Kenneth Halberg
Kenneth Halberg
Kenneth Halberg
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Osmoregulation | With Focus on Fluid<br />
and Solute Dynamics in Tardigrada<br />
PhD Dissertation<br />
KENNETH A. HALBERG<br />
© <strong>Kenneth</strong> A. <strong>Halberg</strong>
FACULTY OF SCIENCE<br />
UNIVERSITY OF COPENHAGEN<br />
Osmoregulation│With Focus on Fluid<br />
and Solute Dynamics in Tardigrada<br />
PhD Dissertation<br />
<strong>Kenneth</strong> A. <strong>Halberg</strong><br />
UNIVERSITAS<br />
HAFNIENSIS<br />
2012<br />
Dissertation submitted Monday the 14th of May 2012.<br />
Supervisor: Associate Professor Nadja Møbjerg, PhD.
Dissertation presented at University of Copenhagen to be publicly examined (provided acceptance in its<br />
current form) in Auditorium 1, August Krogh Building, Universitetsparken 13, Thursday, June 28, 2012<br />
at 14:00 for the degree of Doctor of Philosophy. The examination will be conducted in English.<br />
Abstract<br />
<strong>Halberg</strong>, K. A. 2012. Osmoregulation │With Focus on Fluid and Solute Dynamics in<br />
Tardigrada.<br />
Osmoregulation is the regulated control of water and solute composition in body fluid<br />
compartments. On one hand, the internal composition must be kept within optimal<br />
conditions for metabolic processes in the face of external perturbation. On the other<br />
hand, the nature of the living state demands a continuous traffic of compounds in and<br />
out of the organism. These demands appear to be in fundamental contradiction<br />
however cells and animals achieve so-called “steady-state” by means of an array of<br />
transport proteins, which provide a stringent control on the exchange of water and<br />
solutes across body surfaces. The distinct mechanisms of solute transport have been<br />
studied in most animal groups, but there are still large gaps in our understanding of<br />
how animals cope with osmotic stress. In the present thesis, osmoregulatory<br />
phenomena were studied in vertebrate and invertebrate organism alike, with the main<br />
focus being on fluid and solute dynamics in Tardigrada. For example, the inorganic<br />
ion composition of several species was investigated, which revealed that tardigrades<br />
contain roughly similar relative contributions of inorganic ions to total osmotic<br />
concentration, when compared to closely related animal groups. Moreover, it was<br />
inferred that cryptobiotic tardigrades (species able to enter a state of latent life)<br />
contain a large fraction of organic osmolytes. The mechanisms of organic anion<br />
transport in a marine species of tardigrade was investigated pharmacologically, and<br />
compared to that of insects. These data showed that organic anion transport is<br />
localized to the midgut epithelium and that the transport is both active and transporter<br />
mediated with a pharmacological profile similar to that of insects. Tardigrades survive<br />
in a variety of osmotic environments (semi-terrestrial, limnic and marine habitats),<br />
why the ability to volume and osmoregulate was examined. These studies demonstrated<br />
an ability to regulate total body volume during both hypo- and hyperosmotic<br />
conditions, and that the ability to hyper-regulate could be a general theme among<br />
members of eutardigrades. Thus, the work presented herein, have contributed to<br />
establishing tardigrades as an important experimental group in which central<br />
physiological questions may be answered, including aspects of osmotic and ionic<br />
regulation.<br />
Keywords: osmoregulation, volume regulation, organic anion transport, hyper-regulate,<br />
inorganic ions, organic osmolytes, tardigrade, insect,<br />
<strong>Kenneth</strong> A. <strong>Halberg</strong>, The August Krogh Centre, Department of Biology,<br />
Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark<br />
© <strong>Kenneth</strong> A. <strong>Halberg</strong> 2012
“Beautiful is what we see,<br />
More beautiful is what we perceive,<br />
Most beautiful is what we do not understand”<br />
- Niels Stensen
List of Papers<br />
This thesis is based on the following papers and manuscripts, which are referred to<br />
in the text by their Roman numerals.<br />
I. <strong>Halberg</strong>, K. A., Larsen, K. W., Jørgensen, A., Ramløv, H. & Møbjerg,<br />
N. Cryptobiotic tardigrades contain large fraction of unidentified<br />
organic solutes: A comparative study on inorganic ion composition in<br />
Tardigrada.<br />
II.<br />
<strong>Halberg</strong>, K. A., & Møbjerg, N. (2012). First evidence of epithelial<br />
transport in tardigrades: A comparative investigation of organic anion<br />
transport. Journal of Experimental Biology, 215: 497-507.<br />
III. Møbjerg, N. M., <strong>Halberg</strong>, K. A., Jørgensen, A. Persson., D., Bjørn M,<br />
Ramløv H & Kristensen R. M. (2011). Survival in extreme<br />
environments – on current knowledge of adaptations in tardigrades.<br />
Acta Physiologica, 202: 409-420.<br />
IV. Haugen, B.M., <strong>Halberg</strong>, K.A., Jespersen, Å., Prehn, L.R. & Møbjerg,<br />
N. (2010). Functional characterization of the vertebrate primary ureter:<br />
Structure and ion transport mechanisms of the pronephric duct of<br />
axolotl larvae (Amphibia). BMC developmental Biology, 10: 56.<br />
V. <strong>Halberg</strong>, K. A., Persson, D., Ramløv, H., Westh, P., Kristensen, R. M.<br />
& Møbjerg, N. (2009). Cyclomorphosis in Tardigrada: Adaption to<br />
environmental constraints. Journal of Experimental Biology, 212:<br />
2803-2811.<br />
Additionally, the following papers and manuscripts were prepared during the<br />
course of my PhD studies, but are not included in the thesis:<br />
VI.<br />
VII.<br />
<strong>Halberg</strong> K. A., Jørgensen, A. and Møbjerg, N. (in prep.). Surviving<br />
without water: Tun formation in tardigrades is an active process<br />
mediated by the musculature<br />
<strong>Halberg</strong> K. A., Persson, D., Jørgensen, A. Kristensen, R. M. and<br />
Møbjerg, N. (submitted). Population dynamics of a marine tardigrade:<br />
Temperature limits geographic distribution of Halobiotus crispae.<br />
Marine Biological Research
VIII. Persson, D., <strong>Halberg</strong> K. A., Jørgensen A., Møbjerg N. & Kristensen<br />
R. M. (in review). Neuroanatomy of Halobiotus crispae (Eutardigrada:<br />
Hypsibiidae): Tardigrade brain structure suggests inclusion into<br />
Panarthropoda. Journal of Morphology.<br />
IX. Persson, D., <strong>Halberg</strong> K. A., Jørgensen A., Ricci C., Møbjerg N. &<br />
Kristensen R. M. (2010). Extreme stress tolerance in tardigrades:<br />
Surviving space conditions in low earth orbit. Journal of Zoological<br />
Systematics and Evolutionary Research, 49: 90-97.<br />
X. <strong>Halberg</strong>, K. A., Persson D., Møbjerg N., Wanninger A. & Kristensen<br />
R. M. (2009). Myoanatomy of the Marine Tardigrade Halobiotus<br />
crispae (Eutardigrada: Hypsibiidae). Journal of Morphology, 270:<br />
996-1013.<br />
Lastly, following paper provides important background knowledge for the work<br />
presented herein:<br />
XI. Møbjerg, N., A. Jørgensen, J. Eibye-Jacobsen, K. A. <strong>Halberg</strong>, D.<br />
Persson & R. M. Kristensen (2007). New Records on cyclomorphosis<br />
in the marine eutardigrade Halobiotus crispae (Eutardigrada:<br />
Hypsibiidae). Journal of Limnology, 66 (suppl. 1): 132-140.
Reprint and publication is made with permission from the respective copyright<br />
holders.<br />
Paper II, V © The Company of Biologists.<br />
Paper IV, is copyright of the authors.<br />
Paper III © Wiley-Blackwell<br />
Statement of authorship<br />
Paper I: KAH was deeply involved in study design. KAH participated in<br />
extracting animals and ion chromatography. KAH performed nanoliter osmometry.<br />
KAH performed the data analysis, prepared the figures, participated in discussions<br />
and interpretation of the data, and drafted the manuscript.<br />
Paper II: KAH performed the major part of the experimental work and data<br />
analysis. He participated in planning of experiments, data interpretation, prepared<br />
the figures and drafted the manuscript.<br />
Paper III: KAH performed cell counts and provided images of tardigrades. He<br />
helped draft parts of the manuscript.<br />
Paper IV: KAH participated in immunostaining experiments, performed CLSM<br />
and prepared the 3D images. KAH participated in discussions and interpretation of<br />
the data.<br />
Paper V: KAH participated in planning of experiments, sampling, staging,<br />
scanning electron microscopy, DSC experiments, experiments on cold hardiness<br />
and osmotic stress tolerance, volume measurements, hemolymph sample<br />
collections, and nanoliter osmometry. He furthermore participated in discussions<br />
and interpretation of data, prepared the figures and drafted the manuscript.<br />
Front cover: Scanning elecron micrographs of the tardigrades Rictersius coronifer<br />
(top left), Halobiotus crispae (middle right), and Echiniscus testudo (middle<br />
bottom).
Contents<br />
Preface.......................................................................................................................... 9<br />
Introduction ................................................................................................................ 11<br />
Maintaining a stable internal environment .......................................................... 11<br />
Osmoregulators and osmoconformers ................................................................. 12<br />
Osmoregulatory organs........................................................................................ 12<br />
Filtration-Reabsorption systems ................................................................ 13<br />
Secretion-Reabsorption systems ................................................................ 14<br />
Phylum Tardigrada..................................................................................................... 17<br />
General morphology ............................................................................................ 18<br />
Classification ....................................................................................................... 19<br />
Ecology ................................................................................................................ 21<br />
Fluid and solute dynamics – an overview .................................................................. 23<br />
Inorganic ion composition ................................................................................... 23<br />
Organic anion transport ....................................................................................... 25<br />
Volume and osmoregulation................................................................................ 26<br />
Conclusions and future perspectives .......................................................................... 27<br />
Dansk sammenfatning ................................................................................................ 29<br />
Acknowledgements .................................................................................................... 30<br />
References .................................................................................................................. 32
Preface<br />
The primary aim of this thesis was to address several aspects of the fluid and<br />
solute dynamics in tardigrades, and hereby provide new insight into the general<br />
stress biology of these enigmatic creatures. This was done by adopting an<br />
integrative approach, i.e. applying advanced methods in biology, analytic<br />
biochemistry and physical chemistry, which offered functional data from different<br />
disciplines to be obtained. The experimental work was carried out mainly at The<br />
August Krogh Centre, University of Copenhagen; however, additional<br />
experimental work was performed at the Danish Natural History Museum,<br />
University of Copenhagen and at the Department of Nature, Systems and Models,<br />
Roskilde University. Overall this thesis has contributed to converting tardigrades,<br />
from an almost exclusive taxonomic phenomenon into an established and<br />
important experimental group, in which central physiological questions can be<br />
investigated.<br />
This PhD-thesis comprises a short introduction to osmotic and ionic<br />
regulation in Metazoa, accompanied by a brief review on the general morphology,<br />
classification and ecology of tardigrades. Moreover, an overview of the results<br />
presented as well as conclusions and future perspectives are presented. Five papers<br />
and manuscripts form the basis of this thesis, of which four are published in peer<br />
review journals (Papers II, III, IV and V), and one is prepared for submission<br />
(Paper I). I am the first author of three (Paper I, II and V) and second author on<br />
two (Papers III and IV) of these papers. Five additional papers and manuscripts<br />
were prepared during the course of my PhD studies, but are not included in the<br />
thesis. This work was funded by the 2008 Faculty of Science, University of<br />
Copenhagen Freja-Programme.<br />
Copenhagen, the 14 th of May 2012<br />
<strong>Kenneth</strong> A. <strong>Halberg</strong><br />
9
Introduction<br />
Maintaining a stable internal environment<br />
“Life is as a thing of macromolecular cohesion in salty water” (Gilles & Delpire,<br />
1997). Albeit a crude statement, it frames the fact that the ability to control salt<br />
and water balance is a fundamental prerequisite for both cellular and animal life.<br />
Indeed, the internal environment must usually be kept within relatively narrow<br />
limits, as substantial deviations in cell composition are incompatible with the<br />
optimal function of macromolecules (lipids, proteins, RNA), and may ultimately<br />
modify the rate and extent of cellular reactions (Zhao, 2005). The overall<br />
mechanism by which animals conserve a proper osmotic balance between cells,<br />
extracellular fluid and the environment is termed osmoregulation.<br />
The basis for osmoregulation lies in the strict control of the ionic<br />
composition and the osmotic pressure of the intra- and extracellular compartments<br />
through the regulated accumulation and loss of inorganic ions and organic<br />
compounds (Dawson & Liu, 2009). This regulation is achieved through the<br />
coordinated activity of an array of transporter proteins (both energy-consuming<br />
and passive), which collectively maintain the steady-state condition of cells and<br />
animals (Essig, 1968). During steady-state conditions, compositions of the intraand<br />
extracellular compartments are maintained in a non-equilibrium state (Dawson<br />
& Liu, 2009). This uneven distribution of solutes is important for keeping an<br />
optimal milieu for metabolic processes (Zhao, 2005). Accordingly, the<br />
extracellular fraction of the body fluids of animals are typically high in Na + and Cl -<br />
, and relatively low in the other major ions (e.g. K + , Ca 2+ and Mg 2+ ), while the<br />
intracellular environment of most organisms is low in Na + but high in K + , PO 4<br />
3-<br />
and proteins (e.g. Dawson & Lui, 2009; Paper I). As such, the plasma membrane<br />
of cells must maintain ionic, but not osmotic, differences, while specialized<br />
excretory organs – e.g. antennal glands of crustaceans, Malpighian tubules of<br />
insects and tardigrades, rectal glands of sharks and rays, gills and intestine of<br />
teleost fishes, salt glands of birds and reptiles, the kidneys of vertebrates etc. –<br />
often maintain both ionic and osmotic differences between animals and their<br />
environments (Riegel, 1970; Peaker, 1971; Paper IV, Beyenbach & Piermarini,<br />
2011; Reilly et al., 2011; Whittamore, 2012). In general, mechanisms that allowed<br />
organisms to respond and adapt to an osmotic challenge over the course of<br />
11
evolution, has been fundamental to the invasion of new osmotic hostile habitats,<br />
and such ecological divergence in turn is an important mechanism for the<br />
speciation process (Schluter, 2009). Accordingly, if the ability to osmoregulate<br />
had not evolved, life on the planet would look quiet different from how we know<br />
it.<br />
Osmoregulators and osmoconformers<br />
Generally, animals are divided into two broad categories in terms of their<br />
responses to osmotic stress: osmoregulators, which maintain an internal osmolarity<br />
different from that of the external environment, and osmoconformers, which<br />
conform to the external medium in which they are immersed (Fig. 1). Most<br />
vertebrates are strict osmoregulators (e.g. Paper IV), as they maintain ionic and<br />
osmotic balance within narrow limits; although hagfish, a basal group of<br />
vertebrates, are a notable exception (Sardella et al., 2009). Conversely, marine<br />
invertebrates are typically categorized as osmoconformers, as many of them<br />
appear to be in osmotic balance with sea water over a range of salinities (Fig. 1).<br />
However, there are numerous obvious exceptions including the marine tardigrade<br />
Halobiotus crispae as well as several members of Crustacea (e.g. Sarver et al.,<br />
1994; Normant et al., 2005; Paper V). In fact, the terms ‘strict osmoregulator’ and<br />
‘strict osmoconformer’ must be used with caution, as typical osmoregulators are<br />
forced to conform during the most extreme conditions (e.g. Dowd et al., 2010),<br />
whereas animals otherwise described as osmoconformers actually maintain slight<br />
differences between the internal and external environment (e.g. van Weel, 1957).<br />
Although achieved through different mechanisms, both osmoregulating and<br />
osmoconforming animals may tolerate wide ranges of external salinities, thus<br />
termed euryhaline species, while animals intolerant of large changes are called<br />
stenohaline species.<br />
Osmoregulatory organs<br />
Osmoregulatory organs are specialized organs involved in maintaining ionic and<br />
osmotic homeostasis in the face of osmotic perturbation, as well as in excreting<br />
endobiotic and exobiotic waste products (Dawson & Lui, 2009; Papers II, IV).<br />
The specific organs mediating these processes may vary between different groups<br />
of animals (see above); however, the molecular basis and specific mechanism of<br />
solute and water transport often show a highly convergent/homologous pattern<br />
across different types of epithelia (e.g. Paper II). In general, two types of systems<br />
12
Fig. 1 Examples of osmotic performance of representative species from various groups<br />
exposed to sea/brackish water, expressed as the relation between internal (extracellular) and<br />
external (habitat) osmolality. These data show that osmoconformaty (∆osm=0) is present in<br />
invertebrate and vertebrate species alike, albeit strong hypo-regulators and the ability to<br />
produce a hyperosmotic urine appears restricted to vertebrates. It should be emphasized that<br />
the selected species not necessarily represent the osmotic performance of the entire group, as<br />
large difference may exist between even closely related species. For original data see:<br />
Robertson, 1949 a ; van Weel, 1957 e ; Dice, 1968 d ; Liggins and Grigg, 1985 j ; Diehl, 1986 b ;<br />
Normant et al., 2005 f ; Sardella et al., 2009 g ; Paper V c ;Reilly et al., 2011 h ; Whittamore,<br />
2012 i ).<br />
have evolved by which the initial process of urine formation takes place i) the<br />
filtration-reabsorption type and ii) the secretion-reabsorption type.<br />
Filtration-Reabsorption systems<br />
The kidneys of vertebrates (fish, amphibians, reptiles, birds and mammals), and<br />
the functional analogs of crusteans and molluscs, maintain extracellular fluid<br />
homeostasis by producing urine through the filtration of plasma (ultrafiltration),<br />
which is subsequently modified by selective reabsorption and secretion of ions,<br />
organic molecules and water (Anderson, 1960; Schmidt-Nielsen, 1963; Riegel,<br />
13
1970; Møbjerg et al., 2004; Paper IV; Whittamore et al., 2012). In vertebrates,<br />
three temporally and spatially different kidney generations, the pronephroi,<br />
mesonephroi and metanephroi, successively maintain fluid and electrolyte<br />
homeostasis during morphogenesis, with the pronephroi constituting the functional<br />
kidneys of fish and amphibian larvae (Paper IV). The functional unit of the<br />
vertebrate kidney is the nephron, which is composed of a filtration unit and a renal<br />
tubule (Anderson, 1960; Møbjerg et al., 2004; Paper IV). The filtration process<br />
that takes place in the filtration unit (the glomerulus and Bowman’s capsule) is<br />
‘passive’ i.e. entirely driven by the hydrostatic pressure generated by the heart,<br />
whereas the reabsorption and secretion processes take place over specialized<br />
epithelia of the renal tubule (Schmidt-Nielsen, 1963). The primary membrane<br />
transporter for energizing vertebrate tissue is the Na/K-ATPase (e.g. Paper IV).<br />
Accordingly, vertebrate kidneys may produce both dilute, iso-osmotic and<br />
concentrated urine relative to the body fluids (Paper IV; Whittamore et al., 2012),<br />
which has been a dominant factor in allowing vertebrates to penetrate into all types<br />
of habitats on Earth. Filtration-reabsorption systems are capable of processing<br />
large volumes of fluids, but are energetically costly, as any substance (e.g.<br />
glucose) that has been filtered remains in the urine unless subsequently reabsorbed<br />
