revealed that erotardigades (Echiniscus) had slightly higher points both hydrated and desiccated Milnesium tardigradum (Hengherr et al. 2009). Much higher supercooling survive doses of c-radiation of more than 5 kGy, and up to points of )6.7 C and )7.4 C were originally observed 8 kGy of heavy ion radiation (Horikawa et al. 2006). in respectively Richtersius coronifer and Bertolanius These and previous studies indicate that hydrated animals nebulosus (Westh & Kristensen 1992). These high are just as good or even better at tolerating radiation. This supercooling points are likely the result of the presence indicates that radiation tolerance is not due to biochem- of ice-nucleating agents initiating the freezing process ical protectants associated with the cryptobiotic state, but (Westh et al. 1991). Figure 5 shows thermograms of H. suggests that tardigrades rely on efficient and yet uniden- crispae kept in seawater with a salinity of 20 ppt tified mechanisms of DNA repair (Jo¨nsson et al. 2005, (Fig. 5a) and distilled water (Fig. 5b) prior to differen- Horikawa et al. 2006). Several recent studies involved tial scanning calorimetry (DSC). Water content in the with the BIOPAN 6/Foton-M3 mission in 2007, funded animals kept at 20 ppt and in distilled water was by the European Space Agency, have investigated the respectively 73% and 81%. The latter group of tardi- impact on survival of tardigrades exposed to space grades had a higher supercooling point as compared to conditions (Jo¨ nsson et al. 2008, Rebecchi et al. 2009a, the group kept in 20 ppt saltwater, illustrating the Persson et al. 2011). During this mission, cryptobiotic expected correlation between water content and crys- tardigrades (as well as nematodes and rotifers) were sent tallization temperature in species without ice-nucleating into low earth orbit and exposed to space vacuum and agents (see e.g. Hengherr et al. 2009). Thus, the extent cosmic radiation. The three studies revealed discrepan- of supercooling and thereby the ability to avoid freez- cies in survival rates likely reflecting differences between ing, is coupled to osmoregulation. tardigrade species and experimental setups, however; Halobiotus crispae has a large capacity to tolerate they unanimously conclude that tardigrades can survive perturbations in ambient salinity making it an ideal the rigors of space, and that M. tardigradum is likely the model for the study of osmoregulation and volume most resistant species with embryos as well as adults regulation in tardigrades. Experiments on this species tolerating space conditions (see discussion in Persson revealed tolerance to a wide range of salinities, with et al. 2011). Detailed knowledge of the life history of this specimens of the Greenlandic population remaining predatory tardigrade is available as a result of a compre- active in solutions ranging from distilled water to ) hensive study by Suzuki (2003). saltwater with osmolalities up to 2000 mOsm kg 1 (Halberg et al. 2009b). During experiments with trans- Osmoregulation. Experiments on the marine eutardi- fers to strong hypotonic solutions, active stage H. grade Halobiotus crispae have revealed an extraordinary crispae swell with up to 60% before regulating back to

2011 The Authors 416 Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x Acta Physiol 2011, 202, 409–420 N. Møbjerg et al. Æ Adaptation to extreme environments in tardigrades

(a) 15 (a) 1400

) H. crispae –1 1200 Cooling

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20 ppt. Hemolymph osmolality (m 0 0 0 200 400 600 800 1000 1200 1400 –40 –35 –30 –25 –20 –15 –10 –5 0 5 (b) 1400 (b) 15

)

–1 1200 R. coronifer Cooling

1000

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200 Osmotic performance Isoosmotic line

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Hemolymph osmolality (m 0 ppt. 0 0 200 400 600 800 1000 1200 1400 0 External Osmolality (mOsm kg–1) –40 –35 –30 –25 –20 –15 –10 –5 0 5 Temperature (°C) Figure 6 Osmoregulation in eutardigrades. (a) Measured he- molymph osmolality of active stage Halobiotus crispae from Figure 5 Supercooling in Halobiotus crispae. (a) Supercooling Vellerup Vig, Denmark as a function of external osmolality. in H. crispae kept in seawater with a salinity of 20 ppt. Figure modified from Halberg et al. (2009b). (b) Hemolymph Thermogram modified from Halberg et al. (2009b). (b) Halo- osmolality of Richtersius coronifer from O¨ land, Sweden as a biotus crispae kept in distilled water. Cooling and subsequent function of external osmolality. External solutions with osmotic reheating of a sample containing 46 specimens of H. crispae ) concentrations of 100, 200, 300 and 500 mOsm kg 1 were from Vellerup Vig, Denmark kept in distilled water for 45 min prepared from artificial seawater salt (Tropic Marin, Dr Biener prior to the transfer into pans for differential scanning calo- GmbH, Germany). Each point on the graph represents rimetry (DSC). Freezing ()12.2 C) and melting ()1.2 C) mean Æ SD of hemolymph osmolality measurements (nanolitre temperatures were estimated as onsets of peaks of respectively osmometry; Clifton Technical Physics, Hartford, NY, USA) exothermic and endothermic events using DSC7 software ) made in tardigrades kept in demineralized water and the differ- (cooling rate 2 C min 1) (DSC7; Perkin Elmer Inc., Wellesley, ent salt water solution for 30 min. Five animals were used for MA, USA). hemolymph determination at each of the experimental solutions. Arrow indicates the upper lethal line for R. coronifer. near control values (Fig. 4e). Similarly, specimens transferred into hypertonic solutions shrink and there- after respond by regulating body volume. In a series of concentrations, R. coronifer will become inactive and experiments with transfers into saltwater solutions with eventually die, exhibiting an upper lethal limit of ) ) a salinity between 2 ppt (62 mOsm kg 1) and 40 ppt around 500 mOsm kg 1 (Fig. 6b). In solutions ranging ) (1245 mOsm kg 1) the active stage tardigrades hyper- from demineralized water to salt water with an osmo- ) regulated at all times (Fig. 6a) (Halberg et al. 2009b). lality of 500 mOsm kg 1, the tardigrade, however, Hyperregulation is likely a general feature of at least the maintains a consistent osmotic gradient of around ) eutardigrades, as our data on R. coronifer reveal that 170 mOsm kg 1 above that of the external environment this species also keeps its body fluids hyperosmotic as (Fig. 6b). In comparison, H. crispae exposed to the compared to the surroundings (Fig. 6b). Our data same salinity range maintains an osmotic gradient of ) indicate that this limno-terrestrial cryptobiont is less around 300 mOsm kg 1 above that of the surroundings tolerant of high salinity solutions than the littoral H. (Fig. 6a). These data on two different species of crispae. When exposed to water with increasing salt tardigrades indicate that eutardigrades have a relatively

2011 The Authors Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 417 Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420 high water turnover, and that they excrete a hypo- varieornatus. 11th International Symposium on Tardigrada. osmotic fluid – the likely organs involved in this Tu¨ bingen, Germany. 3–6 August 2009, Conference Guide, p. excretion being Malpighian tubules and the gut system 37 (Abstract). (Fig. 4) (Møbjerg & Dahl 1996, Halberg et al. 2009b). Bavan, S., Straub, V.A., Blaxter, M.L. & Ennion, S.J. 2009. A P2X receptor from the tardigrade species Hypsibius dujar- At present the composition of tardigrade extracellular dini with fast kinetics and sensitivity to zinc and copper. fluids (as well as intracellular fluids) are unknown. BMC Evol Biol 9, 17. Obviously, information on the composition of the Bertolani, R. 1970a. Mitosi somatiche e costanza cellulare hemolymph is much needed for our understanding of numerica nei Tardigradi. Atti Accad Naz Lincei Rend Ser 8a, tardigrade physiology and future discussions on how 739–742. tardigrades regulate their body fluids and especially Bertolani, R. 1970b. Variabilita` numerica cellulare in alcuni which osmolytes they are regulating. It is likely that the tessuti di Tardigradi. Atti Accad Naz Lincei Rend Ser 8a, osmotic pressure obtained through hyperregulation is 442–445. partly built by organic solutes, which in turn may act as Bertolani, R. 1982. Cytology and reproductive mechanisms in bioprotectants or may be important for e.g. the above tardigrades. In: D.R. Nelson (ed.) Proceedings of the Third mentioned ability to supercool. International Symposium on the Tardigrada, pp. 93–114. In summary, recent research has shown that tardi- East Tennessee State University Press, Johnson City, TN. 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2011 The Authors 420 Acta Physiologica 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x

Paper IV

Haugan et al. BMC Developmental Biology 2010, 10:56 http://www.biomedcentral.com/1471-213X/10/56

RESEARCH ARTICLE Open Access FunctionalResearch article characterization of the vertebrate primary ureter: Structure and ion transport mechanisms of the pronephric duct in axolotl larvae (Amphibia)

Birgitte M Haugan, Kenneth A Halberg, Åse Jespersen, Lea R Prehn and Nadja Møbjerg*

Abstract Background: Three kidney systems appear during vertebrate development: the pronephroi, mesonephroi and metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model. Results: We investigated structural as well as physiological properties of the pronephric duct. The key elements of our methodology were: using histology, light and transmission electron microscopy as well as confocal laser scanning microscopy on fixed tissue and applying the microperfusion technique on isolated pronephric ducts in combination with single cell microelectrode impalements. Our data show that the fully differentiated pronephric duct is composed of a single layered epithelium consisting of one cell type comparable to the principal cell of the renal collecting duct system. The cells are characterized by a prominent basolateral labyrinth and a relatively smooth apical surface with one central cilium. Cellular impalements demonstrate the presence of apical Na+ and K+ conductances, as well as a large K+ conductance in the basolateral cell membrane. Immunolabeling experiments indicate heavy expression of Na+/K+- ATPase in the basolateral labyrinth. Conclusions: We propose that the pronephric duct is important for the subsequent modification of urine produced by the pronephros. Our results indicate that it reabsorbs sodium and secretes potassium via channels present in the apical cell membrane with the driving force for ion movement provided by the Na+/K+ pump. This is to our knowledge the first characterization of the pronephric duct, the precursor of the collecting duct system, which provides a model of cell structure and basic mechanisms for ion transport. Such information may be important in understanding the evolution of vertebrate kidney systems and human diseases associated with congenital malformations.

Background nephron, and it is composed of a filtration unit and a During the development from embryo to adult life verte- renal tubule. Urine is produced by the filtration of blood brates use a succession of kidney forms to maintain extra- in the filtration unit, followed by the selective reabsorp- cellular fluid homeostasis and simultaneously rid the tion and secretion of ions, organic molecules and water body of nitrogenous wastes [1,2]. Three spatially and across highly specialized epithelia of the renal tubule [4]. temporally different kidney generations form from the Nephrons open into a ureter, and in the meso- and meta- intermediate mesoderm in an anterior to posterior direc- nephros they do so via a collecting duct system [5,6]. This tion i.e. the pronephroi, mesonephroi and metanephroi system is the site for the important final adjustment of [3]. The functional unit in these paired kidneys is the the urine. Vertebrate kidneys may produce urine, which is either hypoosmotic (diluted), isoosmotic or hyperos- * Correspondence: [email protected] 1 Department of Biology, University of Copenhagen, Universitetsparken, DK- motic (concentrated) relative to the body fluids [7-10]. 2100 Copenhagen, Denmark This ability is a function of i) the evolutionary state of the Full list of author information is available at the end of the article

© 2010 Haugan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Haugan et al. BMC Developmental Biology 2010, 10:56 Page 2 of 9 http://www.biomedcentral.com/1471-213X/10/56

nephrons and ii) the regulation of filtration and of the mary ureter is important for urine dilution in the axolotl. transport of inorganic ions, organic molecules and water The single cell type found in the ureter shows the charac- across the renal epithelia. teristics of the vertebrate collecting duct system principal The first kidneys to form - the embryonic pronephric cell and our data indicate that it reabsorbs Na+ and kidneys - are the functional kidneys of fish and amphib- secretes K+. ian larvae [11-16]. These are very simple kidneys com- posed of a single nephron. A characteristic of vertebrate Results and Discussion kidney organogenesis is the development of a pronephric Identification of functional pronephroi and pronephric duct in association with each pronephros [3]. These ducts ducts are the first or primary ureters of vertebrate kidney sys- We determined the interval in which the axolotl pro- tems. They form the collecting duct system of the meso- nephros and pronephric duct is functional by investigat- and metanephroi, and they, and their derivates, are the ing kidney structure in freshly dissected larvae and in key players in the induction of the nephrogenic mesen- larvae prepared for histology (Figure 1 and 2). The pro- chyme, which forms these latter kidney generations. Few nephroi of axolotl larvae are paired organs located on functional studies exist on the pronephros and functional each side of the dorsal aorta in the most anterior part of studies of the duct are virtually lacking. Molecular studies the body cavity. They are visible from the outside on the have been directed at mapping genes expressed in differ- dorsal side of the larva as two small bulges behind the ent segments of the pronephric nephron, and several gills (Figure 1A). Each of the two kidneys are composed of recent reviews have highlighted the potential use of this a filtrating unit - a glomus originating from the dorsal embryonic kidney in drug and human kidney disease aorta, and a single convoluted renal tubule opening into assessment [17-23]. To date, there has been little focus on the coelom via two ciliated nephrostomes (Figure 1B). the role of the pronephric duct in urine modification and The pronephros is fully functional when the axolotl larva it remains to be shown whether transepithelial transport processes are present in this structure. Ultrastructural investigations have shown duct cells with the characteris- tics of an epithelium involved in active transport e.g. many mitochondria and surface expansions of the baso- lateral cell membranes [14,24]. In addition, gene expres- sion assays have indicated high expression of transporters known to be involved in ion transport, such as the Na+/ K+-ATPase and the ROMK channel [17,18,25-27]. Collec- tively, these data suggest that the pronephric duct may play an important role in regulation of extracellular fluid homeostasis. Therefore we ask the question: Is the duct involved in urine modification? In amphibians the pronephros is a large organ, which is functional for a considerable time, before it degenerates. We investigate structural and functional characteristics of the pronephric duct in the freshwater amphibian Ambys- toma mexicanum (axolotl). Members of the Ambystoma genus have been used as models for the study of pro- nephric structure, function, development and evolution for more than a century [24,28,29] and the formation and caudal migration of the pronephric duct in the axolotl has Figure 1 Axolotl larva stage 45 and 52. A. Dorsal view of recently hatched axolotl larva (stage 45; forelimb present as limb buds). The been thoroughly investigated [30-32]. Numerous func- pronephroi can be seen as two small bulges behind the gills. B. Sche- tional studies exist on the mesonephric collecting duct matic representation of the pronephric nephron in the stage 52 larva system of both urodele and anuran amphibians, which as revealed from light- and transmission electron microscopy on serial provide detailed information on the transport character- section of plastic embedded tissue. The pronephros consists of an ex- istics of these segments [Reviewed in [13]]. Our histologi- ternal glomus (gl) and a single renal tubule, which opens into the coelom (co) via two ciliated nephrostomes (ne). In this late larval stage cal examinations and dissections of axolotl larvae the tubule is divided into two ciliated tubules (ci), two proximal tubule indicate that the pronephros is functional from the time branches (pt), a common proximal tubule, a ciliated intermediate seg- of hatching to larval stage 54. We investigate duct mor- ment (is) and a distal tubule (dt). The distal tubule continues as the pro- phology and cellular transport mechanisms present in nephric duct (pd), which leaves the confines of the pronephros and larvae with functional pronephroi, and show that the pri- empties into the cloaca. Haugan et al. BMC Developmental Biology 2010, 10:56 Page 3 of 9 http://www.biomedcentral.com/1471-213X/10/56

A

dt pt

nc

ne

is gl

co 25 mm

B 50 mm C

pt wd mv mr

pd bl nu

co

mu

15mm 5 mm

Figure 2 Histology of pronephros and pronephric duct. A. Cross section of a stage 54 larva (forelimb completely developed) revealing the filtration unit and the convoluted pronephric tubule. Araldite section, 1.5 μm, stained with toluidine blue. Blood is filtered in the external glomus (gl) and the filtrate enters the coelom (co) before it is taken up into the renal tubule via ciliated nephrostomes (ne). In this late larval stage the tubule is character- ized by possessing a ciliated intermediate segment (is). nc, notochord; dt, distal tubule; pt, proximal tubule. B. Longitudinal section of pronephric duct (stage 52 larva). Araldite section, 2 μm, stained with toluidine blue. The pronephric duct (pd) leaves the confines of the pronephros. co, coelom; mu, muscle; pt, proximal tubule. INSERT: The Wolffian duct (wd) at the level of the caudal part of the mesonephros in a stage 54 larva. The duct epithelium consists of two cell types: principal cells and intercalated, mitochondria-rich cells (mr). C. Transmission electron microscopy of pronephric duct shown in figure 2B. The duct is composed of a single cell type characterized by a relative smooth apical surface with few microvilli (mv) and a well developed basal labyrinth (bl) formed by the highly invaginated basal and to some extent lateral cell membranes. nu, nucleus. Haugan et al. BMC Developmental Biology 2010, 10:56 Page 4 of 9 http://www.biomedcentral.com/1471-213X/10/56

hatches from the egg at stage 44. This was confirmed by junctions. The nucleus is regularly shaped, centrally the appearance of blood cells in the capillaries of the glo- placed and contains a nucleolus and patches of hetero- mus, which from embryonic stage 36 had a fully devel- chromatin. There is a conspicuous basal labyrinth, and oped endothelium and a visceral layer with podocytes. lateral infoldings are seen as well (Figure 2C). The cyto- From stage 44 the cilia of the nephrostomes points plasm contains many mitochondria in addition to a Golgi toward the lumen of the renal tubule, indicating a passage complex and endoplasmatic reticulum. The morphology of fluid from the coelom. At this stage the kidney consists of the duct changes at the level of the mesonephros in late of an external glomus and a renal tubule with the follow- larval stages 52-54, revealing the presence of intercalated, ing morphologically determined segments: two nephros- mitochondria-rich cells (Figure 2B, insert). tomes, each connected to a branch of proximal tubule, a common proximal tubule and a distal tubule. The pro- Ion transport mechanisms in the pronephric duct of larvae nephric duct runs caudally as an extension of the distal stage 46-54 tubule opening into the cloaca. From the time of hatching We examined if the pronephric duct participates in final the kidney was observed to increase in overall size due to urine modification with the aid of glass microelectrodes further segmentation of the renal tubule i.e. the develop- and ion substitution experiments in isolated and perfused ment of a ciliated intermediary segment; present from ducts dissected from 22 larvae. Figure 3A is a frequency stage 52 (Figure 1B and 2A). The fully segmented pro- distribution of the membrane potential (Vm) of 64 nephric tubule consists of the following morphological impaled cells. The data show a broad distribution with an defined segments: two nephrostomial tubules, two proxi- mal tubule branches, a common proximal tubule, a cili- ated intermediary segment and a distal tubule (Figure 25 A 2A). At stage 52 the mesonephros was clearly visible and functional as judged by glomerular maturation and pres- 20 ence of blood cells in the mesonephric glomerular capil- laries. Hence, comparable to the situation found in 15 anuran amphibians [14,33] the two kidney generations functionally overlap in axolotl larvae. At stage 54 the pro- 10 nephros reaches its maximal size. Gonadal primordia were observed medioventral to the mesonephros (not Number of cells 5 shown). These were undifferentiated and sex determina- tion was not possible. During the transition from stage 54 0 to latter stages, characterized by fully developed hind -110 -90 -70 -50 -30 -10 limbs, overall pronephric tubule and glomus volume V (mV) decreased, marking pronephric degeneration. Pronephric m structure in stage 52-54 larvae of Ambystoma mexicanum 0 0 resembled the description by Christensen (1964) of the B C functional pronephros in A. punctatum [24]. -20 -20 high K Pronephric duct structure Structural examination of the pronephric duct in larvae -40 high K -40 with functional pronephroi revealed that the duct con- m sists of a single cell type (Figure 2B and 2C). Thus, the m V (mV) -60 V-60 (mV) heterocellularity with intercalated mitochondria-rich cells interposed between principal cells, characteristic of -80 -80 the collecting duct system of latter kidney generations, is not seen at this point (Figure 2B and 2C). This is compa- rable to the situation in the green toad, Bufo viridis - a -100 2 min -100 terrestrial anuran amphibian [14]. The pronephric duct cells in A. mexicanum are approximately 20 μm high in Figure 3 Voltage recordings from single cells in isolated and per- fused ducts. A. Frequency distribution of the membrane potential early larval stages, but decrease in height to 10-15 μm in (Vm) in 64 cells from pronephric ducts dissected from axolotl larvae in stage 52 and 54 larvae (Figure 2C). They have a relatively the stage 46-54. B. Original voltage trace from single cell. Effect of rais- + smooth apical surface. A single central cilium is present ing bath [K ] from 3 to 20 mmol/l. Vm depolarized indicating the pres- (not shown) in addition to small and sparse microvilli, ence of a basolateral K+ conductance. C. Summary data illustrating the + which are more numerous at the point of the apical cell effect on Vm of the bath K concentration step (n = 29). Haugan et al. BMC Developmental Biology 2010, 10:56 Page 5 of 9 http://www.biomedcentral.com/1471-213X/10/56

average Vm between -75 and -80 mV. Transport charac- shows the result of these substitution experiments. Vm teristics of the duct did not seem to differ between early hyperpolarized upon a decrease in luminal Na+ concen- and late larval stages. As shown in figure 3B and 3C, rais- tration from 102 to 7 mmol/l and depolarized upon an ing the K+ concentration in the basal solution from 3 to increase in luminal K+ concentration from 3 to 20 mmol/ 20 mmol/l resulted in a large, reversible depolarization of l. This indicates that the luminal (apical) cell membrane + + + Vm, revealing the presence of a large basolateral K con- possesses Na as well as K channels. In order to identify ductance. This indicates the presence of K+ channels in an ion pump, which can provide the driving force for the basolateral cell membrane. We examined whether the luminal uptake of Na+ as well as K+ secretion, we isolated duct has luminal electrogenic transporters or channels in pronephric ducts and performed immunolabeling with experiments with luminal K+ and Na+ steps. Figure 4 an antibody directed against the Na+-K+-ATPase α-sub- unit. As shown in figure 5, the Na+-K+-ATPase is highly expressed in the pronephric ducts from these larvae, and 0 is entirely localized to the lateral and highly invaginated A basolateral cell membranes. Electrophysiological studies performed on the meso- -20 nephric collecting duct system of amphibians, have indi- cated that principal cells in aquatic urodeles, have a large high K apical Na+ conductance and no, or very small, K+ conduc- -40 tance. However, in terrestrial anurans, K+ secretion through apical K+ channels seems a major task of the principal cells [34-39]. In the current study we provide m