(Schmidt-Nielsen, 1963). The advantage of such a system; however, is that new<br />
potentially toxic compounds are eliminated without the need for developing<br />
distinct secretory pathways for each new compound, which may be necessary for<br />
the secretion-reabsorption type system (Paper II).<br />
Secretion-Reabsorption systems<br />
The Malpighian tubules of insects, and possibly tardigrades (Møbjerg & Dahl,<br />
1996; Papers II, III, V), are the functional analogs of the vertebrate kidney, but<br />
constitute a secretion-reabsorption system that produces urine in a fundamentally<br />
different way than the filtration-reabsorption-systems (Beyenbach & Piermarini,<br />
2011). In the absence of blood vessels (i.e. a closed circulatory system), the<br />
hemolymph of insects is circulated at pressures insufficient for filtration, and the<br />
Malpighian tubules thus form the (primary) urine entirely by secretion (Beyenbach<br />
& Piermarini, 2011). The formation of the primary urine is generally initiated in<br />
the distal segments (blind-ended tip) of the Malpighian tubule, and is essentially<br />
iso-osmotic (consisting mainly of KCl and NaCl) to the hemolymph (Williams &<br />
Beyenbach, 1983). The subsequent reabsorption of water, ions and metabolites in<br />
proportions that maintain extracellular homeostasis (a process analogous to that of<br />
vertebrates) takes place in downstream structures i.e. proximal tubule, hindgut and<br />
rectum (O’Donnell & Maddrell, 1995; Coast, 2007). The final urine composition is<br />
14
adjusted in the rectum (Coast, 2007) and may be either strongly hypo- or<br />
hyperosmotic depending on the species and its physiological status (Maddrell &<br />
Phillips, 1975; Reynolds & Bellward, 1989). In contrast to vertebrate epithelia, the<br />
V-type H + -ATPase is considered ubiquitous in energizing insect epithelia<br />
(Beyenbach & Piermarini, 2011); although the Na/K-ATPase is still expressed and<br />
functionally relevant for tubular function (Torrie et al., 2004; Paper II). In fact,<br />
energized by the H + -ATPase, some of the highest fluid secretion rates per unit area<br />
membrane from any tissue have been reported from hematophagous insects (e.g.<br />
Aedes aegypti, Rhodnius prolixus) after a blood meal (Williams & Beyenbach,<br />
1983; Maddrell & Phillips, 1975). In addition to playing a key role in<br />
osmoregulation, new properties of the Malpighian tubules of insects have emerged<br />
in recent years, which suggest that Malpighian tubules are involved in such diverse<br />
functions as renal detoxification, metabolism of toxins and immune system<br />
responses (Dow & Davies, 2006; Paper II).<br />
As holds for insects, ultrastructural studies on the Malpighian tubules of<br />
tardigrades indicate that they function as secretion-reabsorption systems involved<br />
in fluid and solute transport (Weglarska, 1987; Møbjerg and Dahl, 1996; Peltzer et<br />
al., 2007). They are positioned at the transition zone between the midgut and<br />
rectum of eutardigrades (Fig. 2), and the positional conformity between insects<br />
and eutardigrades has been used as a strong argument for their homology (Greven,<br />
1982; Møbjerg and Dahl, 1996). However, at present no functional data exist<br />
relating the Malpighian tubules of tardigrades to an osmoregulatory role.<br />
Accordingly, functional studies on the fluid and solute dynamics of tardigrades are<br />
greatly needed (Papers I, II, III, V), and due to the close affinity to the<br />
euarthropod complex (Aguinaldo et al., 1997), would be highly useful in<br />
understanding and reconstructing the evolution of osmoregulation in Insects and<br />
other arthropods.<br />
15
Fig. 2 Structure and organization of the Malpighian tubules of the marine eutardigrade<br />
Halobiotus crispae. dm, dorsal Malpighian tubule; dp, distal part; is, initial segment; mg,<br />
midgut; mv, microvilli,; nu, nucleus; pp, proximal part; re, rectum; From: Møbjerg & Dahl,<br />
1996.<br />
16
Phylum Tardigrada<br />
Tardigrades, also known as water bears, are among the smallest multi-cellular<br />
animals on the planet (0.1-1.2 mm). They were discovered in 1773 by the German<br />
pastor J. A. E. Goeze, who described them as “kleiner Wasserbär”, or little water<br />
bear, due to their strong resemblance to a tiny bear (Ramazzotti & Maucci 1982).<br />
Not long after the current name “Tardigrada” was given by the Italian naturalist<br />
Spallanzani in 1776 (Lat. tardus – slow, grado – walker). In response to<br />
unfavorable environmental conditions, many species of tardigrades have the ability<br />
to enter the ametabolic state of suspended animation, also known as cryptobiosis,<br />
in which the organism is neither dead nor alive (Møbjerg et al., 2011; Fig. 3). The<br />
animal can remain in this state for as much as 20 years (Jørgensen et al., 2007), yet<br />
once external conditions again become favorable, the tardigrade resumes activity<br />
unaffected. This incredible ability is shared with selected species of nematodes,<br />
rotifers and arthropods (Glasheen & Hand, 1988; Crowe & Maddin, 1974; Ricci et<br />
al., 2003). In 1962, Tardigrada was recognized as a phylum by Ramazzotti in Il<br />
Phylum Tardigrada. Presently, there are more than 1000 described species<br />
(Guidetti & Bertolani 2005; Degma & Guidetti, 2007; Degma et al., 2012);<br />
however, it has been estimated that several thousand species remain undescribed<br />
(Paper III).<br />
Tardigrada is included in the invertebrate superclade Ecdysozoa<br />
(Aguinaldo et al. 1997); however, their precise phylogenetic position is still being<br />
debated. Both molecular and morphological investigations produce conflicting<br />
conclusions, and it is currently unclear whether the group is more closely related to<br />
the nematodes and nematomorphs or to arthropods and onychophorans (Aguinaldo<br />
et al. 1997; Dunn et al. 2008; Zantke et al., 2008; Edgecombe 2010; Rota-Stabelli<br />
et al., 2010; Cambell et al., 2011). Regardless, this group is closely related to one<br />
of the two most species-rich and economically important groups Nematoda or<br />
Arthropoda, and thus maintains a central position in relation to the two major<br />
invertebrate model organisms, i.e. Caenorhabditis elegans Maupas, 1900 and<br />
Drosophila (Sophophora) melanogaster Meigen, 1830 (Gabriel et al. 2007;<br />
Goldstein and Blaxter 2002).<br />
17
Fig. 3 Scanning electron micrographs showing the external morphology of Richtersius<br />
coronifer (Eutardigrada) A. lateral view of the active, hydrated state B. Dorsal view of the<br />
dehydrated, cryptobiotic state C. Ventral view of the dehydrated, cryptobiotic state. The<br />
pictures illustrate the morphological changes associated with entry into an ametabolic state<br />
(i.e. cryptobiosis), which include the retraction of head and limbs into the body cavity, and the<br />
formation of a compact shape – the tun. From: Paper VI.<br />
General morphology<br />
Tardigrades are bilaterally symmetric micrometazoans with a body divided into<br />
five separate body segments, i.e. a cephalic segment, containing a mouth, eyespots<br />
18
and sensory organs (papillae cephalica or cirri and clavae), and four trunk<br />
segments (Nelson, 2002; Fig. 3A). The first three trunk segments each bear a pair<br />
of lateroventrally directed legs, while the terminal trunk segment bears a pair of<br />
posterioventrally directed legs (Figs. 3, 4). The legs typically terminate in 4 to 13<br />
claws or suction discs (Nelson, 2002). Tardigrades are ventrally flattened with a<br />
convex dorsal side, and are covered by a segmented cutinous cuticle, which is<br />
periodically shed during molting – formation of the new cuticle is maintained by a<br />
single layer of epidermal cells (Nelson, 2002). The digestive system consists of a<br />
foregut, midgut and a hindgut with a pair of stylets and stylet glands flanking the<br />
buccal tube. Three glands (the Malpighian tubules) are positioned at the transition<br />
zone between the midgut and hindgut in eutardigrades (Weglarska, 1987; Møbjerg<br />
and Dahl, 1996; Peltzer et al., 2007). Tardigrades posses a hemocoel-type of fluidfilled<br />
body cavity, i.e. an open circulatory system as seen in arthropods and<br />
nematodes, which likely functions in circulation and respiration. The somatic<br />
musculature is composed of structurally independent muscle fibers, which can be<br />
divided into a dorsal, ventral, dorsoventral, and a lateral musculature in addition to<br />
a distinct leg musculature (Schmidt-Rhaesa & Kulessa, 2007; Fig. 4). Moreover,<br />
the buccopharyngeal muscles, intestinal muscles and cloacal muscles comprise the<br />
animal’s visceral musculature. Whereas cross striation of the somatic musculature<br />
is especially pronounced in Arthrotardigrada, the somatic muscles of Eutardigrada<br />
are described as an intermediate between smooth and obliquely striated (Walz,<br />
1974). The nervous system of tardigrades consists of an (at least) three lobed brain<br />
(Fig. 5) and a ventral nerve cord with four fused paired ganglia that shows a clear<br />
segmental organization.<br />
Classification<br />
Originally based on morphological characters, tardigrades are divided into two<br />
main evolutionary lines, represented by the extant lineages Eutardigrada and<br />
Heterotardigrada. The validity of a third class, Mesotardigrada, is currently<br />
considered dubious (Ramazzotti and Maucci, 1983).<br />
Heterotardigrada consists of the orders Arthrotardigrada and Echiniscoidea<br />
with arthrotardigrades possessing the most plesiomorphic characters.<br />
Arthrotardigrada consists exclusively of marine species (Renaud-Mornant 1982;<br />
Jørgensen et al. 2010), and are morphologically the most diverse group. They are<br />
present in all oceans from intertidal zones to abyssal depths, and inhabit various<br />
types of sediment. In contrast, the Echiniscoidea comprises both limno-terrestrial,<br />
limnic as well as marine species with the majority of the described species<br />
belonging to the family Echiniscidae. Taxonomically, the main characters<br />
19
Fig. 4 Tardigrade musculature as revealed by fluorescently coupled phalloidin in combination<br />
with confocal laser scanning microscopy three-dimensional reconstruction A. Lateral view of<br />
Halobiotus crispae (Eutardigrada), Paper VIII B. Ventral view of Echiniscoides sigismundi<br />
(Heterotardigrada), unpublished data.<br />
separating the two groups from Eutardigrada include a separate gonopore, a closed<br />
three-lobed anus as well as well-developed cephalic-, trunk-, and leg appendages<br />
(Guidetti and Bertolani, 2005).<br />
The eutardigrades are divided into the two orders; Apochela and Parachela.<br />
Both orders contain mainly limno-terrestrial species, albeit with a few exceptions –<br />
20
Fig. 5 Conceptual drawing based on immunofluorescent and ultrastructural data, showing an<br />
interpretation of the brain structure of Halobiotus crispae. A. Lateral view B. Frontal view.<br />
clg, claw gland; co, connective; dc, dorsal commissure; ey, eye; g0, sub-pharyngeal ganglion;<br />
gI, first ventral trunk ganglion; ic, inner connective; il, inner lobe; lgg, leg ganglion; mg,<br />
median ganglion; mo, mouth opening; oc, outer connective; ol, outer lobe; pc, papilla<br />
cephalica; pb, pharyngeal bulb; st, stylet; t, temporalia; vll, ventrolateral lobe. From: Paper<br />
VIII.<br />
most notably the marine genus Halobiotus (see Paper III). In general,<br />
eutardigrades are cylindrically shaped with a more or less distinct segmentation,<br />
and exhibit a relatively uniform morphology (Fig. 3A). The key characters of<br />
eutardigrades include a cloaca (combined gonopore and anus), the presence of<br />
Malpighian tubules and a strong reduction of cephalic sensory structures (Guidetti<br />
and Bertolani, 2005). Structures such as the bucco-pharyngeal apparatus and claw<br />
shape are important taxonomic characters within Eutardigrada.<br />
Ecology<br />
Tardigrades occupy a range of moisture regimes and often constitute a major<br />
component of meiofaunal communities in terrestrial, limnic and marine<br />
ecosystems throughout the globe. However, they are distinctly aquatic organisms,<br />
requiring a film of water to be active. Tardigrades are predominantly egg-laying,<br />
with both sexual and parthenogenetic modes of reproduction described (Bertolani,<br />
2001). Molting occurs continuously throughout their lifecycle, which may be<br />
between 3-30 months (Nelson, 2002). Populations of tardigrades have been<br />
studied in a variety of habitats; including mosses, lichens, leaf litter and soil, and<br />
21
Fig. 6 Population dynamics of the marine tardigrade Halobiotus crispae showing a unimodal<br />
pattern of maximal frequency. Graphic representation of sampling data (2006-2012)<br />
comparing the temporal pattern in abundance of H. crispae to abiotic parameters<br />
(temperature, (─ ─); salinity, (- - -); pH, (- ─ -), and the seasonal appearance of the different<br />
cyclomorphic stages (shown on top), from the locality of Vellerup Vig, Isefjord, Denmark.<br />
Light grey area is the period in which exuvia containing eggs were found, and thus indicates<br />
the period of sexual reproduction. From: Paper VII.<br />
the life history and population dynamics have received some attention (Martinez,<br />
1975; Morgan 1977; Guidetti et al. 1999; Uhía and Briones 2002; Jönson 2003;<br />
Suzuki 2003). Tardigrade population dynamics may show both unimodal and<br />
bimodal patterns of annual variation (Martinez, 1975; Morgan, 1977; Fig. 6),<br />
albeit the specific pattern appears to be both species and habitat specific. Factors<br />
such as temperature, moisture and food availability have been suggested to be<br />
correlated with population density (Hallas & Yeates, 1972; Morgan, 1977).<br />
However, other factors including competition, predation and parasitism may play a<br />
role in controlling population density and diversity (Nelson, 2002). Tardigrades<br />
possess an amazing reproductive capacity, as indicated by the large changes in<br />
animal density on short temporal scales (Morgan, 1977).<br />
22
Fluid and solute dynamics – an overview<br />
Inorganic ion composition<br />
Knowledge of the composition as well as concentrations of dissolved particles in<br />
internal fluids of an organism, and how these change during various exposures, is<br />
fundamental to the understanding of its basic physiology. However, practically<br />
nothing is known about these aspects in tardigrades, which has been a major<br />
obstacle to the study of the fluid and solute dynamics in these animals. In Paper I,<br />
the inorganic ion content of five different species (Echiniscus testudo, Milnesium<br />
tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus<br />
crispae) covering both a large phylogenetic and habitat spectrum was analyzed<br />
(Fig. 7A). These data demonstrated that Na + and Cl - are the principle inorganic<br />
ions in tardigrade fluids, albeit substantial concentrations of K + , NH + 4 , Ca 2+ ,<br />
Mg 2+ , F - 2-<br />
3-<br />
, SO 4 and PO 4 were also detected. Moreover, tardigrades appear to<br />
contain roughly similar relative contributions of the respective inorganic ions to<br />
total osmotic concentration, when compared to selected species of arthropods,<br />
nematodes and onychophorans (Hobson et al., 1952; Sutcliffe, 1962; Campiglia,<br />
1975; Wilder et al., 1998; Normant et al., 2005), albeit a large relative contribution<br />
of calcium appears characteristic of tardigrade fluids. Inorganic ions destabilize<br />
macromolecules and affect the rate and extent of metabolic reactions at high<br />
concentrations, which invariably leads to impairment of cellular function (Zhao,<br />
2005; Yancey, 2005). However, apparently R. coronifer does not exclude<br />
inorganic ions during dehydration (Fig. 7B), which suggests a concomitant<br />
accumulation of organic solutes. Moreover, as evidenced by the differences<br />
between the calculated osmotic concentrations of the known ions and the<br />
measured total osmolarity in the different species (Fig. 7A), it was inferred that<br />
cryptobiotic tardigrades (in steady-state) contain a large fraction of unidentified<br />
organic osmolytes. Organic osmolytes can be divided into a few major categories<br />
(sugars, polyols, amino acids and various derivatives), and several of these groups<br />
possess known protective functions in relation to osmotic stress (Hincha and<br />
Hagemann, 2004; Yancey, 2005). Accordingly, the future detection and analysis of<br />
such compounds are likely to provide new insight into the biochemistry and<br />
physiology of superior tardigrade adaptations.<br />
23
Fig. 7 Ionic contributions to total osmotic concentration A. Concentrations (mM) of the<br />
respective cations and anions measured in each investigated species, as well as the<br />
corresponding total osmotic concentration (mOsm/kg). The blank area represents the osmotic<br />
deficit (OD). B. Concentrations (mg/l) of the respective cations and anions measured in<br />
hydrated, active specimens compared to dehydrated cryptobiotic animals of Richtersius<br />
coronifer. Data are expressed as mean ± s.d. From: Paper I.<br />
24
Organic anion transport<br />
The ability to excrete endogenous<br />
waste products as well as environmental<br />
toxins is an essential step to<br />
avoid these compounds reach toxic<br />
levels and to keep metabolic reactions<br />
within optimal conditions. One of the<br />
better known systems involved in such<br />
an excretion is the organic anion<br />
transport system, the function of<br />
which has been studied in several<br />
vertebrate (fish, amphibians, reptiles,<br />
birds and mammals) and invertebrate<br />
(nematodes and insects) model<br />
organisms (George et al., 1999;<br />
Dantzler, 2002; Dow and Davies,<br />
2006). In Paper II, these data were<br />
expanded as the sites, characteristics<br />
and pharmacological profile of the<br />
transepithelial transport of chlorophenol<br />
red (CPR), a prototypical<br />
Fig. 8 Tentative cellular model for the<br />
transepithelial transport of organic anions in<br />
tardigrades. From: Paper II.<br />
substrate of the classic organic anion secretion pathway, was investigated in the<br />
tardigrade Halobiotus crispae Kristensen, 1982 and compared to corresponding<br />
data from the desert locust Schistocerca gregaria Forskål, 1775. This was done by<br />
introducing a new method for quantifying non-fluorescent dyes. Our study<br />
revealed i) that tardigrades posses an organic anion transport system, ii) that it was<br />
localized to the midgut epithelium, and iii) that organic anion secretion was both<br />
active and transporter mediated, with possible members of the SLC21/SLCO<br />
transport families mediating the basolateral entry step in tardigrade midgut cells.<br />
Transport by insect Malpighian tubules showed a similar pharmacological profile,<br />
but higher concentrations of CPR were achieved. Based on the observed transport<br />
characteristics in the presence and absence of transport inhibitors, a tentative<br />
cellular model for the transepithelial transport of CPR in tardigrades was<br />
suggested (Fig. 8). Specifically, a large lumen positive potential generated by the<br />
H + -ATPase could provide the driving force for accumulation of anions in the<br />
lumen, although the exact coupling between electrochemical gradients generated<br />
by the pumps and transport of ions is unknown. This study was the first to provide<br />
evidence for epithelial transport in tardigrades.<br />
25
Volume and osmoregulation<br />
Tardigrades survive in a variety of osmotic environments and must protect the<br />
internal tissues from the vagaries and extremes of the external environment. The<br />
secondary marine species Halobiotus crispae colonizes habitats characterized by<br />
especially large fluctuations in external salinity, why the ability to respond to an<br />
osmotic challenge was investigated in this species (Paper V). Animals were<br />
exposed to both hypo- and hyperosmotic media and the subsequent changes in<br />
total body volume and internal osmotic pressure were recorded. These data<br />
revealed that H. crispae is able to regulate total body volume back to control<br />
values when immersed in hypotonic solutions, yet was unable to do so in<br />
concentrated media. Instead a new steady-state was achieved significantly below<br />
control conditions. Animal activity was only markedly affected in very dilute<br />
media, suggesting a possible effect on neuro-muscular function at low salt<br />
concentrations. Conversely, when analyzing the concomitant changes in<br />
hemolymph concentrations, it appeared that H. crispae is a euryhaline<br />
osmoconformer, in which the hemolymph osmotic pressure is largely governed by<br />
the external environment. However, expressing the hemolymph osmolality<br />
measured during exposure to dilute as well as concentrated media as a function of<br />
external salinity revealed that H. crispae is in fact a strong hyper-regulator (Fig.<br />
9A). Expanding these studies to include the limno-terrestrial species Richtersius<br />
coronifer Richters, 1903 (Paper III) showed that this is could be a general feature<br />
of all eutardigrades (Fig. 9B), which was further supported by data presented in<br />
Paper I. The ability to hyper-regulate indicates the excretion of dilute urine. The<br />
three glands positioned at the transition zone between the midgut and rectum of<br />
eutardigrades, are generally considered to have an excretory function. This<br />
assumption is based on the probable homology of the Malpighian tubules of<br />
eutardigrades and insects (Greven, 1982; Møbjerg and Dahl, 1996), and on several<br />
ultrastructural studies that shows that the epithelium is likely involved in fluid and<br />
solute transport (Weglarska, 1987; Møbjerg and Dahl, 1996; Peltzer et al., 2007).<br />
However, data in support of an osmoregulatory function of both the rectum<br />
(Dewel and Dewel, 1979) and midgut (Paper II) also exists, which emphasizes the<br />
need for functional studies on these organs at both the molecular and cellular level.<br />
26
Fig. 9 Osmotic performance of A. Halobiotus crispae and B. Richtersius coronifer during<br />
exposure to media of varying osmotic strength. From: Paper III.<br />
27
Conclusions and future perspectives<br />
This dissertation has provided new insight into the fluid and solute dynamics of<br />
metazoans, particularly relating to one of the most enigmatic groups on the planet<br />
– the tardigrades. Using a multi-disciplinary approach, crucial information was<br />
provided on both organs and systems of several species, representing vertebrates,<br />
arthropods and tardigrades, and general patterns in especially tardigrade<br />
physiology have emerged (e.g. Paper III). By comparing our data on tardigrades<br />
to several evolutionary related groups, including nematodes, onychophorans and<br />
arthropods, basic physiological principles have been discovered (e.g. Paper II),<br />
which emphasizes the importance of comparative physiology.<br />
In this respect, future work on tardigrade physiology could encompass an<br />
extension of the work presented herein. Additional species from different habitats<br />
and evolutionary lineages should be investigated, in order to further explore the<br />
diversity as well as common trends in tardigrade biology. In particular, studies on<br />
marine cryptobionts (e.g. Echiniscoides sigismundi) and additional noncryptobiotic<br />
species would help clarify whether the osmotic deficits observed in<br />
cryptobiotic animals (Fig. 5; Paper I) in fact are related to cryptobiotic ability or<br />
alternatively to habitat preference. Regardless, large scale analyses and<br />
characterization of the organic solutes of cryptobiotic tardigrades should be<br />
performed, which surely would provide an enhanced resolution of several aspects<br />
relating to tardigrade stress responses. Other studies could include a<br />
characterization of the volume and osmoregulatory capacity of heterotardigrade<br />
species. Our results on eutardigrades show a capacity to hyper-regulate over a<br />
broad range of external salinities (Fig. 7); however, do heterotardigrades without<br />
Malpighian tubules posses the same ability True limnic species (e.g. Bertolanius<br />
nebulosus) should also be investigated. In addition to the suggested whole animal<br />
experiments, studies on the molecular and cellular level are needed to fully<br />
understand how fluid and electrolyte homeostasis is achieved in these animals. For<br />
example, electrophysiological investigations using single cell glass microelectrode<br />
impalements on dissected native tissue would help characterize the cellular<br />
transporters involved in e.g. urine formation. Collectively, these studies would be<br />
important to elucidate how tardigrades functionally have solved colonizing every<br />
major type of habitat on the planet, and perhaps be useful in reconstructing how<br />
osmoregulation has evolved in metazoans.<br />
28
Dansk sammenfatning<br />
Osmoregulering er kontrollen af kropsvæskernes sammensætning af vand og<br />
opløste stoffer. Foruden at opfylde de optimale betingelser for metaboliske<br />
processer under indflydelse af eksterne påvirkninger, skal denne<br />
sammensætning samtidig imødekomme den kontinuerlige transport af stoffer<br />
ind og ud af den levende organisme. Disse krav forekommer umiddelbart<br />
fundamentalt uforenelige, men celler og dyr opnår et såkaldt ”steady-state” ved<br />
hjælp af et spektrum af transportproteiner, der udøver streng kontrol over<br />
udvekslingen af vand og opløste stoffer over forskellige kropsoverflader. De<br />
forskellige transportmekanismer ansvarlige for denne kontrol er blevet<br />
undersøgt i de fleste dyregrupper, men vores viden om hvorledes dyr håndterer<br />
osmotisk stress, er stadig ufuldstændig. I dette arbejde er osmoregulatoriske<br />
fænomener blevet undersøgt i såvel vertebrater som invertebrater, omend der<br />
hovedsageligt er blevet fokuseret på dynamikkerne af vand og opløste stoffer i<br />
Tardigrada. Eksempelvis blev sammensætningen af uorganiske ioner undersøgt<br />
i flere forskellige arter af bjørnedyr, hvilket afslørede, at de relative bidrag af<br />
uorganiske ioner til den totale osmotiske koncentration overordnet set er ens i<br />
bjørnedyr og nært beslægtede dyregrupper. Desuden blev det udledt, at<br />
kryptobiotiske bjørnedyr (arter, der er i stand til indtræde i et stadie af latent<br />
liv) indeholder en stor andel af organiske osmolytter. Mekanismerne for<br />
aniontransport blev undersøgt farmakologisk i en marin art af bjørnedyr, og<br />
sammenlignet med de tilsvarende mekanismer i insekter. I bjørnedyret blev den<br />
organiske aniontransport lokaliseret til epitelet i midttarmen, og viste sig at<br />
være en aktiv transport, med en farmakologisk profil svarende til den i insekter.<br />
Bjørnedyr kan overleve et bredt spektrum af osmotiske miljøer (semiterrestrisk,<br />
limnisk og marine habitater), hvorfor evnen til at volumen- og osmoregulere<br />
blev undersøgt. Disse studier demonstrerede at bjørnedyr kan regulere den<br />
samlede kropsvolumen under såvel hypo- som hyperosmotiske forhold, samt<br />
indikerede at hyperregulering kan være en gennemgående tendens hos<br />
eutardigrader. Indeværende arbejder har altså bidraget til at fastslå bjørnedyr<br />
som en vigtig eksperimentel gruppe, hvori centrale fysiologiske spørgsmål kan<br />
besvares, heriblandt aspekter af ionregulering og osmoregulering.<br />
29
Acknowledgements<br />
During the course of my PhD studies I was fortunate to have the generous help<br />
of many great people, which has made this work possible.<br />
First and foremost, I thank my supervisor Nadja Møbjerg for being an<br />
unwavering source of support, encouragement and guidance during past years,<br />
but also for constantly challenging me to develop intellectually as well as a<br />
researcher. I truly appreciate all you have done for me, and hope to be able to<br />
continue our collaboration in the future.<br />
I also greatly appreciate the support of Reinhardt Møbjerg Kristensen<br />
(close to being my second supervisor) who initially introduced me to strange<br />
world of tardigrades, and who took me under his wing, offering me guidance<br />
and support (and equally important coffee). You have had enormous impact on<br />
my education as a young researcher, and I have sincerely enjoyed working with<br />
you.<br />
I have had the great fortune of working with many wonderful<br />
colleagues at University of Copenhagen, both at The August Krogh Center and<br />
the Natural History Museum who all are warmly thanked. Thanks to you it has<br />
been a pleasure coming to work every day. I am especially grateful to Erik<br />
Hviid Larsen with whom I shared many interesting discussions, and who was<br />
an endless source of inspiration. Aslak Jørgensen is thanked for great<br />
collaborations and discussions over the years as well as for critical review of<br />
my thesis. Dennis Krogh Persson is thanked for being a good colleague, but<br />
more importantly, a good friend. Since we started this journey in research<br />
together, I have come to depend on our collaborations and camaraderie, which<br />
has been an invaluable help along the way (by the way, it is your turn to buy<br />
cake). I thank Jette Lyby Michelsen for technical assistance over the years.<br />
During my visits to Roskilde University I was fortunate to collaborate<br />
with several great people. I especially thank Hans Ramløv for allowing me to<br />
come work in his laboratory (on several occasions), which scientifically turned<br />
out to be quite fruitful. I have particularly enjoyed our conversations and<br />
discussions, and I thank you for both your encouragement and support. I also<br />
thank Kristine Wulff Larsen for unparalleled project teamwork as well as our<br />
many conversations on everything and anything. It was an absolute pleasure,<br />
30
which I hope to repeat some time in the future. Peter Westh is thanked for<br />
allowing me to work in his laboratory and for his enormous expertise and help.<br />
Perhaps most importantly I thank the help and support of family and<br />
friends. I especially thank my parents and parent in-laws for their invaluable<br />
support during difficult times, and for helping me and my family in more ways<br />
than I care to mention. You’re the best! Lastly I thank my girlfriend Iben Rønn<br />
Veland for helping me, supporting me, tolerating me and loving me, but mostly<br />
for taking care of our son when I was working – none of this was possible<br />
without your help! I also thank my little son Hannibal for tolerating that I had<br />
to work sometimes, and couldn’t be home when you wanted me to. I am<br />
looking forward to making up for lost time. I love you both more than you<br />
know. Thank you.<br />
This work was funded by the 2008 Faculty of Science, University of<br />
Copenhagen Freja-Programme.<br />
31
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35
36<br />
UNIVERSITAS<br />
HAFNIENSIS<br />
2012
Paper I
Inorganic ion composition in Tardigrada: cryptobionts contain large<br />
fraction of unidentified organic solutes<br />
<strong>Kenneth</strong> Agerlin <strong>Halberg</strong> 1,* , Kristine Wulff Larsen 2 , Aslak Jørgensen 3 , Hans Ramløv 2 and<br />
Nadja Møbjerg 1<br />
1 Department of Biology, the August Krogh Centre, University of Copenhagen, Universitetsparken 13, DK-<br />
2100 Copenhagen Ø, Denmark, 2 Department of Nature, Systems and Models, University of Roskilde,<br />
Universitetsvej 1, DK-4000 Roskilde, Denmark and 3 Laboratory of Molecular Systematics, Natural History<br />
Museum of Denmark, University of Copenhagen, Sølvgade 83, DK-1307 Copenhagen K, Denmark<br />
*Author for correspondence (kahalberg@bio.ku.dk)<br />
Submitted June 2012<br />
SUMMARY<br />
Tardigrades are a group of micrometazoans known to tolerate extreme environmental stress. Significant<br />
efforts have been devoted to the field, however; mechanisms explaining the extreme adaptations found<br />
among tardigrades is still lacking. Here we present data on the inorganic ion composition and total<br />
osmotic concentration of five different species of tardigrades (E. testudo, M. tardigradum, R. coronifer, M.<br />
cf. hufelandi and H. crispae) using high-performance anion-exchange chromatography and nanoliter<br />
osmometry. Quantification of the ionic content indicates that Na + and Cl - are the principle inorganic ions<br />
in tardigrade fluids, albeit substantial concentrations of K + , NH + 4 , Ca 2+ , Mg 2+ , F - 2-<br />
, SO 4 and PO 3- 4 also<br />
were detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor<br />
compared to that of the external medium (Na + , ×70-800; K + , ×20-90; Ca 2+ and Mg 2+ , ×30-200; Cl - , ×20-50;<br />
SO 2- 4 , ×30-150), whereas in the marine species H. crispae Na + , Cl - 2-<br />
and SO 4 are almost in ionic<br />
equilibrium with (brackish) salt water, while K + , Ca 2+ and Mg 2+ are slightly concentrated (×2-10).<br />
However, there is an anion deficit of ~120 mEq/l in M. tardigradum and H. crispae, indicating that there<br />
are ionic components that remain unidentified in these species. Body fluid osmolality ranged from 361±49<br />
in R. coronifer to 961±43 mOsm/kg in H. crispae. Concentrations of most inorganic ions are largely<br />
identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not<br />
exclude large amounts of ions during dehydration. The large osmotic and ionic gradients maintained by<br />
both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in<br />
Tardigrada. Moreover, our data indicates that cryptobiotic tardigrades contain a large fraction of<br />
unidentified organic osmolytes, the identification of which is expected to provide increased insight into<br />
the phenomenon of cryptobiosis.<br />
Key words: tardigrades, inorganic ions, ion chromatography, nanoliter osmometry, organic osmolytes,<br />
cryptobiosis<br />
INTRODUCTION<br />
Tardigrades are a group of minute multi-cellular<br />
animals that are known to tolerate extreme<br />
environmental stress (Guidetti et al., 2010;<br />
Møbjerg et al., 2011). This capacity derives<br />
mainly from their ability to enter a state latent of<br />
life, i.e. cryptobiosis, in which their resistances to<br />
adverse environmental conditions are greatly<br />
increased (Møbjerg et al., 2011). In recent years,<br />
intensive research efforts have been devoted to the<br />
field, as the translational output associated with a<br />
detailed understanding of their complex stress<br />
biology is expected to include new methods for<br />
preserving and stabilizing biological materials<br />
(Wełnicz et al., 2010). Several advances have been<br />
made in our understanding of tardigrade stress<br />
responses, especially regarding i) the role of<br />
selective carbohydrates (trehalose), ii) the
K. A. <strong>Halberg</strong> and others<br />
differential expression of stress proteins (heat<br />
shock proteins and late embryogenesis abundant<br />
proteins), and iii) the identification of antioxidant<br />
defenses and DNA repair mechanisms<br />
(see reviews by Guidetti et al., 2010; Wełnicz et<br />
al., 2010; Møbjerg et al., 2011). However, a<br />
general mechanism explaining the extreme<br />
adaptations found among tardigrades is still<br />
lacking. Consequently, new approaches that may<br />
provide increased insight into the superior stress<br />
adaptations of tardigrades are greatly needed.<br />
Knowledge of the composition as well as<br />
concentrations of dissolved particles in internal<br />
fluids is fundamental to the understanding of<br />
basic physiological processes, such as fluid and<br />
electrolyte homeostasis, signal transduction and<br />
solute transport. Accordingly, such data have<br />
been provided for most major groups of animals<br />
(cnidarians, echinoderms, annelids, molluscs,<br />
crustaceans, insects, chelicerates, tunicates, fish,<br />
amphibians and mammals) more than half a<br />
century ago (Macallum, 1910; Robertson, 1949,<br />
1954; Sutcliffe, 1962; Hronoski & Armstrong,<br />
1977). However, practically nothing is known<br />
about the chemical composition of tardigrades,<br />
which has been a major obstacle to the<br />
understanding of the fluid and solute dynamics in<br />
these animals (<strong>Halberg</strong> et al., 2009b; Møbjerg et<br />
al., 2011; <strong>Halberg</strong> & Møbjerg, 2012). Questions<br />
relating to this area of research are especially<br />
important to address, if we wish to unravel the<br />
biological mechanisms mediating the unique<br />
tolerance to extreme desiccation (anhydrobiosis)<br />
– the most widespread form of cryptobiosis in<br />
Tardigrada.<br />
In the present study, we use a combination of<br />
high-performance anion-exchange chromatography<br />
(HPAEC) and nanoliter osmometry, to<br />
identify and quantify inorganic cations and anions<br />
present in tardigrade homogenates, and to<br />
measure the total osmotic concentrations of five<br />
different species of tardigrades, covering both a<br />
broad phylogenetic and habitat spectrum. Our<br />
study indicates that tardigrades possess powerful<br />
ion-retentive and osmoregulatory capacities, and<br />
that (only) cryptobiotic species contain a large<br />
fraction of organic solutes.<br />
MATERIALS AND METHODS<br />
Tardigrade sampling<br />
Specimens of Richtersius coronifer Richters,<br />
1908, Macrobiotus cf. hufelandi C.A.S. Schultze,<br />
1834 and Milnesium tardigradum Doyère, 1840<br />
were extracted from moss collected at Öland,<br />
Sweden, while Echiniscus testudo Doyère, 1840<br />
was found in moss collected at Nivå, Denmark.<br />
These species were extracted by washing the<br />
respective moss samples with tap water through<br />
six different sieves of progressively smaller mesh<br />
size, so as to concentrate the tardigrades and to<br />
remove large debris. Specimens of Halobiotus<br />
crispae Kristensen, 1982 were isolated from<br />
marine algae and sediment collected from<br />
Vellerup Vig, Denmark according to the method<br />
of <strong>Halberg</strong> and Møbjerg (2012). The total number<br />
of animals used in all experiments was 2220 R.<br />
coronifer, 326 M. tardigradum, 630 M. cf.<br />
hufelandi, 426 E. testudo and 268 H. crispae.<br />
Inorganic cation and anion analysis<br />
The dominant cations and anions present in the<br />
different tardigrade species were determined by<br />
HPAEC using a Metrohm chromatography system<br />
(830 IC interface, 818 IC pump, 819 IC<br />
conductivity detector, columns C4 - 150/4.0<br />
(cations) and A supp 5 150/4.0 (anions); Metrohm,<br />
Herisau, Switzerland). The eluents (mobile<br />
phases) were made according to manufacturer’<br />
instructions: For cations, the eluent consisted of<br />
0.7 mM C 7 H 5 NO 4 + 1.7 mM (65%) HNO 3 . For<br />
anions, it consisted of 3.2 mM Na 2 CO 3 + 1.0 mM<br />
NaHCO 3 . The eluents were filtered (mesh size: 45<br />
µm) prior to use. The analysis settings employed<br />
were a flow-rate of 0.9 ml/min (cations) and 0.7<br />
ml/min (anions) with a pressure of ~6.4 MPa.<br />
Cation analyses were performed non-suppressed,<br />
whereas anion detection was conducted using<br />
chemical suppression. Fluka multi-element cation<br />
and anion standards (Sigma-Aldrich, St. Louis,<br />
MO, USA) were used to construct calibration<br />
curves for the respective ions bracketing the<br />
concentration range of interest. Based on these<br />
calibration curves, the ion chromatography (IC)<br />
software (IC Net 2.3, Metrohm, Herisau,<br />
Switzerland) calculated the ion concentrations of<br />
all subsequent samples (mg/l), which were<br />
recalculated to a different unit of concentration<br />
(mM) and adjusted according to the appropriate<br />
dilution factor (see below). Representative<br />
chromatograms of both the cationic and anionic<br />
fractions are shown for all investigated species<br />
(Figs. 1, 2). The empirically determined elution<br />
order and retention times of the investigated ions<br />
were Na + (t R = 5.37 min), NH 4 + (t R = 6.03 min),
Inorganic ion composition in Tardigrada<br />
Fig. 1. Representational chromatograms revealing the principal inorganic cations present in each investigated species:<br />
1, sodium; 2, ammonium; 3, potassium; 4, calcium; 5, magnesium; nOAp, negative organic acid peak. Stitched square<br />
indicates an unidentified compound (t R = 10.36 min) that increases app. two-fold in absolute concentration in<br />
dehydrated animals of Richtersius coronifer (data not shown). Column, Metrohm C4-150/4.0; mobile phase, 0.7 mM<br />
dipicolinic acid + 1.7 mM (65%) nitric acid; flow-rate, 0.9 ml/min; Conductivity detector without suppression.<br />
Injection volume, 60 µl.<br />
K + (t R = 7.73 min), Ca 2+ (t R = 18.12 min), Mg 2+<br />
(t R = 23.17 min), F - (t R = 4.05 min), Cl - (t R = 6.01<br />
min), PO 4 3- (t R = 14.74 min) and SO 4 2- (t R = 16.58<br />
min).<br />
Sample preparation<br />
Following extraction, specimens were washed<br />
repeatedly with ddH 2 O (Halobiotus crispae was<br />
washed in filtered salt water; SW, 20 ‰), and<br />
subsequently transferred, using an Irwin loop, to<br />
sample tubes containing cation eluent (75-100<br />
µl); samples dissolved in cation eluent allows for<br />
a more precise quantification of cations due to<br />
increased signal to noise ratio (pers. comm.;<br />
Metrohm Nordic, Denmark). Prior to transfer,<br />
surface water was removed by blotting the<br />
animals with tissue paper in an attempt to avoid<br />
unwanted dilution of the samples. Pilot<br />
experiments revealed that this process was critical<br />
to acquire reproducible data, and was accordingly<br />
performed as fast and uniform as possible. A total<br />
of 40-225 animals were transferred to each test<br />
tube and the sample was subsequently<br />
homogenized using a sterile plastic pestle; great<br />
care was taken to ensure complete homogenization<br />
(visually confirmed at 50× magnification), and the<br />
pestle was subsequently rinsed with a small<br />
volume of cation eluent to ensure total transfer of<br />
ions to the test tube. The number of animals per<br />
sample (N) varied according to species size and<br />
availability (Tables 1, 2). The entire sample was<br />
then centrifuged (10 min at 5600 rpm) to remove<br />
solid particles (e.g. cuticle fragments), and the<br />
supernatant was filtered (mesh size: 0.20 µm)<br />
using a single use syringe filter (Sartorius AG,<br />
Göttingen, Germany). The samples were<br />
subsequently frozen at -20 ºC, if not quantified<br />
immediately. A total of five to seven samples were<br />
prepared for each species (Table 2).