V-60 (mV) evidence for a principal cell, which has the characteristics of the mammalian collecting duct principal cell, i.e. with low Na luminal Na+ as well as K+ conductances. -80 low Na

5 min -100 A

0 0 B C

-20 low Na -20 high K

-40 -40 25ìm

B m m

V-60 (mV) V-60 (mV)

-80 -80

-100 -100 DAPI 75ìm Na+-K+-ATPase Figure 4 Electrophysiological response to luminal fluid ex- change. A. Original voltage trace from single cell. Effect of changing + + luminal [Na ] from 102 to 7 mmol/l and luminal [K ] from 3 to 20 Figure 5 Na+-K+-ATPase expression. A. The Na+-K+-ATPase is highly + mmol/l. Vm hyperpolarized upon the decrease in luminal [Na ] and de- expressed in the pronephric ducts and entirely localized to the lateral polarized upon an increase in luminal [K+]. This indicates that the lumi- and highly invaginated basolateral cell membranes. B. Na+-K+-ATPase nal (apical) cell membrane possesses Na+ as well as K+ conductances. expression in duct counterstained with DAPI. Images are three-dimen- B. Summary data illustrating the effect on Vm of concentration steps in sional reconstructions of the original CLSM z-series, showing a median luminal Na+ (n = 6) and K+ (n = 5). longitudinal section of the pronephric duct. Haugan et al. BMC Developmental Biology 2010, 10:56 Page 6 of 9 http://www.biomedcentral.com/1471-213X/10/56

Conclusions collecting duct system- which provides detailed informa- We show that the pronephric duct, which is the first or tion on cell structure and the basic mechanisms for ion primary ureter in all vertebrates, participates in urine transport. adjustment in the axolotl. The cells constituting the duct are on the ultrastructural as well as cell physiological level Methods comparable to principal cells found in the first segments Animals of the mammalian collecting duct system [5,6,14,40-42]. Specimens of the Mexican axolotl Ambystoma mexica- Notably, the pronephric duct lacks intercalated, mito- num (Shaw and Nodder, 1798) came from the animal sta- chondria-rich cells. We propose that the duct is impor- ble of the August Krogh Building, part of the Campus tant for urine dilution through NaCl reabsorbtion, and Animal Research Facility at University of Copenhagen. that it in addition participates in the regulation of K+ Staging were performed according to [46] for embryos homeostasis. Figure 6 provides a model of the ion trans- and the larvae were designated according to the degree of port mechanisms, which we suggest are present in the limb development as defined by the Ambystoma Genetic duct cell. In this model a Na+-K+-ATPase in the basolat- Stock Center, University of Kentucky; http://www.ambys- eral cell membrane pumps Na+ out of the cell and thereby toma.org. Larvae used for experiments, were in the stage provides the driving force for apical uptake of Na+ 44 to 54. They were euthanized by decapitation, followed through channels. The epithelial sodium channel (ENaC) by brain destruction, and were subsequently either pre- is a likely candidate mediating this apical Na+ uptake [43- pared for histology or pronephric ducts (o.d. 50-70 μm, 45]. Na+ exits the cell through the pump. K+ is secreted dissected length 300-1000 μm) were free hand dissected through apical channels, and is recycled for the pump at 6°C and prepared for microperfusion experiments or across the basolateral cell membrane. It is highly probable immunolabeling. The pronephric ducts were dissected that ROMK channels, known to mediate K+ secretion in from the region in front of the mesonephros. Dissections the mammalian collecting duct system [42], and shown to were performed in media containing (in mmol/l): 75 be expressed in amphibian pronephric ducts [18], medi- NaCl, 20 NaHCO3, 3.0 KCl, 1.8 CaCl2, 1.0 MgSO4, 0.8 ate K+ secretion across the apical cell membrane. The Na2HPO4, 0.2 NaH2PO4, 5.5 glucose, 3.3 glycine, 0.4 PVP, active transport of Na+ would create a lumen-negative 5.0 HEPES, titrated to pH 7.8 with NaOH. transepithelial potential, and Cl- would presumably follow Histology, Light and Transmission Electron Microscopy passively through the paracellular pathway of this epithe- Dissections were performed for every developmental lium [45]. This is to our knowledge the first characteriza- stage of post-hatched axolotls from stage 44 to 57. In tion of the pronephric duct-the precursor of the addition, we examined pronephric development in embryos. Light microscopic imaging of live and dissected larvae was performed using Zeiss Stemi 2000-CS and Leica MZ 16 microscopes equipped with an Infinity X Digital Camera (DeltaPix, Denmark). For histology, a total of 31 larvae in the stage 44-54 and 19 embryos from Na+ Na+ stage 21 to 44 were used. Specimens were fixed for 12 ATP hours at room temperature in an aldehyde fixative con- + K+ K taining: 1.2% glutaraldehyde, 1% paraformaldehyde, 0.05 mol/l sucrose and 0.05 mol/l sodium cacodylate buffer (pH 7.4) and subsequently rinsed and stored in 0.05 mol/l sodium cacodylate buffer with 0.05 mol/l sucrose. Follow-

ing 1 hour's post fixation in 2% OsO4 with 0.1 mol/l sodium cacodylate, specimens were dehydrated through a graded series of ethanol and propylenoxide and embed- ded in Araldite. For light microscopy, 1.5 μm sections were cut with glass knives on a Leica ultramicrotome EM UC6 and stained with toluidine blue. Ultrathin sections for transmission electron microscopy were cut on the Figure 6 Suggested model for ion transport mechanisms in the cells of the vertebrate primary ureter. A Na+-K+-ATPase in the baso- same microtome with a Diatome diamond knife and sub- lateral cell membrane pumps Na+ out of the cell and thereby provides sequently stained with uranyl acetate and lead citrate. the driving force for apical uptake of Na+ through channels. K+ is secret- Transmission electron microscopic images were acquired ed across the apical cell membrane through channels and recycled for using JEOL 100SX and JEOL JEM 1011 transmission elec- the pump across the basolateral cell membrane. tron microscopes. Kodak negatives obtained from the Haugan et al. BMC Developmental Biology 2010, 10:56 Page 7 of 9 http://www.biomedcentral.com/1471-213X/10/56

JEOL 100SX were digitized using an Epson Perfection electrode puller (Narishige, Japan). Impalements were 4990 Photo scanner. The JEOL JEM-1011 was equipped achieved by placing the microelectrode tip against the with a GATAN digital camera. Digital images were opti- basal surface of the cell and gently taping the microman- mized for contrast and color using CorelDraw X4. ipulator (Leitz, Germany) on which the electrodes were mounted. Voltage recordings were made by a WPI Duo Microperfusion and cellular impalements 773 electrometer (World Precision Instruments, USA) Pronephric ducts were transferred to a bath chamber and digitized by a PowerLab/4S data acquisition system mounted on an inverted microscope and perfused in vitro (ADInstruments, Australia). The recording of Vm was with a set of pipettes made to fit the diameter of the accepted if the impalement was achieved by a sudden tubules [37-39]. The tubule perfusion system used (Luigs change in the potential read by the electrode and if the & Neumann, Germany) was designed for accurate adjust- impalement was stable. ment and movement of concentric pipettes [47]. Holding The results are based on 64 cell impalements made in and perfusion pipettes were hand made from glass tubing 22 pronephric ducts dissected from 22 axolotl larvae in (Drummond Scientific Company, PA, USA; holding the stage 46-54. Figures were made in Origin 7.5 (Micro- pipettes: o.d. 2.1 mm, i.d. 1.6 mm; perfusion pipettes: o.d. cal, USA) and CorelDRAW X4. 1.2 mm, i.d. 1.0 mm) in a microforge equipped with a microscope (SM II/1 Puller from Luigs & Neumann, Ger- Immunolabeling, Confocal Laser Scanning Microscopy and many). The tubules were perfused with a single-barrelled 3D reconstruction perfusion pipette containing a small glass capillary (o.d. For identification and localization of the Na+-K+-ATPase, 0.3 mm, i.d. 0.2 mm, Drummond Scientific Company, PA, three separate immunolabeling experiments were con- USA) connected to a manual Hamilton valve (Hamilton ducted with equal results covering larvae in stages 46-54. Co., NV, USA) with a four-way distribution system. Fluid In each experiment, pronephric ducts were isolated from exchange during luminal perfusion experiments was four to five specimens and subsequently fixed on ice for made through this capillary, the tip of the capillary being approximately 60 minutes in 3% paraformaldehyde buff- placed close to the opening of the perfusion pipette, ered to pH 7.4 with 0.1 mol/l sodium cacodylate. After ensuring fast fluid exchange in the tubule. Luminal perfu- being rinsed in PBS (perfusion solution), the tissue was sion was performed either by hand through a syringe incubated overnight at 4°C in PBS containing 10% goat connected to one of the ports in the valve or it was grav- serum (Invitrogen, CA, USA), 1% triton-X and the Na+- ity-driven through a port connected to a fluid filled reser- K+ ATPase monoclonal mouse antibody α5-IgG (10 μg/ voir. The pressure applied was adjusted by monitoring ml). The α5 antibody developed by D.M. Fambrough was tubule diameter, ensuring that the tubule neither col- obtained from the Developmental Studies Hybridoma lapsed nor over expanded. The bath was perfused at 8 ml/ Bank developed under the auspices of the NICHD and min and fluid exchange was performed through beakers maintained by The University of Iowa, Department of attached to the outside of the Faraday cage, which sur- Biology (Iowa City, IA 52242). Following an extensive rounds the microperfusion set-up. wash in PBS, the pronephric ducts were incubated with Perfusions of tubule bath and luminal fluid were carried Alexa-488-conjugated goat anti-mouse IgG (1:100, Invit- out at room temperature with a perfusion solution con- rogen, CA, USA) overnight at 4°C. Following rinses in taining (in mmol/l): 75 NaCl, 25 NaHCO3, 3.0 KCl, 1.8 PBS, the tissue was mounted on glass coverslips in Vecta CaCl2, 1.0 MgSO4, 0.8 Na2HPO4, 0.2 NaH2PO4. The per- shield (Vector Laboratories Inc., CA, USA). In some fusion solution was equilibrated with 1.8% CO2 in O2 and preparations, the renal tubules were incubated in DAPI had a measured pH of 7.8. Experimental solutions with (1:250, Invitrogen, CA, USA) for approximately 5 min different sodium and potassium concentrations were pre- and washed in PBS, prior to mounting. Image acquisition pared from this control solution. In the high K+ solution, was performed on a Leica DM RXE 6 TL inverted micro- the K+ concentration was raised to 20 mmol/l by equimo- scope equipped with a Leica TCS SP2 AOBS confocal lar substitution with Na+. In the low Na+ solution with a laser scanning unit, using the 488 nm line of an argon/ [Na+] of 6.8 mmol/l, Na+ was replaced by choline or N- crypton laser. The image series were processed and methyl-D-glucamine (NMDG+) titrated with HCl. edited in the 3-D reconstruction software IMARIS (Bit- Pronephric duct cells were impaled across the basal cell plane AG, Zürich, Switzerland). The confocal images are membrane with KCl (1-3 mol/l) filled glass microelec- based on 150-170 optical sections of a Z-series per- trodes (R ≈ 100 MΩ) and the membrane potential formed at intervals of 0.3-0.5 μm. All control prepara- electrode tions were negative for immunostaining. (Vm) was recorded with respect to the grounded bath. The microelectrodes were pulled from borosilicate glass Abbreviations with filament (Clark Electromedical, UK) on a vertical bl: basal labyrinth; ci: ciliated tubule; co: coelom; dt: distal tubule; gl: glomus; is: ciliated intermediate segment; nc: notochord; ne: nephrostome; nu: nucleus; Haugan et al. BMC Developmental Biology 2010, 10:56 Page 8 of 9 http://www.biomedcentral.com/1471-213X/10/56