K. A. <strong>Halberg</strong> and others<br />
Fig. 2. Representational chromatograms revealing the principal inorganic anions present in each investigated species:<br />
6, fluoride; 7, chloride; 8, phosphate; 9, sulfate; W, negative water peak. Stitched square indicates an unidentified<br />
compound (t R = 7.24 min) that increases app. two-fold in absolute concentration in dehydrated animals of Richtersius<br />
coronifer (data not shown). Column, Metrohm A supp 5 150/4.0; mobile phase, 3.2 mM sodium carbonate + 1.0 mM<br />
sodium hydrogen carbonate; flow-rate, 0.7 ml/min; Conductivity detector with chemical suppression. Injection<br />
volume, 60 µl.<br />
The ionic concentration and composition of<br />
the external media from the different habitats i.e.<br />
moss water (MW) and SW, were additionally<br />
determined. Moss samples were rehydrated in<br />
ddH 2 O for several hours, and MW samples were<br />
subsequently collected from between the leafcovered<br />
stems. SW samples were prepared by<br />
diluting SW (1:200) collected at the locality.<br />
Samples were quantified in triplets using both<br />
vapor pressure osmometry (Vapro 5520, Wescor<br />
inc., UT, USA) and HPAEC (Table 3).<br />
In order to document whether changes in<br />
inorganic ion content occur during dehydration<br />
from an active to a cryptobiotic state, samples of<br />
dehydrated Richtersius coronifer were<br />
additionally prepared. Groups of 75 animals were<br />
transferred to each sample tube, and excess water<br />
was removed by blotting the animals with tissue<br />
paper. The animals were subsequently allowed to<br />
dehydrate over the ensuing 24 h at ambient<br />
temperature and humidity. Following complete<br />
dehydration, tissue paper saturated with ddH 2 O<br />
was used to rinse the surface of the animals, and a<br />
dry tissue paper was used to remove excess<br />
moisture. This was done in order to remove<br />
potential solutes extruded on the surface of the<br />
animals – the surface of some animals was<br />
inaccessible due to animal clumping, and therefore<br />
could not be rinsed. A volume of 90 µl of cation<br />
eluent was added, and the animals were<br />
immediately homogenized. The samples were then<br />
prepared as described above with six samples<br />
prepared in total (Table 2). Data (mg/l) from this<br />
experiment was directly compared to that of<br />
hydrated animals (Table 4; Fig. 3B), as both sets<br />
of samples contained near identical number of<br />
animals per unit volume (i.e. 0.81 and 0.83<br />
animals/µl eluent respectively), which<br />
circumvented the need for recalculations (see<br />
below). In order to test whether R. coronifer<br />
actually produced viable tuns during the<br />
abovementioned conditions, post-cryptobiotic
Inorganic ion composition in Tardigrada<br />
survival was assessed. Using four groups of 50<br />
specimens, a survival rate of 94 ± 4% (mean ±<br />
s.d.) was observed, which is comparable to the<br />
maximally reported survival rate of R. coronifer<br />
dehydrated on Whatman filters (Persson et al.,<br />
2010).<br />
Calculation of ion concentrations<br />
The IC software expressed the integration of<br />
peaks as a concentration (mg/l), which was<br />
recalculated using the respective molecular<br />
weights of each compound to a different<br />
concentration (mM) prior to adjusting for the<br />
dilution factors.<br />
The volume of each investigated species was<br />
calculated according to an adjusted method of<br />
<strong>Halberg</strong> et al. (2009b). In brief, micrographs were<br />
taken of N = 20 animals of each species, using a<br />
digital camera (C-5050, Olympus, Japan)<br />
mounted on an Olympus BX 51microscope<br />
(Olympus, Japan), and median length (h) and<br />
width (2r) of the trunk and legs were measured.<br />
Approximating the geometric shape of the trunk<br />
and legs as a cylinder, and adjusting the volume<br />
of liquid according to the gravimetrically<br />
measured water content (based on Westh and<br />
Kristensen, 1992; <strong>Halberg</strong> et al., 2009b), the fluid<br />
volume of an individual tardigrade of each<br />
species was calculated using equation (1).<br />
(Eq 1) V individual = π(r 2 trunkh trunk + 8r 2 legh leg ) × W<br />
Where V individual is the volume of an individual<br />
tardigrade, r is the radius and h the length of the<br />
trunk and hind legs respectively, while W (0.72 i.e.<br />
mean fractional water content of R. coronifer and<br />
H. crispae) is the gravimetrically measured<br />
fractional water content. Using these data (Table<br />
1), the total tardigrade test volume was calculated<br />
by multiplying the volume of an individual with<br />
the number of animals included in the sample<br />
according to equation (2).<br />
(Eq 2) V total = V individual × N<br />
V total is the total tardigrade test volume, and N the<br />
number of animals included in the sample. Lastly,<br />
the concentrations of the dominant cations and<br />
anions in the investigated species of tardigrades<br />
were calculated by multiplying the measured ion<br />
concentrations with the dilution factor, which was<br />
calculated according to equation (3).<br />
(Eq 3) D = F / V total<br />
Where D is the dilution factor and F is the final<br />
volume (i.e. volume of cation eluent the<br />
tardigrades were transferred to + V total ). Sample<br />
information for the respective species is listed in<br />
Table 2.<br />
Nanoliter osmometry<br />
The total osmotic concentration of tardigrades<br />
from each investigated species was estimated<br />
using nanoliter osmometry. This was done in order<br />
to determine the fraction that the identified<br />
Table 1. Volume estimations. Mean values of length (h) and width (2r) of the trunk and legs (N = 20 animals), as well<br />
as the calculated volume (Eq 1), of each investigated species. W is the average of the gravimetrically determined<br />
water content (72%) of Richtersius coronifer (Westh and Kristensen, 1992) and Halobiotus crispae (<strong>Halberg</strong> et al.,<br />
2009). Data are expressed as mean ± s.d.
K. A. <strong>Halberg</strong> and others<br />
inorganic ions constitute of the total osmotic<br />
concentration in each species, and to provide an<br />
independent verification of our HPAEC data, i.e.<br />
total osmotic concentration should be higher than<br />
the accumulated concentration of the respective<br />
inorganic ions (Table 3). Using the same<br />
procedure for removing excess water as described<br />
above, individual specimens were transferred into<br />
sample oil wells (loading oil type B; cST=1250 ±<br />
10%; Cargille laboratories, Cedar grove, NJ<br />
07009, USA) of a calibrated nanoliter osmometer<br />
(Clifton Technical Physics, Hartford, NY, USA),<br />
and the osmolality (mOsm/kg) was determined by<br />
freezing point depression (FPD = 1.858<br />
°C/Osmol). Six to ten animals of each species<br />
were used in this experiment (Table 3).<br />
Statistics<br />
Significant changes in the individual inorganic<br />
ion concentrations between active and<br />
cryptobiotic animals of Richtersius coronifer<br />
were tested using an unpaired, two-sample t-test<br />
with significance levels of P>0.05 (not<br />
significant, NS), P
Inorganic ion composition in Tardigrada<br />
Figure 3. Graphical representation of the respective ionic contributions to total osmotic concentration. A.<br />
Concentrations (mM) of the respective cations and anions measured in each investigated species, as well as the<br />
corresponding total osmotic concentration (mOsm/kg), as measured by nanoliter osmometry (see also Table 3). The<br />
blank area represents the osmotic deficit (OD), i.e. other solutes. The phylogenetic position and habitat preference of<br />
each species is listed. The light micrographs of the animals are shown to scale (see Table 1 for average species size).<br />
B. Concentrations (mg/l) of the respective cations and anions measured in hydrated, active specimens compared to<br />
dehydrated cryptobiotic animals of Richtersius coronifer (see also Table 4). Data are expressed as mean ± s.d.
K. A. <strong>Halberg</strong> and others<br />
Halobiotus crispae containing the lowest and<br />
highest concentrations respectively (Fig. 3A;<br />
Table 3). There are notable differences in the Na + /<br />
Cl - ratio between the animals, i.e., the ratio is less<br />
than unity in the limno-terrestrial herbivores E.<br />
testudo (0.61), R. coronifer (0.60) and M. cf.<br />
hufelandi (0.33), higher than unity in the limnoterrestrial<br />
predator M. tardigradum (1.19) and<br />
close to unity in the marine herbivore H. crispae<br />
(0.92).<br />
Compared with Na + and Cl - , generally, the<br />
[K + ] is relatively low in all species, ranging from<br />
19-73 mM (Tab. 3). Thus, the Na + / K + ratio is<br />
higher than unity in E. testudo (2.39), M.<br />
tardigradum (2.05), R. coronifer (1.32) and H.<br />
crispae (6.12), however; lower than unity in M.<br />
cf. hufelandi (0.77). Interestingly, the relative<br />
contribution of K + to total osmotic concentration<br />
is low constituting
Inorganic ion composition in Tardigrada<br />
Table 3. Ionic composition and total osmotic concentration of the investigated species of tardigrades and the corresponding external media. Concentrations (mM) of<br />
cations and anions detected in each investigated species, as well as moss water (MS) and 20 ‰ salt water (SW) samples, in addition to the corresponding total osmotic<br />
concentration (mOsm/kg), as measured by nanoliter osmometry or vapor pressure osmometry, respectively. In addition, the osmotic deficits (calculated as the<br />
difference in ionic concentration, and total osmotic concentration), as well as the observed charge deficits (calculated as the difference between positive and negative<br />
charges) are listed; the polarity of the charge deficits is indicated in parenthesis. Numbers noted in brackets indicates the number of samples tested. Data are expressed<br />
as mean ± s.d.
K. A. <strong>Halberg</strong> and others<br />
In the present study we provide data on the ionic<br />
composition of five different species of<br />
tardigrades covering a large phylogenetic<br />
spectrum. Our study is represented by members<br />
of Heterotardigrada (Echiniscoidea) and<br />
Eutardigrada (Apochela and Parachela), four<br />
evolutionary distant families (Echiniscidae,<br />
Milnesiidae, Macrobiotidae and Hypsibiidae), as<br />
well as both limno-terrestrial and marine habitats.<br />
Accordingly, we will discuss the ionic<br />
compositions of the respective species in relation<br />
to systematic position and habitat preference, as<br />
well as make comments on our data in relation to<br />
hemolymph composition in representatives of<br />
phylogenetically related groups (i.e. Arthropoda<br />
and Onychophora).<br />
Echiniscus testudo (Heterotardigrada:<br />
Echiniscidae) belongs to another evolutionary<br />
lineage than the other tardigrades in the present<br />
study. Compared to limno-terrestrial members of<br />
Eutardigrada, the ionic composition of this<br />
heterotardigrade is characterized by a large<br />
contribution of Na + and Cl - (~45%), and a very<br />
low contribution of Mg 2+ 2-<br />
(0.4%), SO 4 (0.6%)<br />
and PO 3- 4 (1.4%), respectively (Tab. 5). The large<br />
contribution of Na + and Cl - to total osmotic<br />
concentration, which is comparable to that seen in<br />
the marine species H. crispae (Table 5), could<br />
reflect the supposed marine origin of tardigrades<br />
(Jørgensen et al., 2010). This hypothesis can be<br />
tested by data on members of the ‘ancient’ and<br />
exclusively marine Arthrotardigrada.<br />
The family Milnesiidae, represented by the<br />
predator Milnesium tardigradum, is currently<br />
considered the sister-group of all other<br />
eutardigrades (Guidetti et al., 2009). M.<br />
tardigradum contains the highest total osmotic- as<br />
well as ionic concentration among the limnoterrestrial<br />
species, with conspicuously high levels<br />
of both K + and Ca 2+ (Table 3). The high [K + ] in<br />
M. tardigradum compared to the phytophagous<br />
species is somewhat surprising, as e.g.<br />
carnivorous insects typically contain low levels of<br />
K + (Sutcliffe, 1962). Conversely, phytophagous<br />
tardigrades are known to feed on bryophytes high<br />
in K + and low in Na + (Smith, 1978), and were,<br />
analogous to phytophagous insects (Sutcliffe,<br />
1962), expected to reflect this relative ion<br />
composition in their extracellular body fluids.<br />
Interestingly, the relative ion contributions to<br />
total osmotic concentration suggests that M.<br />
tardigradum resembles the heterotardigrades more<br />
than the other eutardigrades (Table 5)<br />
Sutcliffe (1962, 1963) argued that definitive<br />
types of hemolymph are related to phylogenetic<br />
position within Insecta. Apart from small<br />
differences in absolute concentrations of Na + and<br />
PO 3- 4 , the ionic compositions and relative<br />
contributions of the different components in<br />
Richtersius coronifer and Macrobiotus cf.<br />
hufelandi (Eutardigrada: Macrobiotidae) are<br />
similar (Tables. 3 and 5). As a testable hypothesis,<br />
these similarities would suggest that the relative<br />
ion composition among the species relates to<br />
phylogeny and systematic position in Tardigrada.<br />
In contrast to the other groups of limno-terrestrial<br />
tardigrades, the inorganic content of R. coronifer<br />
and M. cf. hufelandi is characterized by a<br />
relatively small contribution of Na + (~2-7 times<br />
lower) and Cl - (~2-3 times lower), and conversely,<br />
2-<br />
a relatively large contribution of SO 4 (~4-10<br />
times higher). The physiological significance of<br />
these variations is unknown.<br />
Halobiotus crispae (Eutardigrada:<br />
Hypsibiidae) is a truly marine species, and is the<br />
species with the highest total concentration of both<br />
ions and total solutes measured. Na + and Cl -<br />
account for more than 50% of its total osmotic<br />
concentration. The divalent cations, Ca 2+ and<br />
Mg 2+ , are also detected in high concentrations,<br />
both absolute and relative. In contrast to the<br />
limno-terrestrial species, the total osmotic<br />
concentration of H. crispae is almost exclusively<br />
accounted for by the measured ionic<br />
concentrations, which becomes evident when<br />
considering the charge deficit indicated in Table 3.<br />
The contribution of the total diffusible ions to<br />
the total osmotic concentration of tardigrades is<br />
roughly similar to that of the hemolymph of<br />
arthropods, nematodes and onychophorans (Table<br />
5). In fact, as Na + predominantly is an<br />
extracellular ion, whereas K + and Ca 2+ mainly are<br />
intracellular ions, the ionic composition of<br />
tardigrade hemolymph is expected to resemble<br />
that of closely related animal groups, e.g. other<br />
members of Panarthropoda (see Table 5).<br />
It is relevant to compare concentrations of ions<br />
in tardigrade body fluids with those of the<br />
respective external media (Table 3). The strongest<br />
ability to concentrate ions is seen in limnoterrestrial<br />
species, which hyper-regulate by as<br />
much as ~350-750 mOsm/kg (Fig. 3A). The<br />
marine H. crispae maintains hemolymph osmotic
Inorganic ion composition in Tardigrada<br />
Table 4. Changes in ionic<br />
composition during dehydration.<br />
Concentrations (mg/l) of<br />
the respective cations and<br />
anions measured in hydrated,<br />
active specimens compared to<br />
dehydrated cryptobiotic animals<br />
of Richtersius coronifer. Both<br />
sets of samples contained near<br />
identical number of animals per<br />
unit volume (i.e. 0.8 animals/µl<br />
eluent), and were accordingly<br />
directly comparable. Numbers<br />
noted in brackets indicates the<br />
number of samples tested. Data<br />
are expressed as mean ± s.d.<br />
Significant difference in the<br />
concentration of the respective<br />
inorganic ion concentrations<br />
were tested using an unpaired,<br />
two-sample t-test with<br />
significance levels of P>0.05<br />
(not significant, NS), P
K. A. <strong>Halberg</strong> and others<br />
intracellular osmotic potential so that osmotic<br />
equilibrium between intra- and extracellular<br />
fluids is maintained. Moreover, organic<br />
osmolytes are known to stabilize macromolecular<br />
structures by direct interaction with proteins and<br />
membrane lipids (Crowe et al., 1987; Hincha and<br />
Hagemann, 2004; Yancey, 2005). As evidenced<br />
by the large osmotic deficits, which appears<br />
restricted to cryptobiotic tardigrades (Fig. 3A;<br />
Table 3), it is tempting to suggest that a large<br />
quantity of organic osmolytes are synthesized in<br />
these species, thus enabling the animals to<br />
respond quickly to decreases in external water<br />
potential. Such a strategy seems favorable in light<br />
of the continuous dehydration-rehydration cycles<br />
that may occur in limno-terrestrial habitats,<br />
additionally supported by the short time span<br />
(
Table 5. Total osmotic concentration (mOsm/kg) of the external medium and internal body fluids, as well as the osmotic contribution (%) of the respective ions to the<br />
internal concentration in each of the investigated species of tardigrades. Corresponding data on hemolymph concentration and composition of selected species of<br />
nematodes, crustaceans, insects and onychophorans is included for comparative purposes. TR, terrestrial; ─, not measured.<br />
Inorganic ion composition in Tardigrada
K. A. <strong>Halberg</strong> and others<br />
We would like to thank Anne Lise Maarup for technical<br />
assistance and Reinhardt Møbjerg Kristensen (Natural History<br />
Museum of Denmark) for loan of the Olympus BX 51<br />
microscope. Station Linné (Porten til Alvaret), Ölands Skogsby,<br />
Sweden is warmly thanked for accommodation during sampling<br />
of tardigrades. Funding came from the Carlsberg Foundation<br />
and the Freja-Programme (Faculty of Science, University of<br />
Copenhagen).<br />
FPD<br />
HPAEC<br />
IC<br />
MW<br />
SW<br />
LIST OF ABBREVIATIONS<br />
freezing point depression<br />
high-performance anion-exchange chromatography<br />
ion chromatography<br />
moss water<br />
salt water<br />
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Inorganic ion composition in Tardigrada
Paper II
497<br />
The Journal of Experimental Biology 215, 497-507<br />
© 2012. Published by The Company of Biologists Ltd<br />
doi:10.1242/jeb.065987<br />
RESEARCH ARTICLE<br />
First evidence of epithelial transport in tardigrades: a comparative investigation of<br />
organic anion transport<br />
<strong>Kenneth</strong> Agerlin <strong>Halberg</strong>* and Nadja Møbjerg<br />
Department of Biology, The August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø,<br />
Denmark<br />
*Author for correspondence (kahalberg@bio.ku.dk)<br />
Accepted 31 October 2011<br />
SUMMARY<br />
We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for<br />
quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from<br />
Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, indicating that<br />
organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP<br />
(1mmoll –1 ; 81% reduction), the Na + /K + -ATPase inhibitor ouabain (10mmoll –1 ; 21% reduction) and the vacuolar H + -ATPase inhibitor<br />
bafilomycin (5mmoll –1 ; 21% reduction), and by the organic anions PAH (10mmoll –1 ; 44% reduction) and probenecid (10mmoll –1 ; 61%<br />
reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile,<br />
albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalization of the Na + /K + -ATPase -subunit<br />
in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalization<br />
data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter<br />
mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our<br />
results, we cautiously suggest that apical H + and possibly basal Na + /K + pumps provide the driving force for the transport; the exact<br />
coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit<br />
step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades.<br />
Key words: organic anion transport, Chlorophenol Red, 2,4-dinitrophenol, ouabain, bafilomycin, probenecid, para-aminohippuric acid, tardigrade,<br />
insect, V-type H + -ATPase, Na + /K + -ATPase, Malpighian tubule.<br />
INTRODUCTION<br />
The ability to excrete metabolic waste products as well as<br />
environmental toxins (xenobiotics) is a fundamental prerequisite<br />
for animal life. One of the earliest identified systems involved in<br />
such an excretion is the classic organic anion transport system<br />
(Marshall and Vickers, 1923). In vertebrates, this system is<br />
(especially) well known in the proximal tubule where it rids the<br />
organism of various xenobiotic and endobiotic compounds,<br />
through an ATP-dependent, net transepithelial secretory pathway<br />
(reviewed by Dantzler, 2002; Burckardt and Burckardt, 2003;<br />
Russel, 2010). The importance and understanding of similar<br />
transport activities in various invertebrate phyla, however, is<br />
extremely limited. In this study, we expand on the current<br />
knowledge by investigating organic anion transport in the<br />
tardigrade Halobiotus crispae (Tardigrada), as compared with the<br />
desert locust Schistocerca gregaria (Arthropoda).<br />
The phylum Tardigrada consists of a group of minute, eight-legged,<br />
multicellular animals that, like arthropods and nematodes, belong to<br />
the invertebrate superclade Ecdyzosoa (Aguinaldo et al., 1997). They<br />
are considered essential to our understanding of early metazoan<br />
evolution, yet fundamental questions concerning their basic biology<br />
remain unanswered (Møbjerg et al., 2011). In spite of their small size<br />
(~50–1200mm), tardigrades are relatively complex animals; they are<br />
composed of >1000 cells, and possess a well-developed musculature<br />
and nervous system, as well as a complex alimentary canal and<br />
specialized reproductive and excretory organs (Dewel and Dewel,<br />
1979; Rebecchi and Bertolani, 1994; Møbjerg and Dahl, 1996;<br />
Schmidt-Raesa and Kulessa, 2007; Pelzer et al., 2007; Zantke et al.,<br />
2008; <strong>Halberg</strong> et al., 2009a; Møbjerg et al., 2011; Rost-Roszkowska<br />
et al., 2011). The alimentary canal can be divided into several<br />
morphologically distinct regions i.e. bucco-pharyngeal apparatus,<br />
oesophagus, midgut and hindgut, with the Malpighian tubules (MTs)<br />
of eutardigrades positioned at the junction between these last two<br />
sections. Interestingly, the same basic organizational pattern is found<br />
in insects, and has been used as a strong argument for the homology<br />
of these two organ systems (Greven, 1982; Møbjerg and Dahl, 1996).<br />
Among multi-cellular animals, tardigrades exhibit an extraordinary<br />
ability to resist environmental extremes, and are known to survive<br />
conditions greatly exceeding those encountered in their natural habitat<br />
– even in space (Jönsson et al., 2008; Rebecchi et al., 2008; Persson<br />
et al., 2011). The biochemical and physiological mechanisms<br />
mediating this unique tolerance, however, remain largely unidentified.<br />
Previously, we have shown that the marine eutardigrade H. crispae<br />
tolerates large changes in external salinity surviving periods of osmotic<br />
stress by maintaining haemolymph osmotic pressure above that of<br />
the external medium (<strong>Halberg</strong> et al., 2009b); an adaptive mechanism<br />
likely present in all eutardigrades (Møbjerg et al., 2011). Here, we<br />
identify organs involved in transepithelial transport of organic anions<br />
and investigate the transport characteristics with the aim of providing<br />
a better understanding of the unique biology in these animals.<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
498<br />
K. A. <strong>Halberg</strong> and N. Møbjerg<br />
Organic anion transport has previously been described in other<br />
groups of invertebrates (George et al., 1999; Torrie et al., 2004;<br />
Faria et al., 2010). Notably, the alimentary canal and MTs of insects,<br />
which collectively form the functional analogue of the vertebrate<br />
kidney, have been studied (reviewed by Phillips, 1981; O’Donnell<br />
et al., 2003; Dow and Davies, 2006). As in vertebrates, the excretory<br />
organs of insects transport a wide range of organic solutes and<br />
exogenous toxins through organic anion transporters (Oats), organic<br />
anion-transporting peptides (Oatps), P-glycoproteins (Mdr/P-gp) as<br />
well as multidrug resistance-associated proteins (Mrps) (Maddrell<br />
et al., 1974; Bresler et al., 1990; Lanning et al., 1996; Linton and<br />
O’Donnell, 2000; Torrie et al., 2004; Neufeld et al., 2005; Leader<br />
and O’Donnell, 2005; O’Donnell and Leader, 2006; Chahine and<br />
O’Donnell, 2009; Chahine and O’Donnell, 2010). Organic anions<br />
are divided into type I and type II organic anions, on the basis of<br />
structural and chemical properties (e.g. Wright and Dantzler, 2004).<br />
The different groups of transporters vary in their transport<br />
mechanisms, but overlap in their substrate specificity, as they<br />
transport carboxylates and sulphonates interchangeably (Neufeld et<br />
al., 2005; Chahine and O’Donnell, 2009). Oats and Oatps (solute<br />
carriers belonging to the SLC22 and SLC21/SLCO family) transport<br />
both small (450Da) hydrophobic type II organic anions (Oatps),<br />
whereas Mdr/P-gp and Mrps (ABC transporters, ABCB and ABCC<br />
subfamilies) generally transport large (>500Da) polyvalent type II<br />
organic anions (Wright and Dantzler, 2004; Russel, 2010). From<br />
the insect’s perspective, the clearance of exogenous toxins is of<br />
particular interest, as insects often live in environments with high<br />
xenobiotic exposure, potentially at harmful or lethal concentrations.<br />
Accordingly, they are forced to process naturally occurring plant<br />
toxins (Torrie et al., 2004; Neufeld et al., 2005), as well as<br />
anthropogenic contaminants, such as insecticides (Lanning et al.,<br />
1996; Neufeld et al., 2005; Buss and Callaghan, 2008). The<br />
physiological importance of this detoxification system – and<br />
implicitly the MTs – is emphasized by the fact that transcripts for<br />
these transporters are enriched in the transcriptome of the MTs<br />
(Wang et al., 2004), and because the expression of several Oatp and<br />
Mdr transporters is significantly upregulated upon dietary exposure<br />
to Oatp and Mdr substrates (Mulenga et al., 2008; Chahine and<br />
O’Donnell, 2009; Chahine and O’Donnell, 2010). Indeed, it has been<br />
suggested that organic solute excretion is the most significant<br />
function of the insect MT (Dow and Davies, 2006). Nevertheless,<br />
in spite of significant efforts in the past decades, the mechanisms<br />
underlying transepithelial transport of organic anions is far from<br />
understood, and our knowledge is limited to relatively few taxa.<br />
In this study we examined epithelial transport in tardigrades.<br />
Using a comparative approach, we investigated the sites,<br />
characteristics and pharmacological profile of the net transepithelial<br />
transport of Chlorophenol Red (CPR; 3,3-dichlorophenolsulphone-phthalein),<br />
a pH indicator and a prototypic substrate of<br />
the classic organic anion secretory pathway, in the tardigrade H.<br />
crispae and the desert locust S. gregaria. Our results show that the<br />
tardigrade midgut is the principal site of CPR transport, and that<br />
this transport is active and transporter mediated. Additionally, our<br />
data show that the pharmacological profiles of CPR transport in the<br />
tardigrade midgut and locust MT are surprisingly similar.<br />
MATERIALS AND METHODS<br />
Quantification of CPR accumulation<br />
The quantification of CPR accumulation was performed by<br />
introducing a new method for quantifying non-fluorescent dyes. The<br />
method exploits the optical properties of CPR by relating relative<br />
Difference in red spectrum/green spectrum<br />
relationship (%)<br />
6000<br />
5000<br />
4000<br />
3000<br />
2000<br />
1000<br />
0<br />
pH 6<br />
r 2 =0.994<br />
pH 7–10<br />
r 2 =0.987<br />
0 1 2 3 4 5<br />
[CPR] (mmol l –1 )<br />
pH 11<br />
r 2 =0.996<br />
Fig.1. Standard curves showing the percentage difference in the red<br />
spectrum/green spectrum relationship as a function of Chlorophenol Red<br />
(CPR) concentration at different pH values (pH6, 7–10 and 11). The curves<br />
were fitted by regression analysis using OriginPro 7.5 with a third order<br />
polynomial providing the best fit with r 2 values close to unity.<br />
changes in spectral light to that of dye concentration (Fig.1). As<br />
CPR exhibits a pH-dependent shift in colour (from yellow to violet)<br />
between pH4.6 and 7.0, and a secondary shift (to purple) at pH>10,<br />
a standard curve was constructed at three discrete colours relevant<br />
to our investigation (red, violet and purple corresponding to<br />
pH6–11). This was done in order to correct for potential pH<br />
dependent effects on CPR quantification caused by the tissue. The<br />
standard curves were constructed from optical analysis of<br />
micrographs taken of samples (40ml) with known dye concentrations<br />
(ranging from 0.1 to 5mmoll –1 ) at each respective pH value. The<br />
optical measurements provided relative, arbitrary values for the light<br />
intensities of the red, green and blue spectrum of spectral light from<br />
each sample, and were acquired using the image analysis and<br />
visualization software Imaris 6.4 (Bitplane, Zurich, Switzerland).<br />
The standard curves are expressed as percentage difference in the<br />
red spectrum/green spectrum relationship as a function of CPR<br />
concentration (Fig.1). The curves were fitted by regression analysis<br />
using OriginPro 7.5 (OriginLab, Northampton, MA, USA) with a<br />
third order polynomial providing the best fit with r 2 values close to<br />
unity. The CPR concentration in a given tissue was subsequently<br />
calculated by employing the appropriate standard curve. The<br />
standard curve was chosen by visually comparing the colour of the<br />
accumulated CPR with that of the respective standard curves.<br />
Contrary to our expectations, the midgut of H. crispae was the<br />
only organ in which CPR accumulation was clearly visualized.<br />
No accumulation was observed in the tardigrade MTs. As such,<br />
quantification of dye accumulation was only performed from the<br />
tardigrade midgut, and compared with the accumulation in the<br />
MTs of S. gregaria. Specifically, 6–12 regions from areas of the<br />
tardigrade midgut containing the highest dye intensity were<br />
selected arbitrarily, in addition to a similar number from the<br />
adjacent haemolymph, while four different regions were selected<br />
from each insect MT. Each region selected represented a circle<br />
with a diameter of 5mm for H. crispae and 25mm for S. gregaria.<br />
The average light intensity within these circles was measured by<br />
the Imaris program. Averaging the measured intensity of all<br />
selected regions provided an overall average intensity for the red,<br />
green and blue colour spectrum within each investigated organ<br />
following each exposure. As CPR predominantly appears red (to<br />
violet) in the midgut of H. crispae and in the MTs of S. gregaria,<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Epithelial transport in tardigrades<br />
499<br />
the red spectrum/green spectrum relationship offered an estimate<br />
of dye accumulation, as calculated from the appropriate standard<br />
curve. The final CPR concentration was normalized according to<br />
the background light intensity, i.e. the tardigrade haemolymph or<br />
untreated (control) insect MTs. For tardigrades, only regions<br />
without gut content were selected for quantification, in order to<br />
avoid the influence of gut content on the wavelength of captured<br />
light. Deviations in animal depth did not influence CPR<br />
quantification within the variation encountered in this study. The<br />
MTs of S. gregaria consist of three structurally distinct regions,<br />
i.e. proximal, middle and distal relative to the gut (Garret et al.,<br />
1988). Dye accumulation was estimated from the proximal region<br />
and parts of the middle region within 5mm of the junction with<br />
the gut, as dye accumulation was highest here.<br />
Test solutions<br />
The haemolymph osmolality of H. crispae, kept at a salinity of<br />
20p.p.t., was previously measured by nanolitre osmometry to<br />
~950mOsmkg –1 (<strong>Halberg</strong> et al., 2009b). At present we do not know<br />
the composition of tardigrade extracellular fluids. As such, the<br />
experimental solution (control solution) was prepared from<br />
evaporative reduction of seawater (SW; salinity 20p.p.t., pH8)<br />
collected at the locality, and 35mmoll –1 glucose was added to<br />
alleviate potential variation in experiments caused by differences<br />
in nutrient availability. This yielded a final measured osmolality of<br />
950±3mOsmkg –1 (N3). For S. gregaria an insect saline (control<br />
solution) was prepared containing (in mmoll –1 ): 130 NaCl, 10 KCl,<br />
4 NaHCO 3 , 2 MgSO 4 , 2 CaCl 2 , 6 NaH 2 PO 4 , 35 glucose and 5 Hepes,<br />
titrated to pH7.2, with a measured osmolality of 336±2mOsmkg –1<br />
(N3).<br />
In order to explore the kinetics of CPR transport by MTs of S.<br />
gregaria, a concentration–response curve was constructed over a<br />
5000-fold range of CPR concentrations (1mmoll –1 to 5mmoll –1 )<br />
(Fig.2). These data revealed that CPR transport is saturated at an<br />
external CPR concentration of ~1.6mmoll –1 (V max 1.58mmoll –1 ,<br />
K m 81.8mmoll –1 ; Fig.2). However, the MTs are unable to<br />
concentrate the dye at high concentrations of CPR (>1mmoll –1 ;<br />
Fig.2, inset). Experiments, on both animals, were performed at a<br />
concentration of 1mmoll –1 CPR.<br />
Test solutions were prepared from the two control solutions<br />
containing 1mmoll –1 CPR, and one of the following inhibitors: 2,4-<br />
dinitrophenol (DNP, 1mmoll –1 ), ouabain (10mmoll –1 ), bafilomycin<br />
A 1 (5mmoll –1 ), para-aminohippuric acid (PAH; 10mmoll –1 ) or<br />
probenecid (0.1–10mmoll –1 ). The final osmolality of the solutions<br />
was measured on a Vapro 5520 vapour pressure osmometer (Wescor,<br />
Logan, UT, USA). All solutions were titrated with NaOH to pH8<br />
for H. crispae and pH7.2 for S. gregaria.<br />
Experimental animals<br />
Specimens of H. crispae Kristensen 1982 were collected on 11<br />
February 2008 and 17 January 2010 at Vellerup Vig, Isefjord,<br />
Denmark (55°44.206N, 11°51.258E) (see Fig.3A). Bottom samples<br />
were collected with a mini van Veen grab at a depth of 1–2m (salinity<br />
~20p.p.t., pH8). Rocks, algae and sediment collected with the grab<br />
were freshwater shocked. The debris was decanted into a conical net<br />
(mesh size 63mm) and subsequently transferred to SW from the<br />
locality and kept at 4°C. Animals in the active stage (see Kristensen,<br />
1982; Møbjerg et al., 2007; <strong>Halberg</strong> et al., 2009b) were identified<br />
using a dissection microscope. The tardigrades were kept for a period<br />
of up to 6months at 4°C in SW (salinity 20p.p.t., pH8) and regularly<br />
supplied with fresh substrate, consisting mainly of sediment, organic<br />
debris, filamentous algae and diatoms.<br />
A<br />
B<br />
[CPR]lumen (mmol l –1 )<br />
2.2<br />
2.0<br />
1.8<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
tc<br />
1 μmol l –1 10 μmol l –1 100 μmol l –1 1 mmol l –1 5 mmol l –1<br />
lu<br />
tc<br />
lu<br />
K m =81.8 μmol l –1<br />
V max =1.58 mmol l –1<br />
Lumen/bath ratio<br />
tc<br />
200<br />
150<br />
100<br />
Specimens of S. gregaria Forskål 1775 were acquired from a<br />
specialized animal shop (www.exopark.dk), and kept at room<br />
temperature (RT) with light:dark periods of 16h:8h and fed annual<br />
meadow grass (Poa annua) ad libitum.<br />
Exposure to test solutions<br />
Halobiotus crispae<br />
Evaluation of CPR transport by tardigrade epithelia was performed<br />
on whole animals immersed in the dye solution. Data obtained from<br />
82 animals were used for the study – no distinction was made<br />
between male and female specimens. Initial observations revealed<br />
that the animals did not take up dye through the mouth or cuticle<br />
(CPR non-punctured, Fig.3) and test solutions were therefore<br />
introduced to the haemolymph of single specimens (length<br />
300–500mm) through a small hole made in the cuticle in the anterior<br />
part of the animal. The animal was incubated for a period of 60min<br />
at RT in the respective test solution, and quickly washed in SW<br />
prior to photography. Light micrographs of the specimens were taken<br />
in bright-field at a 40 magnification, using an Olympus DP20<br />
camera mounted on an Olympus BX50 microscope (Olympus,<br />
Hamburg, Germany). Additionally, in order to investigate whether<br />
the test solutions were ingested during the experimental period, intact<br />
non-punctured animals were incubated in the CPR test solution for<br />
a corresponding period. The animals were washed and photographed<br />
as described above.<br />
50<br />
lu<br />
tc<br />
[CPR] bath (mmol l –1 )<br />
lu<br />
0<br />
0 0.5 1<br />
0 1 2 3 4 5<br />
Fig.2. Accumulation of CPR as a function of external CPR concentration in<br />
Malpighian tubules (MTs) of Schistocerca gregaria. (A)Representative light<br />
micrographs of the MTs following exposure to different concentrations<br />
(1mmoll –1 , 10mmoll –1 , 100mmoll –1 , 1mmoll –1 and 5mmoll –1 ) of CPR.<br />
Scale bars, 100mm. lu, lumen; tc, trachea. (B)Luminal CPR concentration<br />
as a function of bath CPR concentration, revealing the kinetic parameters<br />
K m and V max for CPR transport. Each point shows the mean ± s.d. for<br />
N4–6 animals with 3–5 MTs providing the estimate for each animal. The<br />
solid line represents the fit to the Michaelis–Menten equation by non-linear<br />
regression analysis (using error as weight). Insert shows the lumen/bath<br />
ratio of CPR as a function of external CPR concentration.<br />
lu<br />
5<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
500<br />
K. A. <strong>Halberg</strong> and N. Møbjerg<br />
B<br />
[CPR]luminal (mmol l –1 )<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
SW<br />
control<br />
mg<br />
C<br />
(12)<br />
SW control<br />
CPR nonpunctured<br />
mg<br />
(6)<br />
CPR non-punctured<br />
mg<br />
CPR<br />
(13)<br />
1 mmol l –1 CPR<br />
+DNP<br />
–81%<br />
mg<br />
**<br />
(5)<br />
+1 mmol l –1 DNP<br />
+Bafilomycin<br />
+Ouabain +DMSO<br />
–21% –21%<br />
n n<br />
mg<br />
*<br />
+10 mmol l –1 ouabain<br />
mg<br />
*<br />
(6) (8)<br />
+5 μmol l –1 bafilomycin<br />
A<br />
+PAH<br />
–44%<br />
mg<br />
*<br />
*<br />
**<br />
(11)<br />
+10 mmol l –1 PAH<br />
mg<br />
*<br />
Leg 4<br />
+Probenecid<br />
–61%<br />
mg<br />
Leg 1<br />
Leg 2<br />
Leg 3<br />
**<br />
(10)<br />
+10 mmol l –1 probenecid<br />
Fig.3. CPR accumulation in the gut lumen of Halobiotus crispae,<br />
and the effect of inhibitors on dye accumulation. (A)Light<br />
micrograph of H. crispae from a ventral view showing the animalʼs<br />
basic morphology. mg, midgut. Asterisks indicate MTs. Scale bar,<br />
100mm. (B)Representative light micrographs of the midgut (mg)<br />
following exposure to the various test solutions. Seawater control<br />
shows the midgut of a punctured animal following immersion in<br />
seawater, while CPR non-punctured shows the midgut of an intact<br />
animal after immersion in CPR solutions. Estimations of luminal<br />
CPR concentration were performed solely from areas devoid of gut<br />
content, i.e. brown material in the gut. The percentage change in<br />
CPR concentration, compared with experiments on CPR alone, is<br />
noted. Scale bars, 50mm. (C)Corresponding luminal concentrations<br />
of CPR. Data are depicted as means ± s.d. Asterisks refer to a<br />
significant difference from CPR alone (*P
Epithelial transport in tardigrades<br />
501<br />
Microsystems, Wetzlar, Germany) with glass knives and<br />
subsequently stained with Toluidine Blue.<br />
For immunocytochemistry, whole animals of H. crispae and MTs<br />
of S. gregaria were fixed in 3% paraformaldehyde in 0.1moll –1<br />
sodium cacodylate buffer (pH7.4) for 60min and subsequently<br />
transferred to 0.1moll –1 sodium cacodylate buffer. The tissue was<br />
then dehydrated through a graded series of ethanol and xylene,<br />
embedded in paraffin and sectioned into ~10mm sections, or<br />
transferred to PBS and used as whole mounts for immunostaining.<br />
Paraffin sections were deparaffinized through a graded series of xylene<br />
and alcohol, washed in saline (control solution; see ‘Test solutions’<br />
above) and blocked with 10% normal goat serum (Invitrogen,<br />
Carlsbad, CA, USA) for 30min, prior to incubation with primary<br />
antibody. Paraffin sections, as well as whole mounts, were incubated<br />
overnight at 4°C in insect saline (MTs) or PBS (tardigrades) containing<br />
10% normal goat serum, 0.1% Triton-X and primary antibody. The<br />
Na + /K + -ATPase -subunit monoclonal mouse primary antibody 5-<br />