mr: intercalated mitochondria-rich cell; mu: muscle; mv: microvilli; pd: pro- 15. Vize PD: Embryonic kidneys and other nephrogenic models. In The nephric duct; pt: proximal tubule; Vm: membrane potential; wd: Wolffian duct. Kidney: From normal development to congenital disease Edited by: Vize PD, Woolf AS, Bard JBL. London: Academic Press; 2003:1-6. Authors' contributions 16. Vize PD, Seufert DW, Carroll TJ, Wallingford JB: Model systems for the NM conceived and designed the study. BMH and NM fixed larvae and per- study of kidney development: Use of the pronephros in the analysis of formed the dissections for the structural investigation. BMH sectioned speci- organ induction and patterning. Developmental Biology 1997, mens, and made LM and TEM investigations with help from ÅJ and NM. NM 188:189-204. made the microperfusion experiments and microelectrode impalements. KAH, 17. Zhou XL, Vize PD: Proximo-distal specialization of epithelial transport LRP and NM carried out immunostaining experiments and KAH performed processes within the Xenopus pronephric kidney tubules. CLSM and prepared the 3D images. All authors participated in discussions and Developmental Biology 2004, 271:322-338. interpretation of the data. NM wrote the paper with inputs from the other 18. Tran U, Pickney LM, Ozpolat BD, Wessely O: Xenopus Bicaudal-C is authors. All authors read and approved the final version of the manuscript. required for the differentiation of the amphibian pronephros. Developmental Biology 2007, 307:152-164. Acknowledgements 19. Raciti D, Reggiani L, Geffers L, Jiang Q, Bacchion F, Subrizi AE, Clements D, We sincerely thank Mrs. Jette Lyby Michelsen and Mrs. Kristine J.K. Sørensen for Tindal C, Davidson DR, Kaissling B, et al.: Organization of the pronephric technical assistance. Funding came from the 2008 Faculty of Science, Univer- kidney revealed by large-scale gene expression mapping. Genome sity of Copenhagen Freja-Programme and from the Carlsberg Foundation Biology 2008, 9:. (grant numbers: 2004_04_0572; 2006_01_0534; 2008_01_0466). The funders 20. Wingert RA, Davidson AJ: The zebrafish pronephros: A model to study had no role in study design, data collection and analysis, decision to publish, or nephron segmentation. Kidney International 2008, 73:1120-1127. preparation of the manuscript. 21. Vasilyev A, Liu Y, Mudumana S, Mangos S, Lam PY, Majumdar A, Zhao JH, Poon KL, Kondrychyn I, Korzh V, et al.: Collective cell migration drives morphogenesis of the kidney nephron. Plos Biology 2009, 7:101-114. Author Details 22. Wheeler GN, Brandli AW: Simple vertebrate models for chemical Department of Biology, University of Copenhagen, Universitetsparken, DK- genetics and drug discovery screens: Lessons from Zebrafish and 2100 Copenhagen, Denmark Xenopus. Developmental Dynamics 2009, 238:1287-1308. Received: 18 September 2009 Accepted: 27 May 2010 23. Vize PD, Carroll TJ, Wallingford JB: Induction, development and Published: 27 May 2010 physiology of the pronephric tubules. In The Kidney: From normal This©BMC 2010 isarticleDevelopmental an Haugan Open is available Accesset al; Biology licensee from:article 2010, http distributedBioMed://www.biomedcentral.com/1471-213X/10/56 10:56 Central under Ltd. the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. development to congenital disease Edited by: Vize PD, Woolf AS, Bard JBL. London: Academic Press; 2003:19-50. References 24. Christensen AK: Structure of functional pronephros in larvae of 1. Evans DH: Osmotic and ionic regulation: Cells and animals Boca Raton: CRC Ambystoma opacum as studied by light and electron microscopy. Press; 2009. American Journal of Anatomy 1964, 115:257-278. 2. Vize PD, Woolf AS, Bard JBL: The Kidney: From normal development to 25. Eid SR, Brandli AW: Xenopus Na, K-ATPase: primary sequence of the beta congenital disease London: Academic Press; 2003. 2 subunit and in situ localization of alpha 1, beta 1, and gamma 3. Saxén L: Organogenesis of the kidney Cambridge: Cambridge University expression during pronephric kidney development. Differentiation Press; 1987. 2001, 68:115-125. 4. Dantzler WH: Comparative aspects of renal function. In The Kidney: 26. Kumano T, Konno N, Wakasugi T, Matsuda K, Yoshizawa H, Uchiyama M: Physiology and Pathophysiology 2nd edition. Edited by: Seldin D, Giebisch Cellular localization of a putative Na+/H+ exchanger 3 during ontogeny G. New York: Raven Press; 1992:885-942. in the pronephros and mesonephros of the Japanese black 5. Møbjerg N, Jespersen A, Wilkinson M: Morphology of the kidney in the salamander (Hynobius nigrescens Stejneger). Cell and Tissue Research West African caecilian, Geotrypetes seraphini (Amphibia, Gymnophiona, 2008, 331:675-685. Caeciliidae). Journal of Morphology 2004, 262:583-607. 27. Uochi T, Takahashi S, Ninomiya H, Fukui A, Asashima M: The Na+, K+- 6. Kriz W, Kaissling B: Structural and functional organization of the ATPase alpha subunit requires gastrulation in the Xenopus embryo. mammalian kidney. In Seldin and Giebisch's the kidney. Physiology and Development Growth & Differentiation 1997, 39:571-580. pathophysiology Edited by: Alpern RJ, Herbert SC. Burlington, MA: 28. Field HH: The development of the pronephros and segmental duct in Academic Press; 2008:479-563. Amphibia Cambridge, USA: Bulletin of the Museum of Comparative 7. Greger R: Principles of renal transport; concentration and dilution of Zoology; 1891. urine. In Comprehensive human physiology. From cellular mechanisms to 29. Howland RB: On the effect of removal of the pronephros of the integration Volume 2. Edited by: Greger R, Windhorst U. Berlin Heidelberg: amphibian embryo. Proceedings of the National Academy of Sciences of Springer; 1996:1489-1516. the United States of America 1916, 2:231-234. 8. Giebisch G, Windhager E: Urine concentration and dilution. In Medical 30. Drawbridge J, Steinberg MS: Morphogenesis of the axolotl pronephric physiology. A cellular and molecular approach Edited by: Boron WF, duct: A model system for the study of cell migration in vivo. Boulpaep EL. Philadelphia: Saunders; 2005:828-844. International Journal of Developmental Biology 1996, 40:709-713. 9. Larsen EH, Møbjerg N, Nielsen R: Application of the Na+ recirculation 31. Gillespie LL, Armstrong JB: Formation of the pronephros and pronephric theory to ion coupled water transport in low- and high resistance duct rudiment in the Mexican axolotl. Journal of Morphology 1985, osmoregulatory epithelia. Comparative Biochemistry and Physiology 185:217-222. 2007, 148:101-116. 32. Poole TJ, Steinberg MS: Amphibian pronephric duct morphogenesis - 10. Larsen EH, Møbjerg N: Na+ recirculation and isosmotic transport. segregation, cell rearrangement and directed migration of the Journal of Membrane Biology 2006, 212:1-15. Ambystoma duct rudiment. Journal of Embryology and Experimental 11. Brandli AW: Towards a molecular anatomy of the Xenopus pronephric Morphology 1981, 63:1-16. kidney. International Journal of Developmental Biology 1999, 43:381-395. 33. Jaffee OC: Morphogenesis of the pronephros of the leopard frog (Rana 12. Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier pipiens). Journal of Morphology 1954, 95:109-123. AF, Neuhauss SCF, Stemple DL, Zwartkruis F, Rangini Z, et al.: Early 34. Dietl P, Stanton BA: The amphibian distal nephron. In New insights in development of the zebrafish pronephros and analysis of mutations vertebrate kidney function Edited by: Brown JA, Balment RJ, Rankin JC. affecting pronephric function. Development 1998, 125:4655-4667. Cambridge: Cambridge University Press; 1993:115-134. 13. Hillyard SD, Møbjerg N, Tanaka S, Larsen EH: Osmotic and ion regulation 35. Horisberger J, Hunter M, Stanton B, Giebisch G: The collecting tubule of in amphibians. In Osmotic and ionic regulation: Cells and animals 1st Amphiuma II. Effects of potassium adaptation. American Journal of edition. Edited by: Evans DH. Boca Raton: CRC Press; 2009:367-441. Physiology 1987, 253:F1273-F1282. 14. Møbjerg N, Larsen EH, Jespersen A: Morphology of the kidney in larvae 36. Hunter M, Horisberger J-D, Stanton B, Giebisch G: The collecting tubule of Bufo viridis (Amphibia, Anura, Bufonidae). 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37. Møbjerg N, Larsen EH, Novak I: K+ transport in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules. Journal of Experimental Biology 2002, 205:897-904. 38. Møbjerg N, Larsen EH, Novak I: Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules. Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology 2004, 137:585-595. 39. Møbjerg N, Werner A, Hansen SM, Novak I: Physiological and molecular mechanisms of inorganic phosphate handling in the toad Bufo bufo. Pflugers Archiv - European Journal of Physiology 2007, 454:101-113. 40. Koeppen BM: Conductive properties of the rabbit outer medullary collecting duct: outer stripe. American Journal of Physiology - Renal Physiology 1986, 250:F70-F76. 41. Schlatter E: Regulation of ion channels in the cortical collecting duct. Renal Physiology and Biochemistry 1993, 16:21-36. 42. Wang WH, Giebisch G: Regulation of potassium (K) handling in the renal collecting duct. Pflugers Archiv-European Journal of Physiology 2009, 458:157-168. 43. Garty H, Palmer LG: Epithelial sodium channels: Function, structure, and regulation. Physiological Reviews 1997, 77:359-396. 44. Loffing J, Korbmacher C: Regulated sodium transport in the renal connecting tubule (CNT) via the epithelial sodium channel (ENaC). Pflugers Archiv-European Journal of Physiology 2009, 458:111-135. 45. Schlatter E, Greger R, Schafer JA: Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl- transport. Pflugers Archiv-European Journal of Physiology 1990, 417:317-323. 46. Bordzilovskaya NP, Dettlaf TA, Duhan ST, Malacinski GM: Developmental- stage series of axolotl embryos. In Developmental Biology of the Axolotl Edited by: Armstrong JB, Malacinski GM. New York: Oxford University Press; 1989:201-219. 47. Greger R, Hampel W: A modified system for in vitro perfusion of isolated renal tubules. Pflugers Archiv - European Journal of Physiology 1981, 389:175-176.