IgG (10mgml –1 ) was developed by D. M. Famborough, and obtained<br />
from the Developmental Studies Hybridoma Bank (University of<br />
Iowa, Iowa City, IA, USA). This antibody has been used to identify<br />
the Na + /K + -ATPase in numerous excretory tissues, including MTs of<br />
Drosophila melanogaster (Lebovitz et al., 1989; Torrie et al., 2004),<br />
the gills of the blue crab Callinectes sapidus (Towle et al., 2001) and<br />
the pronephros of Ambystoma mexicanum (Haugen et al., 2010).<br />
Following an extensive wash in saline, the tissue was incubated with<br />
(anti-mouse) Alexa Fluor 594 (1:100) secondary antibody (Invitrogen)<br />
overnight at 4°C. The tissue was rinsed with saline then counterstained<br />
with Alexa Fluor 488-conjugated phalloidin (luminal marker; 1:40;<br />
Invitrogen) and DAPI (50mgml –1 ; Invitrogen) for 2h, washed and<br />
mounted on glass coverslips in Vectashield (Vector Laboratories Inc.,<br />
Burlingame, CA, USA). Images were acquired using a Leica DM<br />
RXE 6 TL microscope equipped with a Leica TCS SP2 AOBS<br />
confocal laser scanning unit (Leica Microsystems, Wetzlar, Germany).<br />
The tissue was scanned employing sequential scanning (setting:<br />
between frames) using the 488nm line of an argon/krypton laser and<br />
the 594nm line of a helium laser, in addition to the 405nm UV laser<br />
line. The image series was processed and edited using Imaris software.<br />
Confocal images are based on 240 optical sections of a Z-series<br />
performed at intervals of 0.7mm. Experiments were conducted<br />
multiple times with corresponding results. All control preparations<br />
without primary antibody were negative for immunostaining.<br />
Chemicals<br />
All chemicals were obtained from Sigma-Aldrich (St Louis, MO,<br />
USA). Bafilomycin was dissolved and stored in dimethyl sulphoxide<br />
(DMSO). Inhibitors were allowed to dissolve in the CPR solution<br />
overnight prior to use.<br />
Statistics<br />
Data are expressed as means ± s.d. unless otherwise stated. The<br />
statistical significance of differences between the various exposures<br />
was tested using one-way ANOVA followed by a Tukey multiple<br />
comparisons of means. The statistical tests were performed using<br />
the data analysis program OriginPro 7.5 (OriginLab). Significance<br />
levels were P>0.05 (not significant), P
502<br />
K. A. <strong>Halberg</strong> and N. Møbjerg<br />
[CPR]lumen (mmol l –1 )<br />
A<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
CPR<br />
mg<br />
B<br />
(13)<br />
*<br />
+Bafilomycin<br />
+DMSO<br />
–14%<br />
+DMSO<br />
–21%<br />
CPR<br />
mg<br />
**<br />
*<br />
gm<br />
(5) (8)<br />
C<br />
tc lu lu tc lu<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
D<br />
+DMSO<br />
+5%<br />
**<br />
(11) (10)<br />
+Bafilomycin<br />
+DMSO<br />
–26%<br />
**<br />
(8)<br />
tc<br />
Fig.6. The effect of bafilomycin and dimethylsulphoxide (DMSO) on<br />
CPR accumulation. (A)Representative light micrographs of the<br />
tardigrade midgut (mg) following exposure to the test solutions.<br />
The percentage change in CPR concentration, compared with<br />
experiments with CPR alone, is noted. Scale bars, 50mm.<br />
(B)Corresponding luminal concentration of CPR in midgut lumen.<br />
(C)Representative light micrographs of locust MTs following<br />
exposure to the test solutions. The percentage change in CPR<br />
concentration, compared with experiments on CPR alone, is noted.<br />
Scale bars, 100mm. (D)Corresponding luminal concentration of<br />
CPR in the MTs. Asterisks refer to significant difference from<br />
1mmoll –1 CPR alone (*P
Epithelial transport in tardigrades<br />
503<br />
A<br />
[CPR]lumen (mmol l –1 )<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
+0.1 mmol l –1<br />
CPR –26%<br />
CPR<br />
mg<br />
B<br />
(13)<br />
**<br />
mg<br />
** **<br />
(7)<br />
+1 mmol l –1<br />
–56%<br />
*<br />
mg<br />
**<br />
+10 mmol l –1<br />
–61%<br />
mg<br />
(4) (10)<br />
C<br />
tc<br />
lu<br />
lu<br />
1.6 D ** **<br />
(11) **<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
CPR<br />
+0.1 mmol l –1<br />
–9%<br />
(5)<br />
+1 mmol l –1<br />
–47%<br />
lu<br />
(5)<br />
+10 mmol l –1<br />
–77%<br />
tc<br />
**<br />
lu<br />
(9)<br />
Fig.7. Titration of the inhibitory effect of<br />
probenecid. (A)Representative light<br />
micrographs of the tardigrade midgut (mg)<br />
following exposure to the different<br />
concentrations (0.1, 1 and 10mmoll –1 ) of<br />
probenecid. The percentage change in CPR<br />
concentration, compared with experiments<br />
with CPR alone, is noted. Scale bars, 50mm.<br />
(B)Corresponding luminal concentration of<br />
CPR in midgut lumen. (C)Representative<br />
light micrographs of the insect MTs following<br />
exposure to the different concentrations (0.1,<br />
1 and 10mmoll –1 ) of probenecid. The<br />
percentage change in CPR concentration,<br />
compared with experiments on CPR alone, is<br />
noted. Scale bars, 100mm. (D)Corresponding<br />
luminal concentration of CPR in the MTs.<br />
Asterisks refer to significant difference from<br />
1mmoll –1 CPR alone (*P
504<br />
K. A. <strong>Halberg</strong> and N. Møbjerg<br />
probenecid concentration and CPR accumulation (and activity; data<br />
not shown). At a concentration of 0.1mmoll –1 probenecid, CPR<br />
concentration was reduced by 26% with a luminal concentration of<br />
0.42±0.07mmoll –1 in H. crispae, and by 9% corresponding to a<br />
concentration of 1.2±0.14mmoll –1 in S. gregaria. When applied at<br />
a concentration of 1mmoll –1 , probenecid reduced CPR concentration<br />
by 56% corresponding to a luminal concentration of<br />
0.25±0.05mmoll –1 in H. crispae, and by 47% equivalent to<br />
0.7±0.16mmoll –1 in S. gregaria (Fig.7).<br />
Midgut and MT structure and immunostaining<br />
Our transport studies indicate that CPR transport is active and<br />
transporter mediated in the midgut of H. crispae as well as the MTs<br />
of S. gregaria. It is well established that the vacuolar H + -ATPase<br />
is important for energizing transepithelial transport in insects,<br />
whereas (and in contrast to vertebrate literature) the role of the<br />
Na + /K + -ATPase is controversial (e.g. Torrie et al., 2004; Beyenbach<br />
et al., 2010). The data obtained in the present study using ouabain<br />
would suggest that the Na + /K + -ATPase is important for CPR<br />
transport in both animals. Conversely, ouabain could act as a<br />
competitive inhibitor, being transported by the same transporters as<br />
CPR. We therefore investigated whether the Na + /K + pump is<br />
expressed in the epithelia.<br />
Unfortunately, our attempt to localize Na + /K + -ATPase in<br />
tardigrades was unsuccessful, both on whole mounts and on paraffin<br />
sections, and also in attempts at antigen retrieval.<br />
Our observations on the MTs of S. gregaria generally correspond<br />
with those previously described (Garret et al., 1988), when focusing<br />
on the proximal region and parts of the middle region used in the<br />
present study. In general, we observed a prominent brushborder<br />
(Fig.8B,C) and at least two cell types. One type, a principal-like cell,<br />
has a single nucleus, while the other is a very large double-nucleated<br />
cell (dashed circles in Fig.8). Cross-striated muscle fibers are present<br />
in both the transverse (Fig.8A,B) and longitudinal direction<br />
(Fig.8E,G). The transverse muscle fibers were only observed in the<br />
proximal-most region of the MTs, whereas the longitudinal muscle<br />
extended the entire length of the tubule. Visual observations revealed<br />
that the transverse muscles aid the clearance of luminal content into<br />
the midgut through peristaltic contractions. Our immunocytochemical<br />
analysis shows that the Na + /K + -ATPase is expressed in the MTs of<br />
S. gregaria, and is localized to the basal plasma membranes<br />
(Fig.8D–G), although to a lesser extent in the double-nucleated cells<br />
(Fig.8D,G). The localization was confirmed by counterstaining of<br />
nuclei by DAPI and staining apical microvilli with phalloidin. This<br />
is, to our knowledge, the first study showing immunolocalization of<br />
the Na + /K + -ATPase in S. gregaria MTs. Interestingly, the localization<br />
of the transporter substantiates previously published data on isolated<br />
basal membrane fractions from MTs and hindgut from S. gregaria,<br />
which show a prominent Na + /K + -ATPase activity (Al-Fifi, 2007).<br />
DISCUSSION<br />
Our study is significant in two main aspects: (i) we are the first to<br />
provide evidence for active epithelial transport in tardigrades, and<br />
(ii) our data show that tardigrades possess an organic anion transport<br />
system. Consequently, our study is important for understanding the<br />
evolution of transport systems.<br />
CPR is categorized as a sulphonate, but is known to compete with<br />
substrates for both solute carriers (SLC22 and SLC21/SLCO) and<br />
ABC transporters in a number of organisms (Pritchard et al., 1999;<br />
Linton and O’Donnell, 2000; Chahine and O’Donnell, 2009). In H.<br />
crispae, CPR transport is ATP dependent (strong inhibition by DNP),<br />
probably energized by both the Na + /K + -ATPase and the V-type H + -<br />
ATPase (bafilomycin and ouabain reduce CPR accumulation), and<br />
possibly transporter mediated (inhibited by the prototypic organic<br />
anions PAH and probenecid). The latter suggests that CPR transport<br />
is (at least partly) transcellular. Comparing the transport characteristics<br />
of CPR transport between the tardigrade midgut and the insect MT<br />
reveals a surprisingly similar overall pharmacological profile of the<br />
investigated tissues, albeit with markedly higher concentrations of<br />
CPR observed in the insect MT. In addition to transcellular transport<br />
of the dye, fluid secretion may augment transport of organic anions<br />
by convective secretion through the paracellular pathway, and/or<br />
reduce diffusive back-flux of organic anions from the tubule lumen<br />
to the haemolymph (O’Donnell and Leader, 2006; Chahine and<br />
O’Donnell, 2010). Consequently, without a measure of fluid secretion<br />
rates, and thereby a measure of net CPR secreted, we cannot conclude<br />
whether the difference in relative concentration of CPR in the gut<br />
lumen of H. crispae, compared with the MTs of S. gregaria, is a<br />
consequence of a lower dye transport capacity, or whether it reflects<br />
differences in fluid transport rates. Also, we cannot be certain that<br />
the concentration of CPR in the haemolymph of H. crispae is exactly<br />
the same as that of the surrounding bath – although, the relatively<br />
high dye concentration used along with the small diffusion distances<br />
involved, the relatively long exposure time (60min) and the animal<br />
movements that facilitate fluid exchange, would make potential<br />
concentration differences negligible.<br />
A vast number of papers have investigated various aspects of<br />
organic anion transport in insect MTs. In the present study we<br />
observed a mean CPR concentration of 1.3±0.13mmoll –1 when no<br />
inhibitors were added. This concentration is elevated above the bath<br />
concentration (1mmoll –1 ), albeit by a much smaller factor than that<br />
reported from other insects (Maddrell et al., 1974; Bresler et al.,<br />
1990; Linton and O’Donnell, 2000; Leader and O’Donnell, 2005).<br />
However, when exposed to external CPR concentrations of 1, 10<br />
and 100mmoll –1 , the mean luminal CPR concentrations were<br />
0.16±0.03, 0.73±0.09 and 0.89±0.07mmoll –1 , respectively, which<br />
is a factor of ~160, 70 and 9 above bath concentrations (Fig.2).<br />
When exposed to a bath concentration of 5mmoll –1 CPR, the mean<br />
luminal concentration was 1.79±0.33mmoll –1 , a factor of 2.8 below<br />
external concentrations. Consequently, the luminal CPR<br />
concentration is maximally elevated (~160-fold) above that in the<br />
bathing medium when the latter contains CPR at a concentration of<br />
1mmoll –1 . This concentrative ability is among the highest measured<br />
for insects, and is an additional confirmation of active transport of<br />
CPR by the insect MTs.<br />
Ouabain, a well-characterized, potent inhibitor of the Na + /K + -<br />
ATPase, reduced the CPR concentration in both the tardigrade<br />
midgut and the insect MTs by ~20–21%. A similar 23% reduction<br />
in PAH secretion in the presence of 1mmoll –1 ouabain was reported<br />
from MTs of D. melanogaster (Linton and O’Donnell, 2000), while<br />
fluorescein uptake was reduced in MTs of Blaberus giganteus by<br />
30% and in MTs of Locusta migratoria by 20%, when ouabain was<br />
used at 1 and 0.1mmoll –1 , respectively (Bresler et al., 1990). Our<br />
immunocytochemical investigation on the MTs of S. gregaria<br />
revealed expression of the Na + /K + -ATPase in the basal cell<br />
membranes, although to a lesser extent in the double-nucleated cells.<br />
A basal localization of the Na + /K + -ATPase in principal cells of MTs<br />
of D. melanogaster has previously been reported (Torrie et al., 2004).<br />
The fact that there is expression of the Na + /K + -ATPase in the basal<br />
membranes, and that CPR transport is ouabain sensitive, suggests<br />
that the pump is important for CPR transport. Alternatively, it could<br />
be argued that the inhibitory effects of ouabain are due to competitive<br />
inhibition, rather than to non-competitive inhibition, given the fact<br />
that ouabain is actively transported by members of the SLC21/SLCO<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Epithelial transport in tardigrades<br />
505<br />
subfamily (Oatps) in D. melanogaster (Torrie et al., 2004). Whether<br />
this is ubiquitous in insects is at present not known; however, several<br />
members of the Oatp family are known to transport ouabain in<br />
human tissue (see Hagenbuch and Gui, 2008). Competition studies<br />
using fluorescently labeled ouabain would help in clarifying this<br />
matter. Considering the striking similarities between the<br />
pharmacological profiles of the tardigrade midgut and insect MT,<br />
it seems reasonable to assume that the Na + /K + -ATPase is similarly<br />
present in the basal cell membranes of the tardigrade midgut (in<br />
spite of failed attempts to localize this transporter). In support of<br />
this interpretation is the fact that transcripts for the -subunit of the<br />
Na + /K + -ATPase were found in the expressed sequence tags (EST)<br />
library from Hybsibius dujardini Doyère, 1840 (TardiBASE cluster<br />
ID: HDC01733 TardiBASE; http://xyala.cap.ed.ac.uk/research/<br />
tardigrades/tardibase.shtml).<br />
Bafilomycin is a specific inhibitor of the V-type H + -ATPase and<br />
was found to reduce CPR accumulation in both H. crispae and S.<br />
gregaria. This observation is consistent with the fact that the V-type<br />
H + -ATPase is viewed as being central to the transport activities of<br />
the MT in insects (Weng et al., 2003; Beyenbach et al., 2010) and<br />
perhaps also for transport in tardigrades; the B-, C-, D-, E-, G- and<br />
H-subunits of the V-type H + -ATPase were found in the EST library<br />
of H. dujardini (TardiBASE). Indeed, visual changes to CPR colour<br />
were observed in the midgut of H. crispae (CPR became purple; data<br />
not shown) 10min post-incubation, suggesting an alkalization of the<br />
midgut content. This observation is in accordance with specific<br />
inhibition of an apical V-type H + -ATPase in the tardigrade midgut.<br />
PAH is a prototypical substrate of the classic organic anion transport<br />
system (i.e. the SLC22 subfamily) and was shown to be an effective<br />
inhibitor of CPR transport in both H. crispae and S. gregaria (~40%<br />
reduction in both animals). This finding is a strong indication that<br />
PAH (carboxylate) and CPR (sulphonate) transport are mediated by<br />
a common transporter, or alternatively through two separate transport<br />
systems overlapping in affinity, in both investigated epithelia. Whether<br />
carboxylates (e.g. PAH and probenecid) and sulphonates (e.g. CPR)<br />
are handled by a common transporter (Bresler et al., 1990), or by two<br />
separate transport systems (Maddrell et al., 1974; Linton and<br />
O’Donnell, 2000; Chahine and O’Donnell, 2009), appears to be highly<br />
variable and/or species specific. Indeed, transport of PAH and<br />
probenecid (carboxylates) and CPR and methotrexate (MTX;<br />
sulphonates) are all mediated by one tranporter (OAT1) in humans,<br />
but not in rats (Burckardt and Burckardt, 2003), whereas MTX uptake<br />
is competitively inhibited by CPR and probenecid, but not PAH in<br />
D. melongaster (Cahine and O’Donnell, 2009).<br />
Probenecid is implicitly regarded as a competitive inhibitor of<br />
organic anion transport, and has been shown to compete with both<br />
solute carrier and ABC transporter substrates (Bresler et al., 1990;<br />
Linton and O’Donnell, 2000; Neufeld et al., 2005; Leader and<br />
O’Donnell, 2005). It is typically applied in a concentration of<br />
1mmoll –1 ; here, we examined the effects of probenecid on CPR<br />
transport in the range 0.1–10mmoll –1 . In H. crispae, probenecid<br />
reduced CPR accumulation by 26%, 56% and 61% at a concentration<br />
of 0.1, 1 and 10mmoll –1 , respectively, whereas the corresponding<br />
reduction in CPR concentration was 9%, 47% and 77% in S.<br />
gregaria. In the presence of very high probenecid concentrations<br />
(10mmoll –1 ), we observed an almost total loss of motility in H.<br />
crispae. Indeed, the level of animal movement was comparable to<br />
that in treatments containing DNP. This observation suggests that<br />
the drug affected processes in addition to CPR transport at the given<br />
concentration. In fact, when applied at concentrations of 1mmoll –1<br />
or above, probenecid is reported to induce a range of non-specific<br />
effects presumably initiated by the uncoupling of mitochondrial<br />
Haemolymph<br />
Na + /K + -ATPase<br />
K + OA –<br />
ATP<br />
P Na +<br />
i +ADP<br />
Tardigrade<br />
midgut cell<br />
P i +ADP H+ ATP<br />
V-type H + -ATPase<br />
Lumen<br />
Solute carrier<br />
(SLC21/SLCO)<br />
Nucleus<br />
OA –<br />
Basal lamina<br />
Apical junction<br />
complex<br />
Fig.9. Tentative model of the tardigrade midgut cell derived from the<br />
current study on H. crispae. Based on the pharmacological profile of the<br />
tardigrade midgut epithelium, both the Na + /K + -ATPase and the V-type H + -<br />
ATPase are potential candidates for providing an electrochemical driving<br />
force for the transepithelial movement of organic anions. Transport<br />
characteristics and the presence in tardigrade EST libraries suggest that a<br />
member of the SLC21/SLCO transporter family may mediate the<br />
basolateral entry of organic anions in tardigrades. The exact coupling<br />
between electrochemical gradients generated by the pumps and transport<br />
of ions, as well as the nature of the apical exit step, are not known.<br />
oxidative phosphorylation (Masereeuw et al., 2000). It is therefore<br />
possible that the reduction in CPR transport is non-specific when<br />
the drug is applied in concentrations ≥1mmoll –1 . At lower<br />
concentrations of probenecid (
506<br />
K. A. <strong>Halberg</strong> and N. Møbjerg<br />
ID: HDC00004, HDC02687 and HDC03352) (ABCB and ABCC<br />
protein families) are present and expressed in tardigrades – just as<br />
they are in insects (Maddrell et al., 1974; Lanning et al., 1996;<br />
Bresler et al., 1990; Linton and O’Donnell, 2000; Torrie et al., 2004;<br />
Neufeld et al., 2005; Leader and O’Donnell, 2005; O’Donnell and<br />
Leader, 2006; Chahine and O’Donnell, 2009). Comparing the<br />
transport characteristics of the different groups of transporters reveals<br />
that it is unlikely that members of the ABC transporter family (i.e.<br />
P-gps and Mrps) mediate the basolateral entry of CPR in H. crispae<br />
and S. gregaria. This assumption is based on the fact that P-gp<br />
transporters predominantly recognize large cationic species (Russel,<br />
2010), and because Mrp transporters mainly transport large<br />
(>500Da) polyvalent type II organic anions (Wright and Dantzler,<br />
2004; Russel, 2010) – the apical transporter MRP2, however, was<br />
shown to transport PAH in humans (see below). In addition, PAH<br />
does not compete with MTX (an Mrp substrate) in D. melongaster,<br />
suggesting distinct transport systems for these compounds in insects<br />
(Chahine and O’Donnell, 2009). In contrast, both members of the<br />
SLC22 and SLC21/SLCO protein families (Oats and Oatps) have<br />
been reported to transport all the investigated organic anions,<br />
including probenecid, PAH and CPR (Pritchard et al., 1999; Lee<br />
and Kim, 2004; Torrie et al., 2004). Therefore, it seems probable<br />
that an Oat or Oatp homologue mediates the basolateral entry of<br />
CPR in the tardigrade midgut cell as well as the insect MTs (Fig.9).<br />
At present, our data do not allow us to make assumptions on the<br />
nature of the luminal exit. Evidence suggests that MRP2 is involved<br />
in the efflux of organic anions across the brush-border membrane<br />
in the human kidney proximal tubule (Leier et al., 2000), and a<br />
similar situation could exist in tardigrades and insects. Our data<br />
tentatively suggest that the V-type H + -ATPase, and perhaps also<br />
the Na + /K + -ATPase, provide the driving force for the transepithelial<br />
transport of organic anions in both H. crispae and S. gregaria<br />
(Fig.9). It is likely that a large lumen-positive transepithelial<br />
potential, generated by an apical H + -ATPase provides a significant<br />
driving force for the accumulation of anions in the lumen. However,<br />
the exact coupling between electrochemical gradients generated by<br />
the pumps and transport of the ions is not known.<br />
In future studies, it would be of interest to investigate whether<br />
substrates of Mrps (e.g. Texas Red and MTX) also accumulate in<br />
the midgut of tardigrades, and whether transport of these anions<br />
occurs via a separate or a common transporter to CPR. Similarly,<br />
an understanding of the electrophysiological properties of the<br />
midgut epithelium would be highly relevant in our ongoing struggle<br />
to understand the complex biology of these amazing animals.<br />
ACKNOWLEDGEMENTS<br />
We would like to thank Reinhardt M. Kristensen for the use of the Olympus BX50<br />
stereomicroscope, Jette Lyby Michelsen for technical assistance and Dennis K.<br />
Persson and Aslak Jørgensen for help during sampling.<br />
FUNDING<br />
Funding came from the 2008 Faculty of Science, University of Copenhagen Freja-<br />
Programme and from the Carlsberg Foundation.<br />
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Mulenga, A., Khumthong, R., Chalaire, K. C., Strey, O. and Teel, P. (2008). Molecular<br />
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Pelzer, B., Dastych, H. and Greven, H. (2007). The osmoregulatory/excretory organs of<br />
the glacier-dwelling eutardigrade Hypsibius klebelsbergi Mihelcic, 1959 (Tardigrada).<br />
Mitt. Hamb. Zool. Mus. Inst. 104, 61-72.<br />
Persson, D., <strong>Halberg</strong>, K. A., Jørgensen, A., Ricci, C., Møbjerg, N. and Kristensen, R.<br />
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Phillips, J. (1981). Comparative physiology of insect renal function. Am. J. Physiol. Regul.<br />
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274, 33382-33387.<br />
Rebecchi, L. and Bertolani, R. (1994). Maturative pattern of ovary and testis in<br />
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Rebecchi, L., Altiero, T., Guidetti, R., Cesari, M., Bertolani, R., Negroni, M. and<br />
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THE JOURNAL OF EXPERIMENTAL BIOLOGY
Paper III
Acta Physiol 2011, 202, 409–420<br />
REVIEW<br />
Survival in extreme environments – on the current<br />
knowledge of adaptations in tardigrades<br />
N. Møbjerg, 1 K. A. <strong>Halberg</strong>, 1 A. Jørgensen, 2 D. Persson, 1,2 M. Bjørn, 3 H. Ramløv 3 and<br />
R. M. Kristensen 2<br />
1 Department of Biology, University of Copenhagen, Copenhagen, Denmark<br />
2 Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark<br />
3 Department of Science, Systems and Models, University of Roskilde, Roskilde, Denmark<br />
Received 17 October 2010,<br />
revision requested 13 November<br />
2010,<br />
revision received 6 January 2011,<br />
accepted 10 January 2011<br />
Correspondence: N. Møbjerg,<br />
Department of Biology, August<br />
Krogh Building, Universitetsparken<br />
13, DK-2100 Copenhagen Ø,<br />
Denmark.<br />
E-mail: nmobjerg@bio.ku.dk<br />
Abstract<br />
Tardigrades are microscopic animals found worldwide in aquatic as well as<br />
terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa,<br />
as do the two major invertebrate model organisms: Caenorhabditis elegans<br />
and Drosophila melanogaster. We present a brief description of the tardigrades<br />
and highlight species that are currently used as models for physiological<br />
and molecular investigations. Tardigrades are uniquely adapted to a<br />
range of environmental extremes. Cryptobiosis, currently referred to as a<br />
reversible ametabolic state induced by e.g. desiccation, is common especially<br />
among limno-terrestrial species. It has been shown that the entry and exit of<br />
cryptobiosis may involve synthesis of bioprotectants in the form of selective<br />
carbohydrates and proteins as well as high levels of antioxidant enzymes and<br />
other free radical scavengers. However, at present a general scheme of<br />
mechanisms explaining this phenomenon is lacking. Importantly, recent<br />
research has shown that tardigrades even in their active states may be extremely<br />
tolerant to environmental stress, handling extreme levels of ionizing<br />
radiation, large fluctuation in external salinity and avoiding freezing by<br />
supercooling to below )20 °C, presumably relying on efficient DNA repair<br />
mechanisms and osmoregulation. This review summarizes the current<br />
knowledge on adaptations found among tardigrades, and presents new data<br />
on tardigrade cell numbers and osmoregulation.<br />
Keywords cell numbers, cryptobiosis, evolution, osmoreglation, supercooling,<br />
tardigrade.<br />
Tardigrades, also known as water bears, are microscopic<br />
metazoans (approx. 0.1–1.2 mm). They were<br />
discovered in the 18th Century with the development of<br />
early microscopes and were first described by the<br />
German zoologist Goeze in 1773, who named them<br />
‘kleiner Wasserbär’ or little water bear because of their<br />
strong resemblance to a little bear (see e.g. Ramazzotti<br />
& Maucci 1983, Nelson 2001, Schill 2010). Shortly<br />
after in 1776, the current name, Tardigrada (from Latin<br />
tardigradus, slow-moving), was given by the Italian<br />
natural scientist Spallanzani (see e.g. Rebecchi et al.<br />
2007). Tardigrades are exceptional among metazoans in<br />
their adaptations to the most extreme environments. As<br />
is also known from selected species of arthropods,<br />
nematodes and rotifers, many species have the ability to<br />
enter cryptobiosis; a state of suspended animation<br />
believed to be ametabolic (Keilin 1959, Clegg 2001).<br />
Corti already noted these adaptations in tardigrades in<br />
1774, when he observed that these animals could be<br />
revived after desiccation (Kinchin 1994). In 1962,<br />
Tardigrada was recognized as a phylum by Ramazzotti<br />
in Il Phylum Tardigrada (Ramazzotti 1962). There are<br />
Ó 2011 The Authors<br />
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 409
Adaptation to extreme environments in tardigrades Æ<br />
N. Møbjerg et al. Acta Physiol 2011, 202, 409–420<br />
currently approx. 1000 described species (Guidetti &<br />
Bertolani 2005, Degma et al. 2010), but this is likely far<br />
from the real number of tardigrade species, as especially<br />
the marine arthrotardigrades remain relatively unexplored.<br />
Tardigrada belongs to the invertebrate superclade<br />
Ecdysozoa, however; their precise phylogenetic position<br />
is still debated and it is presently not clear whether the<br />
group is more closely related to arthropods and<br />
onychophorans or to the nematodes and nematomorphs<br />
(Fig. 1) (Aguinaldo et al. 1997, Dunn et al. 2008,<br />
Edgecombe 2010). In any case, as noticed in recent<br />
papers, this group has a central position placed in<br />
between the two major invertebrate model organisms –<br />
the nematode Caenorhabditis elegans Maupas, 1900<br />
and the arthropod Drosophila (Sophophora) melanogaster<br />
Meigen, 1830 (Goldstein & Blaxter 2002, Gabriel<br />
& Goldstein 2007).<br />
Little is known about the physiological mechanisms<br />
underlying adaptations to extreme environmental conditions<br />
in tardigrades. Past centuries of tardigrade<br />
research have mainly focused on species descriptions<br />
and morphological investigations related to phylogenetic<br />
analysis. In recent years, however, research in the<br />
field has taken advantage of new molecular tools and an<br />
increasing number of scientists find the group fasci-<br />
Loricifera<br />
Kinorhyncha<br />
Priapulida<br />
ECDYSOZOA<br />
<br />
Nematomorpha<br />
Nematoda*<br />
Tardigrada*<br />
Arthropoda*<br />
Onychophora<br />
PROTOSTOMIA<br />
Brachiopoda<br />
Annelida<br />
LOPHOTROCHOZOA<br />
Mollusca<br />
Rotifera*<br />
Platyhelminthes<br />
DEUTEROSTOMIA<br />
Chordata<br />
Echinodermata<br />
Cnidaria<br />
Figure 1 Evolutionary position of tardigrades in the Animal Kingdom. Phylogeny of the Metazoa (animals) based on<br />
Aguinaldo et al. (1997) and Dunn et al. (2008) showing selected phyla with emphasis on the position of the Tardigrada. The<br />
inferred position of the tardigrades is based on EST sequences from Richtersius coronifer and Hypsibius dujardini. Ecdysozoa<br />
includes all molting animals and is one of the two protostome superclades. The marked phyla have cryptobiotic species.<br />
Porifera<br />
410<br />
Ó 2011 The Authors<br />
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Acta Physiol 2011, 202, 409–420<br />
N. Møbjerg et al. Æ<br />
Adaptation to extreme environments in tardigrades<br />
nating. In particular, the mechanisms underlying the<br />
ability to enter cryptobiosis have attracted considerable<br />
scientific interest. This interest is no doubt associated<br />
with the suspected translational output related to a<br />
detailed understanding of the complex stress physiology<br />
of tardigrades, i.e. in connection with cryopreservation<br />
and dehydration of biological material. Noticeably, the<br />
phenomenon cryptobiosis touches upon our conception<br />
of life and death; one of the largest enigmas being how<br />
metabolism is restarted after years of suspension. This<br />
review puts focus on physiological and molecular<br />
adaptations to extreme conditions found among tardigrades.<br />
We additionally present a brief description of<br />
the phylum and highlight species that are currently used<br />
as models for these investigations.<br />
Phylum Tardigrada<br />
Tardigrade phylogeny and evolution<br />
About 35 extant animal groups have body plans and<br />
genes that are distinct enough to warrant elevation to<br />
phylum status (Nielsen 2001). The tardigrades, comprising<br />
the phylum Tardigrada, are one of these groups<br />
(Fig. 1). Tardigrades are microscopic invertebrates with<br />
a well developed organization including brain and<br />
sensory organs, muscles, a complex feeding apparatus<br />
and alimentary tract, reproductive and osmoregulatory<br />
organs (see e.g. Rebecchi & Bertolani 1994, Dewel &<br />
Dewel 1996, Møbjerg & Dahl 1996, Eibye-Jacobsen<br />
1997, Jørgensen et al. 1999, Greven 2007, <strong>Halberg</strong><br />
et al. 2009a). They are found worldwide in aquatic as<br />
well as terrestrial environments, but depend on free<br />
water to be in their active, reproducing state. It has been<br />
suggested that tardigrades, like e.g. nematodes, have<br />
eutely, but detailed studies on the subject are still<br />
lacking. Cell counts based on nuclear staining with<br />
DAPI (4¢,6-diamidino-2-phenylindole) in four active<br />
stage adults of the marine eutardigrade Halobiotus<br />
crispae Kristensen, 1982 revealed a total cell number of<br />
around 1060 cells, when excluding gametes (Fig. 2).<br />
This number could represent a slight underestimate of<br />
the total somatic cell number as body cavity cells (also<br />
known as storage cells) may have escaped two of the<br />
specimens, which were punctured prior to the staining<br />
(Fig. 2). We did not observe cell divisions (mitosis)<br />
during the counts. Mitosis has, however, previously<br />
been reported in post-embryonic eutardigrades (Bertolani<br />
1970a,b, 1982).<br />
There are two main evolutionary lines within the<br />
tardigrades, represented by the classes Eutardigrada and<br />
Heterotardigrada (Fig. 3) (see e.g. Jørgensen & Kristensen<br />
2004). The validity of a third class, Mesotardigrada,<br />
is currently uncertain. Mesotardigrada only<br />
contains a single species, Thermozodium esakii Rahm,<br />
1937 originally found in a hot spring in Japan. The type<br />
specimens of T. esakii no longer exist and the type<br />
locality was apparently destroyed in an earthquake<br />
A<br />
P<br />
br<br />
pb<br />
gI<br />
c.gl.<br />
Leg 1<br />
eo<br />
gII<br />
c.gl.<br />
Leg 2<br />
mg<br />
gIII<br />
c.gl.<br />
Leg 3<br />
go<br />
gIV<br />
c.gl.<br />
Leg 4<br />
Figure 2 Cell numbers in Halobiotus crispae. 3-D reconstruction of cell arrangement in the eutardigrade H. crispae based on<br />
confocal laser scanning microscopy of a DAPI stained specimen. In order to obtain an estimate of somatic cell numbers in this<br />
species, specimens were relaxed in CO 2 -enriched water, fixed in 4% paraformaldehyde and ultrasonicated. Two of the four<br />
specimens were additionally delicately punctured with a fine needle. The specimens were subsequently incubated with DAPI and<br />
thoroughly rinsed before mounting. Image acquisition was performed using a Leica DM RXE 6 TL microscope equipped with a<br />
Leica TCS SP2 AOBS confocal laser scanning unit. Cell counts and image processing were performed using the software program<br />
Imaris (Bitplane, Zurich, Switzerland). The total number of somatic cells in H. crispae was estimated at approx. 1060, based on<br />
stainings of four male specimens (mean SD: 1058 53). This number could represent a slight underestimate of the total somatic<br />
cell number as so-called body cavity cells may have escaped the two specimens that were punctured prior to staining (cell counts for<br />
the punctured specimens: 998 and 1088; counts for the non-punctured specimens: 1036 and 1112). The largest number of cells is<br />
clearly present in the anterior part of the animal containing the brain and buccopharyngeal apparatus. A, anterior; P, posterior; br,<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.<br />
Ó 2011 The Authors<br />
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 411
Adaptation to extreme environments in tardigrades Æ<br />
N. Møbjerg et al. Acta Physiol 2011, 202, 409–420<br />
Echiniscoides<br />
Echiniscoidae<br />
Echiniscus<br />
Echiniscidae<br />
Milnesium*<br />
Milnesiidae<br />
Halobiotus<br />
Isohypsibioidea<br />
Richtersius<br />
Paramacrobiotus<br />
Macrobiotoidea<br />
Hypsibius*<br />
Ramazzottius*<br />
Hypsibioidea<br />
Arthrotardigrada<br />
marine<br />
non-cryptobionts<br />
Echiniscoidea<br />
limno-terrestrial<br />
Intertidal<br />
Cryptobionts<br />
Apochela<br />
limno-terrestrial<br />
Cryptobionts<br />
Parachela<br />
limno-terrestrial<br />
cryptobionts<br />
non-cryptobionts<br />
Heterotardigrada<br />
Mesotardigrada ()<br />
Eutardigrada<br />
Tardigrada<br />
*Genome projects<br />
Figure 3 Tardigrade phylogeny. Phylogeny<br />
of tardigrades showing major clades<br />
and position of model/discussed species.<br />
The phylogeny is based on Sands et al.<br />
(2008).<br />
(Nelson 2002). However, a thorough re-sampling for<br />
this species has to our knowledge not been performed.<br />
Tardigrades most likely evolved within the marine<br />
environment, and marine species are especially numerous<br />
within the heterotardigrade order Arthrotardigrada<br />
(Renaud-Mornant 1982, Maas & Waloszek 2001,<br />
Jørgensen et al. 2010). Arthrotardigrades are present<br />
in all oceans from intertidal zones to abyssal depths,<br />
inhabiting different sediment types. In addition, marine<br />
species are found within the other main heterotardigrade<br />
order, Echiniscoidea, represented by the intertidal<br />
Echiniscoides sigismundi (M. Schultze, 1865). This<br />
species may very well be the toughest creature on<br />
Earth, having to endure periods of desiccation and low<br />
oxygen tension as well as large perturbations in salinity<br />
and freezing (Kristensen & Hallas 1980). Nevertheless,<br />
the exact range of this tardigrade’s tolerances remains<br />
to be investigated. It may be hypothesized that an<br />
Echiniscoides-like tardigrade invaded the freshwater/<br />
terrestrial environment and gave rise to the almost<br />
exclusively limno-terrestrial eutardigrades (Kinchin<br />
1994). This is however currently not supported by<br />
molecular data (Sands et al. 2008, Jørgensen et al.<br />
2010; Jørgensen et al. 2011).<br />
The eutardigrades are divided into two orders;<br />
Apochela and Parachela. Two genera within the latter<br />
order, Ramajendas represented by Ramajendas renaudi<br />
(Ramazzotti 1972) in the Southern Hemisphere and<br />
Halobiotus in the Northern Hemisphere, have secondarily<br />
invaded the marine environment (Ramazzotti<br />
1972, Kristensen 1982, Møbjerg et al. 2007). Although<br />
cryptobiosis is common in most eutardigrades, our<br />
recent findings in H. crispae suggest that among these<br />
secondary marine species, adaptations are present that<br />
are quite extraordinary (<strong>Halberg</strong> et al. 2009b). The<br />
tardigrades stay active while experiencing large fluctuations<br />
in abiotic factors, fluctuations that in other<br />
tardigrades would induce cryptobiosis.<br />
Tardigrade genomes<br />
There is a huge variation in the genome size of<br />
tardigrades ranging from about 75–100 Mb in Hypsibius<br />
and Ramazzottius to 800 Mb in Bertolanius<br />
(Gregory 2010, C-values converted from picograms to<br />
base pairs using the conversion 1 pg = 978 Mb according<br />
to Dolezel et al. (2003), Bertolanius was previously<br />
named Amphibolus). For comparison, the genome sizes<br />
of Caenorhabditis elegans and Drosophila melanogaster<br />
are about 100 Mb and 175 Mb respectively (Gregory<br />
2010). The general diploid chromosome numbers of<br />
the eutardigrades are 10–12 and 14 for the heterotardigrade<br />
Echiniscus (Bertolani 1982). Polyploidy with<br />
up to 24 chromosomes is common in eutardigrades<br />
(Bertolani 1982). Three major sequencing projects are<br />
currently ongoing within Tardigrada. However, all<br />
three projects are investigating eutardigrade species;<br />
no data are presently available for the other main<br />
tardigrade group – the heterotardigrades. The international<br />
collaborative Ecdysozoan Sequencing Project is<br />
assembling the genome of Hypsibius dujardini (Doyère,<br />
1840) as part of an investigation into the ancestral<br />
genome of the Ecdysozoa lineage. Prior to this project,<br />
the Edinburgh based TardiBASE project, generated<br />
more than 5000 EST sequences for H. dujardini<br />
(GenBank 2010). The German based FUNCRYPTA<br />
project was focused on investigating cryptobiosis in<br />
Milnesium tardigradum Doyère, 1840 through studies<br />
of gene and protein expression (Förster et al. 2009,<br />
Mali et al. 2010, Schokraie et al. 2010). The project<br />
had in 2010 generated approx. 7000 quality EST<br />
sequences and aimed at advancing the basic understanding<br />
of protein expression in tardigrades through transcriptomic<br />
and proteomic studies (Mali et al. 2010). The<br />
Japanese based Kumamushi Genome Project is assembling<br />
the genome of Ramazzottius varieornatus Bertolani<br />
and Kinchin, 1993 and has preliminarily predicted<br />
412<br />
Ó 2011 The Authors<br />
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Acta Physiol 2011, 202, 409–420<br />
N. Møbjerg et al. Æ<br />
Adaptation to extreme environments in tardigrades<br />
about 20 000 candidate genes in this species (Horikawa<br />
et al. 2008, Katayama et al. 2009). The great challenge<br />
in the years to come will be to correlate the description of<br />
tardigrade genes with studies on the function of these<br />
genes as nicely illustrated in a recent study on a<br />
purinergic P2X receptor from H. dujardini (Bavan et al.<br />
2009).<br />
Adaptation to extreme environments<br />
Tardigrades are extraordinary in their tolerance to<br />
extremes, including limno-terrestrial habitats that frequently<br />
dry out, habitats that freeze and habitats that<br />
experience large fluctuations in e.g. osmotic pressure<br />
and oxygen tension. Cryptobiosis, referred to as a<br />
reversible ametabolic state induced by unfavorable<br />