doi: 10.1186/1471-213X-10-56 Cite this article as: Haugan et al., Functional characterization of the verte- brate primary ureter: Structure and ion transport mechanisms of the pro- nephric duct in axolotl larvae (Amphibia) BMC Developmental Biology 2010, 10:56

Paper V

2803

The Journal of Experimental Biology 212, 2803-2811 Published by The Company of Biologists 2009 doi:10.1242/jeb.029413

Cyclomorphosis in Tardigrada: adaptation to environmental constraints

Kenneth Agerlin Halberg1, Dennis Persson1,2, Hans Ramløv3, Peter Westh3, Reinhardt Møbjerg Kristensen2 and Nadja Møbjerg1,* 1Department of Biology, University of Copenhagen, August Krogh Building, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark, 2Natural History Museum of Denmark, Zoological Museum, Invertebrate Department, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark and 3Department of Nature, Systems and Models, University of Roskilde, Universitetsvej 1, DK-4000 Roskilde, Denmark *Author for correspondence (e-mail: [email protected])

Accepted 9 June 2009

SUMMARY Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice formation proceeds rapidly at a crystallization temperature of around –20°C, revealing extensive supercooling in both stages, while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage specimens (350–500 μm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was approximately 950 mOsm kg–1. Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades. Key words: cyclomorphosis, environmental stress, freeze tolerance, Halobiotus crispae, invertebrate, osmoregulation, tardigrade, volume regulation.

INTRODUCTION active stage, (2) the pseudosimplex 1 (P1) stage and (3) the The phylum Tardigrada comprises a group of hydrophilous micro- pseudosimplex 2 (P2) stage (Møbjerg et al., 2007). The defining metazoans, exhibiting close affinities to the euarthropod complex physiological and biochemical characteristics of the individual stages (Garey et al., 1996; Giribet et al., 1996; Mallatt et al., 2004). They are largely unknown but most likely correlate with dominant abiotic occupy a range of niches in terrestrial, freshwater and marine factors. A ubiquitous factor in all tidal and subtidal habitats is the environments from continental Antarctica (Convey and McInnes, large temporal and spatial fluctuations in external salinity. Yet, 2005) to the icecap of Greenland (Grøngaard et al., 1999) yet are additional adaptations are necessary at high latitudes to ensure winter especially abundant in mosses and lichens, where they constitute a survival due to prolonged exposure to subzero temperatures. In the major component of the cryptic fauna. Along with nematodes and present study, we focus on the adaptive significance of the two main rotifers, selected species of tardigrades exhibit a remarkable cyclomorphic stages in H. crispae, i.e. the active stage corresponding resilience against physical extremes, including low and high to the reproductive stage of other tardigrades and the P1 stage, a temperatures (–253°C to +151°C), ionizing radiation (up to hibernation stage, which is comparable to the cysts found in other 6000Gy), vacuum, high pressure (up to 600MPa) and extreme tardigrades (e.g. Guidetti et al., 2008). We do not deal with the P2 desiccation (Ramløv and Westh, 1992; Westh and Kristensen, 1992; stage, which is a sexual maturation stage that has not yet been Ramløv and Westh, 2001; Schill et al., 2004; Horikawa et al., 2006; reported from other tardigrades. Our preliminary data, however, Jönson and Schill, 2007; Hengherr et al., 2008; Hengherr et al., suggest that this stage has a unique osmoregulatory profile. We show 2009). However, the underlying physiological and biochemical that the transition between the active and P1 stages is associated mechanisms mediating these unique tolerances are still largely with profound changes in the physiology of the animal. The P1 stage unidentified and represent an exciting challenge to contemporary is the only stage at which H. crispae survives internal ice formation. biology. The active stage tolerates large shifts in ambient salinity and we The marine eutardigrade Halobiotus crispae Kristensen 1982 investigate in detail the volume and osmoregulatory capacity of this (Fig.1) colonizes tidal and subtidal habitats at numerous localities stage. Our study presents the first detailed analysis of osmoregulation throughout the northern hemisphere (Møbjerg et al., 2007). This in tardigrades. The data show that active-stage H. crispae hyper- species is characterized by the appearance of seasonal cyclic regulate at any investigated external salinity, which would indicate changes in morphology, i.e. cyclomorphosis (Kristensen, 1982). excretion of dilute urine. This is likely to be a general feature of Three distinct cyclomorphic stages have been recognized: (1) the eutardigrades, which all possess Malpighian tubules.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 2804 K. A. Halberg and others

Fig. 1. SEM investigation of Halobiotus crispae from Vellerup Vig, Denmark. (A) Overview of P1 stage indicating the areas shown in B and C. The thick outer cuticle functionally isolates the animal from the surroundings (scale bar=100 μm). (B) Close-up of the head region of P1. Notice that the mouth is closed by cuticular thickenings (scale bar=25 μm). (C) Close-up of the posterior area of the P1 stage. As shown for the mouth, the cloaca is closed (scale bar=10 μm). (D) Close-up of the head region of the active stage. Note the six peribuccal sensory organs (*) that surround the open mouth (scale bar=25 μm). (E) Close-up of the posterior area of the active stage, revealing the open tri-lobed cloaca (scale bar=10 μm).

MATERIALS AND METHODS point dryer, Bal-Tec Union, Balzers, Liechtenstein), mounted on Tardigrade sampling aluminum stubs, sputter-coated with platinum–palladium (thickness Specimens of Halobiotus crispae were collected at regular intervals ~12nm) using a JEOL JFC-2300HR (JEOL, Tokyo, Japan) and in the period 2005 to 2008 at Vellerup Vig, Isefjord, Denmark examined in a JEOL JSM-6335F Field Emission scanning electron (55°44.209ЈN, 11°51.365ЈE) and at Nipisat Bay, Disko Island, West microscope (JEOL, Japan). Greenland (69°25.934ЈN, 54°10.768ЈE) in August 2006. At Vellerup Vig, bottom samples were collected at an approximate depth of Cold hardiness 1.0–2.5m, while samples from Nipisat Bay were taken in the subtidal Six groups of 10 animals in both the active and P1 stage were zone 3–4cm below low tide. With the exception of the data on transferred to Eppendorf tubes containing 1.5ml of SW from osmotic stress tolerance presented in Fig.3, the obtained results are Vellerup Vig (20ppt). The samples were cooled to a constant based entirely on animals collected at Vellerup Vig. Detailed temperature of –20°C at a cooling rate of approximately 1°Cmin–1 descriptions of the two localities can be found elsewhere (Kristensen, (Block, 1991) and held at the target temperature for a period of 24h. 1982; Møbjerg et al., 2007). Collected samples were freshwater- The animals were thawed at room temperature and the survival shocked, decanted into a conical net (mesh size 62 μm) and assessed successively over the course of 96h. Animals retaining transferred to Petri dishes. These dishes were supplied with fresh locomotory function or responsive to tactile stimuli following this seawater (SW; 18–20ppt; pH8–9) and substrate from the locality. period were considered alive. Tardigrades were localized using a Leica MZ16 stereomicroscope As subzero temperatures may be experienced for longer periods (Leica Microsystems, Wetzlar, Germany), and primarily found on of time in Arctic habitats, the long-term survival at subzero the haptera of various filamentous algae present in the substrate. temperatures was investigated. An additional six groups of 10 Isolated tardigrades from Vellerup Vig were kept at 4°C in SW for specimens in each cyclomorphic stage were frozen to –20°C at a periods of up to 6months by regularly supplying fresh substrate cooling rate of approximately 1°Cmin–1 and kept frozen for a total from the locality. Different cyclomorphic stages (see Fig.1) were of 36days. Survival was assessed as described above. identified using an Olympus BX 51 interference-contrast microscope (Olympus, Tokyo, Japan). Differential scanning calorimetry The quantity and kinetics of ice formation associated with cooling Scanning electron microscopy of H. crispae from Vellerup Vig (20ppt) in active and P1 stages For scanning electron microscopy, specimens were fixed in 2.5% were studied by differential scanning calorimetry (DSC). Groups glutaraldehyde in 0.1moll–1 sodium cacodylate buffer (pH7.4), of 40–75 animals in each respective stage were transferred to 30μl rinsed in the buffer and subsequently postfixed in 1% OsO4 in aluminum DSC pans. In order to avoid dehydrating the animals 0.1moll–1 sodium cacodylate buffer (pH7.4). Following fixation, during the removal of external water, the tardigrades were clumped the specimens were dehydrated through a graded series of ethanol in the central part of the DSC pan and excess water was subsequently and acetone. They were critical point dried (Bal-Tec CPD 030 critical removed with small pieces of delicate task wipes. Sample mass was

THE JOURNAL OF EXPERIMENTAL BIOLOGY Cyclomorphosis in tardigrades 2805 determined gravimetrically to the nearest 0.01mg using a fine-scale salinity. Animal activity was concomitantly assessed. Animals were AT261 Deltarange (Mettler-Toledo, Columbus, OH, USA), yielding allowed a period of 20–40min of acclimatization following a salinity a total mass of 0.16–0.39mg (wet mass). The pans were sealed and change prior to assessment. Individuals responsive to tactile stimuli transferred to a calorimeter (Perkin Elmer DSC 7 equipped with an were considered active. Five groups of specimens in active and P1 Intercooler II mechanical cooling device), with an empty pan as stages were assessed at both hypo- and hyperosmotic salinities. reference. The calorimeter was calibrated with gallium [melting –1 point, Tm=29.78°C; melting enthalpy, ⌬Hm=80.1 J g ], water Volume measurements (Tm=0°C) and n-decane (Tm=–29.66°C). All scans involved cooling Individual adult active-stage specimens of H. crispae (size from 5°C to –40°C and subsequent reheating to 5°C at a cooling 300–500μm) from Vellerup Vig (20ppt) were visualized in an rate of 5°Cmin–1. Samples were reweighed following the DSC run Olympus BX 51 microscope (Olympus), photographed using a to ensure that no water loss had occurred. In order to determine the digital camera (C-5050, Olympus) and subsequently exposed for water content of samples following the freeze/thaw cycle, pans were set time periods of 30min, 1, 2, 4, 24 and 48h to saltwater solutions punctured and dried at 80°C to a constant mass (dry mass). A with salinities of 2ppt, 10ppt and 40ppt. The osmotic treatments minimum of three groups of animals in each stage was used (see were conducted in small glass vials containing 4ml of SW at 4°C. Table1). The obtained thermograms (heat flows vs temperature) At the end of each time interval, individuals were transferred, in a were analyzed with respect to crystallization temperature (Tc), drop of the appropriate solution, to glass microscope slides and amount of ice formed during the freezing exotherm (assuming that photographed under cover slips for subsequent estimations of body the latent heat of crystallization is the same as for pure water), Tm volume. During photography, great care was taken to minimize the and the osmotic pressure of the extracellular fluids as calculated by time spent by the animals under the cover slips, in order to avoid the standard DSC 7 software. Ice contents were calculated using evaporative water loss, which would alter the osmotic pressure of the water content and the enthalpy of the freeze exotherm. The the solution. The animals were ensured total freedom of movement. temperature dependence of the enthalpy of crystallization of water Following photography, individuals were returned to the respective was taken into account as previously described (Kristiansen and salinities until the end of the next set time period, when the process Westh, 1991). For hemolymph osmolality calculations, the onset was repeated. At each of the time intervals, 10–14 individuals were melting point measured by the DSC 7 software was determined photographed at each of the SW treatments. Images were analyzed according to the approach of Nicholajsen and Hvidt (Nicholajsen using DP-softTM (Olympus), and total body volume was calculated 2 2 and Hvidt, 1994), in which the established melting point of the body according to the equation: Vtotal=π(r bodyhbody+2r leghleg), where V fluid was derived from a standard curve made from predetermined is the volume of the specimen, r is the measured radius, and h is NaCl solutions. the measured length of the body and hind legs, respectively. In order to assess the behavioral response of H. crispae during Preparation of experimental solutions osmotic shock and to quantify potential mortality related to the Salt water solutions of different osmotic pressure were made by respective treatments, a separate experiment was performed. successive dilution with distilled water or by evaporative reduction Specimens (N=10) were transferred directly to 2ppt, 10ppt, 20ppt of 100% SW from the locality. Measurements of osmotic pressure (control) and 40 ppt, respectively, and animal activity was were made in parallel on a Vapro 5520 vapor pressure osmometer subsequently monitored over the course of 48h at 4°C. Individuals (Wescor, Logan, UT, USA) and on a refractometer (S-1 Shibuya responsive to tactile stimuli at the above-mentioned set time periods Land, Tokyo, Japan). were considered active and alive (see Fig.7). Three groups exposed to each treatment were assessed. Osmotic stress tolerance Animals collected from the Danish as well as the Greenlandic Measurement of hemolymph osmolality population were used for the current experiment. Groups of 20 Hemolymph osmolality was measured in individual tardigrades specimens were transferred to small glass vials containing 4ml of following exposure for 30min, 4 and 48h to the experimental 100% SW at 4°C, and specimens were either exposed to a gradual solutions of 2ppt (62mOsmkg–1), 10ppt (311mOsmkg–1) and 40ppt increase or decrease in salinity. The gradual changes in salinity were (1245 mOsm kg–1). Six animals were used for osmolality performed over the course of 4–5h by periodically replacing small determination in each of the experimental solutions. Six animals volumes of SW with prefixed solutions of either a higher or lower kept at 20ppt (623mOsmkg–1) served as a control. Hemolymph

Table 1. Post-freeze survival and data obtained from differential scanning calorimetry on H. crispae from Vellerup Vig, Denmark (20 ppt) in the active and P1 stages Post-freeze Body-water Post-freeze survival (%) frozen during Osmolality of survival (%) (frozen for Crystallization Melting temp. Water content freezing extracellular Sample (frozen for 24 h) 36 days) temp. (°C) (°C) (%) exotherm (%) fluids (mOsm kg –1 ) Pseudosimplex 1 53.3±15 (6) 12.7±7 (6) –19.6±3.1 (6) –4.29±0.79 (5) 67±4 (5) 59±3 (4) 928±77 (5) Active 0 (6) 0 (6) –21.6±2.1 (3) –4.48±0.15 (3)68±4 (3) 69±5 (3) 975±36 (3) t-test (P<0.05) **NS NS NS* NS All values are expressed as means ± s.d. Parentheses indicate the number of replicate groups examined, each group containing 40–75 animals. The first column refers to the type of cyclomorphic stage investigated. Second and third columns show the survival following cooling to –20°C at 1°C min–1 for 24 h and 36 days, respectively. The temperatures in the fourth and fifth columns are the onsets of the peaks as calculated by the DSC 7 software. Water content was determined gravimetrically using the equation: (wet mass – dry mass)/wet mass. Ice content (seventh column) was calculated using the water content and the enthalpy ( H) of the freezing exotherm. The final column indicates the osmolality of the extracellular fluids determined by melting point depression. Significance level was P>0.05 (NS, not significant), P<0.05 (*, significant).

THE JOURNAL OF EXPERIMENTAL BIOLOGY 2806 K. A. Halberg and others samples (2–3nl) were collected by piercing individual specimens demonstrated to be freeze-tolerant. Post-freeze survival following under immersion oil (type A; 150 centistoke; Cargille Laboratories, 24h exposure to –20°C was 53.3±15%; however, when prolonging Cedar Grove, NJ, USA) using hand-pulled glass capillary tubes the period spent at subzero temperatures to 36days, the survival of (capacity 1 μl; Micro-caps, Drummond Scientific Company, animals in the P1 stage decreased to 12.7±7% (Table1). Animal Broomall, PA, USA). Hemolymph samples were acquired through recovery was monitored over a period of 96 h following the capillary action and subsequently ejected into immersion oil. Care freeze/thaw cycle; however, the majority of animals had resumed was taken to ensure that the measurements were made on fluid activity after a period of 48h. No additional recovery was monitored originating from hemolymph alone and samples containing gut beyond 96h following any of the investigated treatments. contents were discarded. Prior to sample collection, immersion oil Substantial ice formation proceeded rapidly following the first was collected into the capillary tube in order to avoid any evaporative ice nucleation in the freeze-tolerant P1 stage with a mean of water loss. Using an Irvin loop, samples were immediately –19.6±3.1°C; as indicated by the large exotherm in Fig.2. At the transferred in a drop of immersion oil into sample oil wells (type given salinity, the amount of ice formed during the freezing B; 1250 centistoke; Cargille Laboratories) of a calibrated nanoliter exotherm amounted to approximately 60% of the body water osmometer (Clifton Technical Physics, Hartford, NY, USA), and (Table1). The absence of additional small exothermic peaks during the osmolality (mOsmkg–1) was determined by melting point the subsequent cooling to –40°C indicated that no additional ice depression (MDP=1.858°COsm–1). formation occurred following the initial large freezing exotherm. The freeze exotherm lasted less than one minute. The initial Statistics separation of ice (Tc) occurred in the temperature range of –15.4 to Significant differences between experimental and control conditions –23.2°C. were tested using unpaired, two-tailed t-tests, and a significance level Only marginal differences in Tc of animals in the P1 and active of P≤0.05. stage were observed (Table1), suggesting an absence of seasonal variations in ice-nucleating activity in H. crispae. In spite of invariant RESULTS water contents between the two stages, the amount of water Survival at sub-zero temperatures crystallizing during cooling in animals in the active stage was The external morphology of H. crispae in the active and the P1 significantly higher than in animals in the P1 stage; however, the stage is shown in Fig.1. Notably, the P1 stage is characterized by melting points of the two stages remained largely unaltered. The a conspicuous double cuticle, in which both the mouth and cloaca latter indicates that body fluid osmolality at a given external salinity are closed by cuticular thickenings (Fig.1A–C). Post-freeze survival is unaffected by the animal’s transition from the active to the P1 following both short- and long-term exposure to subzero stage. temperatures is listed in Table1. An example of the quantitative kinetics of ice formation associated with the freeze/thaw cycle of Volume- and osmoregulatory capacity P1-stage H. crispae kept at 20ppt is illustrated in Fig.2. The Fig.3 shows the percentage of active animals of H. crispae following collective DSC analysis of the onsets and areas of peaks after the the exposure to gradual changes in the external salinity. When freeze/thaw cycles, together with the water content of the samples, comparing Danish P1 and active-stage H. crispae, the active stage provide the remaining results listed in Table1. displayed a larger tolerance towards the more concentrated SW Whereas animals in the active stage and P2 stage (data not shown) solutions and were slightly more tolerant of the very dilute solutions. were intolerant of freezing, animals in the P1 stage were Indeed, a significantly higher percentage of active-stage specimens

15 Osmolality (mOsm kg–1) 0 500 1000 1500 2000 2500 Cooling 100 100

80 * 80 7.5 * Endotherm 60 60 *

Exotherm 40 40

Heating Activity (%) t flow (mW) al heat flow Differenti * 20 20 Active, Vellerup 0 P1, Vellerup * Active, Nipisat * –40 –35–30 –25 –20 –15 –10 –5 0 5 0 0 Temperature (°C) 0102030405060708090 Fig. 2. Representative thermogram displaying the exothermic and Salinity (ppt) endothermic events associated with the cooling and heating of a sample containing P1-stage Halobiotus crispae from Vellerup Vig, Denmark Fig. 3. Osmotic stress tolerance of Halobiotus crispae in the P1 and active (20 ppt). The crystallization and melting temperatures were estimated as stages. Active-stage (ᮀ) and P1-stage (᭡) specimens from the Danish the onsets of peaks (–17.5°C and –4.5°C, respectively) using the DSC 7 population at Vellerup Vig. Active-stage (᭺) specimens from the software (see also Table 1). The very low crystallization temperatures Greenlandic population at Nipisat Bay. Data are means ± s.e.m. from five measured exclude the presence of physiologically relevant ice-nucleating independent experiments. *, significantly different from Vellerup Vig P1 agents. stage (P<0.05).

THE JOURNAL OF EXPERIMENTAL BIOLOGY Cyclomorphosis in tardigrades 2807 remained active in the salinity spectrum of 0–3ppt and 45–60ppt, passive throughout the treatment (Fig. 7A). Nevertheless, as compared with the animals in the P1 stage, and in general seemed following a gradual return to 20 ppt, all animals regained less affected by the impositions of osmotic stress. Our preliminary locomotory functions. No mortality was observed in any of the data on P2 from Vellerup Vig show that this stage tolerates very treatments. dilute solutions better than the other stages, yet is the least tolerant Upon immersion of individual specimens into a less severe hypo- of increases in salinity, becoming inactive at around 50ppt. As a osmotic media of 10ppt (311mOsmkg–1) a similarly significant comparison, the active stage from Greenland (Nipisat Bay), living increase in total body volume was observed; reaching a mean value in a more exposed habitat compared with the Vellerup Vig of 132±11% after 0.5h exposure (Fig.5A,B). However, following population, displayed an even higher tolerance to concentrated SW, this initial increase, total body volume stabilized, and a RVD was with observed activity at 80ppt. observed after 1–2h exposure. After 4h incubation, total body In the following, we investigate in detail volume and volume was 110±8%, which was not significantly different from osmoregulation in active-stage H. crispae from Vellerup Vig kept the control situation. An effect on the locomotory functions was at a control salinity of 20ppt (Figs4–8). When exposed to a severe observed initially, as some animals displayed sluggish movements, hypo-osmotic shock of 2 ppt (63 mOsm kg–1), the animals yet only a limited number of animals were passive during this exhibited a large significant increase in body volume, resulting experiment (Fig.7B). in a total body volume of 127±11% after merely 0.5h of exposure When transferring H. crispae to a hyperosmotic solution of 40ppt (Fig.4A,B). During this time period, animals became bloated and (1245mOsmkg–1), a significant decrease in total body volume was rigid and most specimens lost locomotory functions (Fig.7A). observed (Fig.6A,B). Total body volume was significantly reduced This passive uptake of water continued during the initial 2h of to 66±9% following the first 0.5h of immersion and remained largely the exposure, culminating in a total body volume of 162±17%. unaltered during the following hours of the treatment. After 24h, However, after this period of time, a regulatory volume decrease most specimens had displayed a regulatory volume increase, (RVD) was observed. The total body volume of specimens was resulting in a mean total body volume of 82±9%, yet total body considerably reduced to 114±14% following 48h immersion and volume remained significantly different from the control situation was not significantly different from the controls (Fig. 4B). even after 48h. Nevertheless, animal motility was little affected by Additionally, an increase in the number of active animals was the hypertonic shock, neither at the initial transfer nor throughout observed, yet a considerable number of specimens remained the rest of the experiment (Fig.7C).

20 ppt, 10 ppt, 20 ppt, 2 ppt, –1 –1 623 mOsm kg–1 62 mOsm kg–1 623 mOsm kg 311 mOsm kg A A 0h 0.5h 1h 2h 4h 24h 48h 0h 0.5h 1h 2h 4h 24h 48h ) )

–1 200 1400 B 200 1400 B –1 (12) 180 180 (12) 1200 1200 160 (10) 160 (12) (6) (6)(10)(12) 1000 1000 (12) (12) 140 140 (10) (11) 120 800 120 (10) (13) 800

100 600 ume (%) al body vol 100 600

ume (%) al body vol

Tot Tot 80 (6) 80 (6) (6) (6) 400 (6) 400 60 (6) 60

200 200 Hemolymph o s motic pre ssu re (mO m kg 40 Hemolymph o s motic pre ssu re (mO m kg 40 021 3 420 304050 021 3 420304050 Exposure time (h) Exposure time (h)

Fig. 4. (A) Halobiotus crispae in active stage from Vellerup Vig, Denmark Fig. 5. (A) Halobiotus crispae in active stage from Vellerup Vig, Denmark (20 ppt). Light-microscopical images at different time points following (20 ppt). Light-microscopical images at different time points following exposure to 2 ppt (62 mOsm kg–1) of a single specimen (scale bar=100 μm). exposure to 10 ppt (311 mOsm kg–1) of a single specimen (scale (B) Changes in total body volume (᭿) and measured internal osmolality (᭺) bar=100 μm). (B) Changes in total body volume (᭿) and measured internal over a period of 48 h following exposure to an external salinity of 2 ppt. osmolality (᭺) over a period of 48 h following an exposure to an external Data are expressed as means ± s.d. Numbers in parentheses indicate the salinity of 10 ppt. Data are expressed as means ± s.d. Numbers in number of animals used for assessment of body volume and hemolymph parentheses indicate the number of animals used for assessment of body osmolality at each time point. volume and hemolymph osmolality at each time point.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 2808 K. A. Halberg and others

20 ppt, 40 ppt, indicating that H. crispae hyper-regulates during steady-state 623 mOsm kg–1 1245 mOsm kg–1 conditions. This hyperosmotic regulation was independently A confirmed by the DSC investigation in which the hemolymph 0h 0.5h 1h 2h 4h 24h 48h osmolality was measured at 975±36mOsmkg–1 (Table1). The two measurements are not significantly different (t-test, P<0.01). Upon immersion into a hypo-osmotic media of 2ppt (63mOsmkg–1), a large rapid decrease was observed, as hemolymph osmolality was reduced to 296±55mOsmkg–1 following 0.5h exposure. This value remained largely constant throughout the rest of the treatment, resulting in an osmolality of 330±51mOsmkg–1 after 48h (Fig.4B). A similar pattern was associated with a less severe (10 ppt, –1

200 1400 ) 311mOsmkg ) hypo-osmotic treatment. The osmolality of the

B –1 extracellular body fluids changed to 458±42mOsmkg–1 after 0.5h 180 (6) (6) 1200 immersion, yet a slight increase to 584±69 was detected following (6) 48h incubation (Fig.5B). Conversely, hyperosmotic stress (40ppt, 160 –1 1000 1245 mOsm kg ) induced an initial increase in hemolymph –1 140 osmolality to 1224±21mOsmkg after 0.5h (Fig.6B). A steady- (6) –1 800 state value of 1293±43mOsmkg was obtained after 48h. Our study 120 reveals that the active stage tolerates large shifts in hemolymph –1 (11) osmolality, with a final osmolality ranging from 330±51mOsmkg ume (%) al body vol 100 (13) 600 to 1293±43mOsmkg–1 during the investigated treatments. The initial

Tot 80 (12) (11) changes in hemolymph osmotic pressure are very fast (maximally 400 (12) (14) within 0.5h), suggesting a limited resistance to cross-cuticular 60 movement of osmotically active solutes and water. 200 Hemolymph o s motic pre ssu re (mO m kg 40 The osmotic performance after 48h acclimation as a function of 021 3 4 20 304050 the external salinity is summarized in Fig.8. Notably, during control Exposure time (h) and steady-state conditions following exposure to diluted media, H. crispae maintains a consistent osmotic gradient of Fig. 6 (A) Halobiotus crispae in active stage from Vellerup Vig, Denmark 270–330mOsmkg–1 above that of the external environment. This (20 ppt). Light-microscopical images at different time points following exposure to 40 ppt (1245 mOsm kg–1) of a single specimen (scale capacity to hyper-regulate becomes less pronounced in the high- bar=100 μm). (B) Changes in total body volume (᭿) and measured internal osmolality solution, in which the hemolymph osmolality is merely osmolality (᭺) over a period of 48 h following an exposure to an external sustained 50mOsmkg–1 above that of the surroundings. In summary, salinity of 40 ppt. Data are expressed as means ± s.d. Numbers in our data show that H. crispae maintains an osmotic pressure gradient parentheses indicate the number of animals used for assessment of body between the internal and external environment, remaining hyper- volume and hemolymph osmolality at each time point. osmotic during all investigated salinities. This hyper-regulation would indicate (1) the excretion of dilute urine and/or (2) the adaptive synthesis of organic osmolytes and/or (3) the active uptake Notably, hemolymph osmotic pressure differed significantly of salts from the external medium. from the control condition at all time points examined during the various salinity treatments (Figs4–6). Hemolymph osmolality of H. DISCUSSION crispae varied in proportion to the gradient set up by the salinity In Greenland (Nipisat Bay), the transformation of H. crispae into transfer. The body fluids of animals kept under control conditions the P1 stage is correlated with the approach of the long Arctic winter, (20ppt, 623mOsmkg–1) had an osmolality of 926±29mOsmkg–1, and this stage is thus considered a true hibernation stage, which is

ABC2 ppt, 63 mOsm kg–1 10 ppt, 311 mOsm kg–1 40 ppt, 1245 mOsm kg–1 100

80 * 60 * * * 40 * *

% Active animal % Active 20

0 0 0.5 1 2 4 24 48 0 0.5 1 2 4 24 48 0 0.5 1 2 4 24 48 Exposure time (h)

Fig. 7. Halobiotus crispae in active stage from Vellerup Vig, Denmark. Percentage of animals responsive to tactile stimuli following exposure to (A) 2 ppt (62 mOsm kg–1), (B) 10 ppt (311 mOsm kg–1) and (C) 40 ppt (1245 mOsm kg–1) over a period of 48 h. Black columns indicate control condition at a salinity of 20 ppt (623 mOsm kg–1). Gray columns indicate osmotic shock exposure. Three groups of 10 animals were used for the assessment at each salinity. Data are expressed as means ± s.e.m. (*, significantly different from control condition at P<0.05).

THE JOURNAL OF EXPERIMENTAL BIOLOGY Cyclomorphosis in tardigrades 2809

1400 control. Surprisingly, our calorimetric investigation of the freeze-

)

–1 tolerant P1 stage reveals that ice crystallization occurs at 1200 Active stage approximately –20°C, excluding the presence of any physiologically relevant ice-nucleating agents in this stage. In fact, the very low 1000 crystallization temperatures measured in both stages suggest that 800 the capacity for supercooling is maintained throughout the majority of the year, which is in general contrast to the pattern observed in 600 most other freeze-tolerant invertebrates (Block, 1991; Westh and Kristensen, 1992; Ramløv et al., 1996). However, in spite of an 400 invariant melting point and water content between the two stages, the amount of water crystallized during cooling to –40°C appears 200 Osmotic performance Isoosmotic line to be about 60% for animals in the P1 stage and 70% for animals

Hemolymph osmolality (mOsm kg 0 in the active stage (see Table 1). Consequently, the cellular 0 200 400 600 800 1000 1200 1400 dehydration induced by freezing is expected to be much higher in External osmolality (mOsm kg–1) animals in the active stage, compared with that of specimens in the P1 stage. This reduction in ice accumulation in specimens in the Fig. 8. Measured hemolymph osmolality of Halobiotus crispae (active stage P1 stage could potentially be explained by an increased production from Vellerup Vig, Denmark) during steady-state conditions after 48 h of macromolecules [which kinetically inhibits ice formation but has acclimation to 2 ppt (63 mOsm kg–1), 10 ppt (311 mOsm kg–1), 20 ppt negligible effect on the melting temperature (see Westh and –1 –1 (623 mOsm kg ) and 40 ppt (1245 mOsm kg ), respectively. Each point Kristensen, 1992)], as compared with the active stage. Whether this represents the mean ± s.d. of the individual experiments. The broken line indicates the isoosmotic line at which no osmoregulation occurs. observation alone explains the observed freeze tolerance is difficult to determine. Selected cryptobiotic species of tardigrades, nematodes as well as some freeze-tolerant insects tolerate as much as 80% of the body water being converted into ice (Westh and Kristensen, functionally characterized as a movable cyst (Kristensen, 1982; 1992; Ramløv and Westh, 1993; Wharton and Block, 1997; Møbjerg et al., 2007; Guidetti et al., 2008). In Denmark (Vellerup Hengherr et al., 2009). Vig), this stage is dominant during the summer months, presumably The apparent morphological difference between the two stages enabling H. crispae to withstand heat stress and oxygen depletion. is similarly relevant in regard to the observed freeze tolerance. The The active stage, the only stage at which active feeding and sexual P1 stage is formed from an incomplete molt in which both the mouth reproduction occur, is the dominant stage during the Greenlandic and cloaca become sealed by cuticular thickenings (see Fig.1), and summer, whereas this stage is present during late winter and the the gut content is often shed prior to this transition. In nature, ice spring months in Denmark (Møbjerg et al., 2007). nucleation can be initiated by a wide range of exogenous substances (Wharton and Worland, 1998). Consequently, the additional layer Freeze avoidance and freeze tolerance of cuticle could increase the capacity to avoid inoculative freezing Winter temperatures are frequently below the equilibrium freezing in animals in the P1 stage, as has been demonstrated for eggs of point of the surrounding seawater at least in some portions of the the nematode Panagrolaimus davidi Timm 1971 (see Wharton, natural environments of H. crispae, and certain habitats may even 1994). Indeed, the extensive capacity for supercooling in both the become completely frozen for extended periods of the year active and P1 stage, along with the additional layer of cuticle and (Kristensen, 1982). Enduring such hostile surroundings requires the clearing of gut contents in P1, would indicate that H. crispae corresponding cold-tolerance strategies that enable long-term preferentially seek to avoid internal ice formation. Nevertheless, survival. Traditionally, two main options are exploited by animals in the P1 stage tolerate internal ice formation for both shorter ectothermic animals when faced with subzero temperatures, i.e. and longer periods of time. freeze avoidance and freeze tolerance (Lee, 1991). When exposing animals in the P1 stage of H. crispae to temperatures below the Volume and osmoregulation equilibrium freezing point (Tc) of their body fluids, freeze tolerance Our results indicate that active-stage H. crispae is the most tolerant is demonstrated, indicating that winter survival could involve of changes in external salinity. Specimens from the population from extracellular ice formation in this species. However, the finding that Nipisat Bay, Greenland exhibit an increased tolerance towards mortality increases with prolonged exposure to subzero temperatures concentrated SW solutions as compared with animals from Vellerup suggests that the consequent damages accumulate in proportion to Vig, Denmark, suggesting that the ability to tolerate large increases the time spent exposed to freezing conditions. This observation is in salinity is potentiated by living in a more-exposed habitat. The likely explained by the depletion of essential metabolites and is of observed volume regulatory response of active-stage H. crispae particular interest in Arctic habitats in which subfreezing during hypo- and hypertonic treatments differs in a significant way. temperatures have to be endured for long periods of time. When exposed to the hypo-osmotic solutions, the initial increase in In freeze-tolerant organisms, ice formation is usually promoted total body volume was regulated to a new steady state, which was at relatively high subzero temperatures (–2 to –10°C) by ice- not significantly different from the control condition. In fact, a new nucleating agents present in the extracellular fluid (Zachariassen, steady-state value was demonstrated after merely 4h immersion in 1985; Block, 1991; Westh and Kristensen, 1992). The adaptive the external medium of 10ppt. Conversely, during acute exposure advantage of such a strategy is that the process of ice formation to concentrated seawater (40ppt), a partial recovery to normal levels proceeds relatively slowly at relatively high temperatures, enabling was demonstrated; however, total body volume remained the organism to maintain the damage associated with freezing within significantly different from the control condition even after 48h tolerable boundaries. Indeed, both the localization and the amount immersion. These data suggest that the body volume of H. crispae of ice formed in freeze-tolerant organisms are usually under tight is more tightly regulated during exposure to dilute as compared with

THE JOURNAL OF EXPERIMENTAL BIOLOGY 2810 K. A. Halberg and others more concentrated saltwater solutions. Interestingly, this observation exist relating the Malpighian tubules of tardigrades to an seems reflected in an evolutionary context. According to our osmoregulatory role. Nevertheless, several detailed morphological previous study, Halobiotus has evolved within the freshwater genus investigations of the tubules support the hypothesis. These studies Isohypsibius, thus potentially explaining the enhanced volume have provided ultrastructural data, which are in agreement with an regulatory response during exposure to dilute media (Møbjerg et active transporting epithelium involved in solute and fluid transport al., 2007). (Greven, 1979; Weglarska, 1987a; Weglarska, 1987b; Møbjerg and When submitted to osmotic shock of 10 ppt and 40 ppt, Dahl, 1996; Peltzer et al., 2007). As holds for insects, the Malpighian respectively, our data show that H. crispae experience few tubules of tardigrades are considered secretion–reabsorption kidneys. limitations in terms of motility. Active-stage specimens from the In light of the ultrastructural data available on tardigrade Malpighian Nipisat population even retain activity when exposed to a gradual tubules, it seems reasonable to assume that the first steps in urine salinity increase to 60ppt (Fig.3). However, upon direct transfer to formation take place across initial segment cells, characterized by an extreme seawater dilution of 2ppt, animal activity is markedly a conspicuous basal labyrinth, numerous mitochondria and an reduced, probably due to the pronounced increase in hydrostatic enlarged apical surface. There is, moreover, ultrastructural support pressure and concomitant reduction in hemolymph osmotic pressure. for assigning the tardigrade rectum an osmoregulatory function Proper locomotory function in tardigrades relies on the hydrostatic (Dewel and Dewel, 1979). A possible mode of urine formation was pressure of the body cavity (Kinchin, 1994); thus, maintaining an outlined by Dewel and Dewel (Dewel and Dewel, 1979). They appropriate body volume is essential to normal coordination of suggest that isoosmotic urine produced by the Malpighian tubules movement. In addition, as the membrane potentials of animal cells is modified in the rectum through the active reabsorption of solutes, are highly dependent upon extracellular ionic strength (Spyropoulos leading to the excretion of hypo-osmotic urine. Our data seem in and Teorell, 1968), the concomitant changes in hemolymph osmotic favor of such a mechanism of urine formation. Interestingly, pressure could influence animal motility due to inhibition of neuro- preliminary and unpublished data (H.R. research group) on muscular activity. Indeed, the fact that a significant number of Richtersius coronifer (Richters 1903) indicate that this species also animals were observed passive during exposure to 2ppt, while remains hyperosmotic during exposures to a range of external animals remained largely unaffected during exposure to 10ppt, in salinities and it is therefore likely that hyper-regulation, and possibly spite of experiencing comparable average changes in body volume hypo-osmotic urine formation, is a general feature of eutardigrades. (compare Fig.4B and Fig.5B after 0.5h immersion), suggests that However, until functional studies at the cellular and molecular level not only total body volume but also hemolymph osmolality is an are performed, the exact mechanisms involved in osmoregulation important factor in maintaining locomotory functions. in tardigrades remain to be elucidated. Exposing H. crispae to severe osmotic stress reveals that this In conclusion, we show that the transition between the individual species is a euryhaline osmoconformer, in which the hemolymph cyclomorphic stages of H. crispae is associated with profound osmotic pressure is largely governed by the external environment. changes in the physiology of the animal. Our results show that However, when analyzing hemolymph osmotic pressure at steady animals in the active stage tolerate large changes in the external state following 48h exposure to the various salinity treatments as osmotic pressure by regulating their total body volume and by a function of the external osmolality, an interesting pattern emerges. enduring large concomitant changes in hemolymph osmotic H. crispae maintains a large osmotic pressure gradient between the pressure. H. crispae remains hyperosmotic at any investigated internal and external environment, thus distinctly hyper-regulating external salinity, suggesting that this species is a strong hyper- during all investigated salinity treatments – albeit markedly less in regulator. Our study is the first to provide evidence for the volume concentrated seawater. This would imply a large water turnover in and osmoregulatory capacity in Tardigrada. Whereas animals in the this animal, with osmotic water uptake being balanced by the active stage are intolerant of freezing, the P1 stage is demonstrated excretion of dilute urine. to be freeze tolerant. The relatively low crystallization temperature Hyperosmoregulation is known in other euryhaline invertebrates. reveals that extensive supercooling of the body fluids takes place The crayfish Procambarus clarkii Girard 1852 (Arthropoda) during cooling and that no physiologically relevant ice-nucleating produces highly dilute urine and is a strong hyperosmoregulator in agents are present. freshwater (Sarver et al., 1994). However, the excreted urine becomes progressively more concentrated in media of higher ionic We would like to thank the crew onboard the research vessel R/W Porsild (Arctic Station, Qeqertarsuaq, Greenland) who made the collection at the type locality, strength and is nearly isoosmotic when exposed to an external Nipisat, possible. Funding came from the Carlsberg Foundation and from the concentration of 750mOsmkg–1, at which P. clarkii cease to hyper- 2008 Faculty of Science, University of Copenhagen Freja-Programme. regulate (Sarver et al., 1994). Moreover, similar osmoregulatory responses have been reported from nematodes (Fusé et al., 1993; LIST OF ABBREVIATIONS Forster, 1998). Indeed, the internal osmolality of the parasitic DSC differential scanning calorimetry nematode Pseudoterranova decipiens (Krabbe, 1878) was MDP melting point depression maintained 90mOsmkg–1 above that of the external environment RVD regulatory volume decrease SW seawater during exposure to media of widely varying osmolality (Fusé et al., Tc crystallization temperature 1993). Tm melting temperature Three glands positioned at the transition zone between the midgut and rectum of eutardigrades are traditionally ascribed an REFERENCES osmoregulatory function. The term used for these structures, i.e. Block, W. (1991). To freeze or not to freeze? Invertebrate survival of sub-zero Malpighian tubules, was introduced more than a century ago (Plate, temperatures. Funct. Ecol. 5, 284-290. Convey, P. and McInnes, S. J. (2005). Exceptional tardigrade-dominated ecosystems 1889). The positional conformity of the Malpighian tubules in in Ellsworth Land, Antarctica. Ecology 86, 519-527. eutardigrades and in hexapods has been used as a strong argument Dewel, R. A. and Dewel, W. C. (1979). Studies on the tardigrades. J. Morphol. 161, 79-110. in favor of a homology between these structures (Greven, 1982; Forster, S. J. (1998). Osmotic stress tolerance and osmoregulation of intertidal and Møbjerg and Dahl, 1996). However, at present, no functional data subtidal nematodes. J. Exp. Mar. Biol. Ecol. 224, 109-125.

THE JOURNAL OF EXPERIMENTAL BIOLOGY Cyclomorphosis in tardigrades 2811

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