environmental conditions, is a common adaptation<br />
especially among limno-terrestrial tardigrade species<br />
(see e.g. Wright 2001). Four cryptobiosis inducing<br />
physical extremes are traditionally recognized: dehydration<br />
(anhydrobiosis), extremely low temperatures<br />
(cryobiosis), lack of oxygen (anoxybiosis) and high salt<br />
concentration (osmobiosis) (Keilin 1959), with desiccation<br />
induced anhydrobiosis and freezing induced cryobiosis<br />
being the most extensively studied states.<br />
Anhydrobiosis and cryobiosis are not equivalent phenomena<br />
and likely involve different mechanisms for<br />
protection of cells and tissues (Crowe et al. 1990,<br />
1992). Little is known of cryobiosis in tardigrades<br />
(Westh et al. 1991, Ramløv & Westh 1992, Westh &<br />
Kristensen 1992, <strong>Halberg</strong> et al. 2009b, Hengherr et al.<br />
2009, 2010), whereas a great deal of attention has been<br />
paid to anhydrobiosis (Wright et al. 1992, Wright<br />
2001, Rebecchi et al. 2007, Schill 2010). The anhydrobiotic<br />
state is characterized by the formation of a socalled<br />
tun with withdrawn legs and a longitudinally<br />
contracted body (see e.g. Bertolani et al. 2004). Tun<br />
formation is also seen in bdelloid rotifers, whereas<br />
desiccation tolerant nematodes coil into a tight spiral.<br />
Obviously, the ability to pack internal organs during<br />
tun formation is an important adaptation to desiccation.<br />
Importantly, tun formation is an active, regulated<br />
event and not merely an effect of water removal (Crowe<br />
1972). Along this line, our unpublished data show that<br />
when we expose the active state of the cryptobiotic<br />
tardigrade Richtersius coronifer (Richters, 1903) to<br />
water containing high levels of chemical substances, the<br />
tardigrades will respond by contracting their bodies<br />
initiating tun formation thus undergoing chemobiosis–a<br />
cryptobiotic response to environmental toxins. The<br />
formation of the tun is a critical and necessary event<br />
for tardigrades entering anhydrobiosis. Much more<br />
work needs to be done in order to understand the<br />
processes undertaken during transformation to this<br />
ametabolic state, from the molecular to whole organism<br />
level, anatomically as well as physiologically. In the tun<br />
state tardigrades may not only stand long periods of<br />
desiccation and exposure to toxic chemicals but also<br />
very low subzero temperatures, vacuum, high pressure,<br />
radiation, extreme pH, anoxia and to some extent high<br />
temperature (see e.g. Wright 2001, Rebecchi et al.<br />
2007). It has been suggested and to some degree shown<br />
that adaptations to these extremes may involve synthesis<br />
of bioprotectants in the form of selective carbohydrates<br />
and proteins, high levels of antioxidant enzymes<br />
and other free radical scavengers, biological membranes<br />
containing specific phospholipids as well as powerful<br />
DNA repair mechanisms (Westh & Ramløv 1991, Schill<br />
et al. 2004, Jönsson et al. 2005, Rizzo et al. 2010).<br />
In addition to cryptobiotic tardigrade species, we also<br />
find species that form cysts and enter diapause (see e.g.<br />
Møbjerg et al. 2007, Guidetti et al. 2008). Importantly,<br />
it has been shown that tardigrades even in their active<br />
states may be extremely tolerant to environmental stress<br />
(May et al. 1964, Jönsson et al. 2005, Horikawa et al.<br />
2006, <strong>Halberg</strong> et al. 2009b). Virtually nothing is known<br />
about the normal physiology of tardigrades, and<br />
answers to how they tolerate these extremes may<br />
actually be found here. We have recently shown that<br />
the littoral eutardigrade Halobiotus crispae handles<br />
large fluctuation in external salinity and avoids freezing<br />
by supercooling to around )20 °C in its active stage<br />
(<strong>Halberg</strong> et al. 2009b). This species is characterized by<br />
seasonal cyclic changes in morphology and physiology<br />
known as cyclomorphosis, one of the cyclomorphic<br />
stages being freeze tolerant (Kristensen 1982, Møbjerg<br />
et al. 2007, <strong>Halberg</strong> et al. 2009b). The morphological<br />
changes occurring during cyclomorphosis in H. crispae<br />
in some respects resemble the formation of dauer larvae<br />
in C. elegans (see e.g. Cassada & Russell 1975, Burnell<br />
et al. 2005).<br />
Cryptobiosis in tardigrades<br />
Many experiments on tardigrade cryptobiosis have been<br />
performed on the eutardigrade Richtersius coronifer.<br />
R. coronifer, also known as the giant yellow water bear,<br />
has a body length of up to 1 mm (Fig. 4a–c). Both males<br />
and females are present in some populations, but, as<br />
commonly found among eutardigrades, several populations<br />
reproduce by parthenogenesis. R. coronifer lives in<br />
moss in alpine and arctic environments and is furthermore<br />
numerous in moss on carbonated bedrock in dry<br />
Swedish lowland areas known as Alvar (see e.g. Westh<br />
& Kristensen 1992, Jönsson et al. 2001). R. coronifer is<br />
a true cryptobiont, tolerating extreme desiccation as<br />
imposed by e.g. space vacuum conditions (Jönsson et al.<br />
2008, Persson et al. 2011) and it additionally survives<br />
exposures to very low temperatures as encountered by<br />
transfers into liquid nitrogen (approx. )196 °C) in the<br />
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Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 413
Adaptation to extreme environments in tardigrades Æ<br />
N. Møbjerg et al. Acta Physiol 2011, 202, 409–420<br />
(a) (b) (c)<br />
st<br />
pb<br />
eo<br />
A<br />
P<br />
A<br />
P<br />
A<br />
P<br />
mg<br />
st<br />
pb mg<br />
*<br />
*<br />
*<br />
(d) (e) (f)<br />
st<br />
st<br />
st<br />
pb<br />
eo<br />
pb<br />
eo<br />
pb<br />
mg<br />
mg<br />
mg<br />
go<br />
* * * *<br />
*<br />
* * *<br />
*<br />
Figure 4 Cryptobiotic and non-cryptobiotic survival in extreme environments. (a–c): Microscopy of Richtersius coronifer from<br />
Stora Alvar on the Swedish island Öland in the Baltic Sea. Richtersius coronifer has been a model tardigrade for investigations on<br />
cryptobiotic survival. (a) Active state; (b) light microscopy and (c) scanning electron microscopy of the cryptobiotic tun state<br />
induced by desiccation. In this state R. coronifer e.g. tolerates complete desiccation as experienced by vacuum condition and<br />
freezing in liquid nitrogen. (d–f): Microscopy of Halobiotus crispae from the Danish population at Vellerup Vig, Denmark. This<br />
tardigrade is one of a few species of eutardigrades that have secondarily invaded the marine environment. Halobiotus crispae in the<br />
active stage (d) uniquely adapted to cope with profound changes in ambient salinity occurring in tidal and subtidal habitats. Upon<br />
transfers to dilute salt water solutions, active stage H. crispae swell (e) and subsequently regulate their body volume to near control<br />
conditions. Halobiotus crispae is characterized by appearing in different cyclomorphic stages. The pseudosimplex 1 stage (f) is freeze<br />
tolerant. eo, esophagus; mg, midgut; pb, pharyngeal bulb; st, stylet. Asterisks mark the position of three Malpighian tubules<br />
presumably involved in osmoregulation. Scale bars: 50 lm.<br />
tun as well as active hydrated state, however,with the<br />
tuns tolerating considerable longer time of exposure<br />
(Ramløv & Westh 1992, Persson et al. 2011). The<br />
anhydrobiotic state survives temperatures of up to<br />
approx. 70 °C for 1 h, but survival rapidly decreases<br />
when the temperature exceeds 70 °C, and no specimens<br />
survives exposure to 100 °C (Ramløv & Westh 2001).<br />
The increase in life span offered by anhydrobiosis in this<br />
species seems to be restricted to approx. 5 years (Westh<br />
& Kristensen 1992). This is less than the cryptobiotic<br />
life expansion observed for the heterotardigrade Echiniscus<br />
testudo (Doyère, 1840), for which we have<br />
revived specimens from moss cushions dried for approx.<br />
20 years (Jørgensen et al. 2007). What then sets the<br />
limits for cryptobiotic survival Probably accumulated<br />
damage to DNA and other molecules as well as to<br />
tissues and organs obtained during the ametabolic state.<br />
This includes damage obtained through oxidative processes<br />
as well as predation, bacterial and fungal<br />
infections. A recent investigation in Paramacrobiotus<br />
richtersi (Murray, 1911) has shown that regulation of<br />
antioxidant metabolism likely plays an important role<br />
in defense against the potential oxidative damage<br />
associated with dehydration (Rizzo et al. 2010). It has<br />
long been known that there is a positive correlation<br />
between the time spent in the anhydrobiotic state and<br />
the time required for recovering active life after rehydration<br />
(Crowe & Higgins 1967). Recent investigations<br />
indicate that the longer the time spent in anhydrobiosis,<br />
the more damage is inflicted to DNA (Neumann et al.<br />
2009, Rebecchi et al. 2009b), which would explain the<br />
prolonged recovery time, indicating that considerable<br />
repair of DNA (and other molecules) occurs in the postanhydrobiotic<br />
state.<br />
Trehalose accumulation. Current knowledge suggests<br />
that specific carbohydrates and proteins are important<br />
in protecting the cells from damage encountered during<br />
414<br />
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Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Acta Physiol 2011, 202, 409–420<br />
N. Møbjerg et al. Æ<br />
Adaptation to extreme environments in tardigrades<br />
entry and exit of cryptobiosis. Bioprotectants may<br />
interact directly with macromolecular structures such<br />
as membranes, DNA and proteins, or they may act as<br />
osmolytes during osmotic or dehydration stress. As has<br />
been shown for other cryptobiotic invertebrates, noticeably<br />
in the cysts of the brine shrimp Artemia franciscana<br />
Kellogg, 1906, some, but not all, cryptobiotic<br />
tardigrades accumulate the dissacharide trehalose during<br />
desiccation (Clegg 1965, Westh & Ramløv 1991,<br />
Hengherr et al. 2008, Jönsson & Persson 2010). Trehalose<br />
has been proposed to act as a molecular stabilizer<br />
replacing water and to further stabilize cellular structure<br />
through the formation of amorphous glasses, a<br />
process known as vitrification (Clegg 2001). The highest<br />
trehalose concentrations measured in anhydrobiotic<br />
tardigrades ranges from 2.3% d.w. in R. coronifer<br />
and Macrobiotus krynauwi Dastych and Harris, 1995<br />
to 2.9% d.w. in Macrobiotus islandicus Richters, 1904<br />
(Westh & Ramløv 1991, Jönsson & Persson 2010).<br />
These trehalose values are, however, relatively low<br />
when compared with the above mentioned crustacean,<br />
which accumulates the disaccharide in concentrations of<br />
around 15% d.w. Moreover, trehalose levels are barely<br />
measurable and do not increase during dehydration in<br />
the cryptobiotic Milnesium tardigradum (see Hengherr<br />
et al. 2008, Jönsson & Persson 2010). The latter<br />
investigations, analysing trehalose contents in eutardigrades<br />
as well as heterotardigrades, show that<br />
trehalose accumulation does not represent a universal<br />
protective mechanism enabling tardigrades to undergo<br />
cryptobiosis.<br />
Expression of heat-shock proteins. Cryptobiosis has<br />
been suggested to rely on the synthesis of molecular<br />
chaperones such as heat-shock-proteins, which may<br />
assist folding of newly synthesized proteins, control<br />
their final intracellular location, as well as protect them<br />
from stress-associated denaturation and aid in renaturation<br />
(Clegg 2001). During entrance into anhydrobiosis<br />
Ramløv & Westh (2001) described the upregulation of<br />
a protein with a molecular weight of approx. 71 kDa in<br />
R. coronifer and proposed that this protein might<br />
belong to the heat-shock-protein (Hsp70) family. Schill<br />
et al. (2004) subsequently investigated RNA expression<br />
patterns of three Hsp70 isoforms in active and anhydrobiotic<br />
states of M. tardigradum as well as during<br />
entrance into and exit out of anhydrobiosis. Only one<br />
of these isoforms (isoform 2) was significantly induced<br />
by the transition from the active to cryptobiotic state<br />
and interestingly, it showed a considerable increase in<br />
expression during the post-cryptobiotic phase, while<br />
the other isoforms were down regulated during cryptobiosis<br />
as well as in transitional states. Jönsson &<br />
Schill (2007) used an immuno-westernblot method to<br />
quantify the induction of Hsp70 in R. coronifer in<br />
response to desiccation, ionizing radiation and heating.<br />
They reported elevated levels of Hsp70 following both<br />
heat and radiation treatment as well as in tardigrades<br />
rehydrated after a period of desiccation. Noticeably,<br />
the authors found that tardigrades in the desiccated<br />
state had reduced Hsp70 levels as compared to the nontreated<br />
control group and accordingly suggested that<br />
Hsp70 may be involved in the repair processes after<br />
desiccation rather than acting as a biochemical stabilizer<br />
in the dry state. Based on the M. tardigradum EST<br />
library, several additional heat-shock proteins have<br />
been identified, including two a-crystalline heat-shock<br />
proteins, and the relative abundance of the transcripts<br />
coding for these stress proteins have been investigated<br />
during phases of dehydration and rehydration (Reuner<br />
et al. 2010). The results obtained suggested a limited<br />
role for heat-shock proteins in the desiccation tolerance<br />
of M. tardigradum. The authors found a variable<br />
pattern of expression with most of the candidate genes<br />
being down regulated, and only one of the genes (Mthsp90),<br />
being significantly upregulated in the dehydrated<br />
state (Reuner et al. 2010). Comparable studies<br />
in Paramacrobiotus richtersi did not show evidence for<br />
an increased expression of either Hsp70 or Hsp90<br />
between hydrated and dehydrated animals (Rizzo et al.<br />
2010). Additionally, specimens of P. richtersi sent into<br />
space (Foton-M3 mission) revealed no significant<br />
change as compared to ground controls in the expression<br />
of these heat-shock proteins (Rebecchi et al.<br />
2009a). These contrasting results indicate that Hsp<br />
expression is species specific. A general role in the<br />
cryptobiotic survival of tardigrades can at present not<br />
be attributed to these stress proteins. Nevertheless, a<br />
synergistic effect between HSPs and other bioprotectants<br />
such as trehalose or LEA (late-embryogenesis<br />
abundant) proteins might exist (see e.g. Goyal et al.<br />
2005). Notably, LEA proteins have been described in<br />
many other desiccation tolerant organisms from plants<br />
to arthropods and might analogously be functionally<br />
important in tardigrades (see e.g. Schill 2010, Warner<br />
et al. 2010). Indeed, a putative LEA protein has<br />
recently been detected in M. tardigradum (Schokraie<br />
et al. 2010). Consequently, future studies on these and<br />
other bioprotectants might offer additional clues in our<br />
search to understand the phenomenon cryptobiosis.<br />
Obviously, cryptobiosis is far from understood and<br />
much more research is needed in order to understand<br />
the mechanisms underlying this state. Interestingly, a<br />
preliminary study from the Kumamushi Genome<br />
Project on R. varieornatus suggests that comparative<br />
metabolome profiling of active and anhydrobiotic<br />
states may provide a novel insight into anhydrobiosis<br />
(Arakawa et al. 2009). Additionally, clues to cryptobiotic<br />
survival may be found in the extreme tolerance<br />
seen in active state tardigrades.<br />
Ó 2011 The Authors<br />
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Adaptation to extreme environments in tardigrades Æ<br />
N. Møbjerg et al. Acta Physiol 2011, 202, 409–420<br />
Non-cryptobiotic survival in tardigrades<br />
While tardigrades are well known for their abilities to<br />
cope with extreme environmental conditions by entering<br />
cryptobiosis, little focus has been on their ability to<br />
sustain metabolism and remain active during fluctuating<br />
external circumstances. In this context it is interesting to<br />
note that tardigrades are genuine aquatic animals – they<br />
are dependent on free water to be in their active feeding<br />
and reproducing states. Truly terrestrial conditions are<br />
only survived following entry into the cryptobiotic<br />
state. Furthermore, limno-terrestrial tardigrades that<br />
have the ability to enter the tun state may do so in<br />
response to environmental challenges (e.g. exposure to<br />
chemical substances) that in marine species would not<br />
force the animals into cryptobiosis. In the following we<br />
discuss what is currently known about the physiology<br />
and stress tolerance of tardigrades in their active states.<br />
Radiation tolerance. Investigations on radiation tolerance<br />
in Richtersius coronifer have revealed that exposure<br />
to c-radiation at doses up to 1 kGy does not affect<br />
survival of desiccated nor hydrated animals, with<br />
hydrated animals tolerating doses of up to 5 kGy (Jönsson<br />
et al. 2005). Horikawa and co-workers revealed that<br />
both hydrated and desiccated Milnesium tardigradum<br />
survive doses of c-radiation of more than 5 kGy, and up to<br />
8 kGy of heavy ion radiation (Horikawa et al. 2006).<br />
These and previous studies indicate that hydrated animals<br />
are just as good or even better at tolerating radiation. This<br />
indicates that radiation tolerance is not due to biochemical<br />
protectants associated with the cryptobiotic state, but<br />
suggests that tardigrades rely on efficient and yet unidentified<br />
mechanisms of DNA repair (Jönsson et al. 2005,<br />
Horikawa et al. 2006). Several recent studies involved<br />
with the BIOPAN 6/Foton-M3 mission in 2007, funded<br />
by the European Space Agency, have investigated the<br />
impact on survival of tardigrades exposed to space<br />
conditions (Jönsson et al. 2008, Rebecchi et al. 2009a,<br />
Persson et al. 2011). During this mission, cryptobiotic<br />
tardigrades (as well as nematodes and rotifers) were sent<br />
into low earth orbit and exposed to space vacuum and<br />
cosmic radiation. The three studies revealed discrepancies<br />
in survival rates likely reflecting differences between<br />
tardigrade species and experimental setups, however;<br />
they unanimously conclude that tardigrades can survive<br />
the rigors of space, and that M. tardigradum is likely the<br />
most resistant species with embryos as well as adults<br />
tolerating space conditions (see discussion in Persson<br />
et al. 2011). Detailed knowledge of the life history of this<br />
predatory tardigrade is available as a result of a comprehensive<br />
study by Suzuki (2003).<br />
Osmoregulation. Experiments on the marine eutardigrade<br />
Halobiotus crispae have revealed an extraordinary<br />
tolerance to perturbations in body volume and osmotic<br />
pressure (<strong>Halberg</strong> et al. 2009b). The tidal habitat in<br />
which this tardigrade lives is characterized by large<br />
fluctuations in abiotic factors; most noticeably, alterations<br />
are seen in salinity and temperature (Møbjerg et al.<br />
2007). One way of coping with these extremes would be<br />
to enter cryptobiosis. However, H. crispae is not a<br />
cryptobiont. In the active stage the tardigrade handles<br />
extremes by expending energy on active regulatory<br />
mechanisms. In addition, the so-called pseudosimplex 1<br />
(P1) stage of this animal is freeze tolerant (<strong>Halberg</strong> et al.<br />
2009b); it is not, however, a cryobiont as the P1 stage does<br />
not tolerate gradual freezing to )80 °C. Cryobiosis is<br />
defined by the apparent absence of a lower lethal<br />
temperature (Wright 2001). The P1 stage is distinctly<br />
characterized by a double cuticle and closed mouth and<br />
cloaca (Kristensen 1982, Møbjerg et al. 2007).<br />
Halobiotus crispae kept at a salinity of 20 ppt have an<br />
extensive ability to supercool (avoid freezing) down to<br />
around )20 °C, enabling active stage animals to withstand<br />
subzero temperatures without freezing (<strong>Halberg</strong><br />
et al. 2009b). Similar supercooling points have been<br />
reported from limno-terrestrial eutardigrades (Macrobiotus,<br />
Paramacrobiotus and Milnesium), whereas heterotardigades<br />
(Echiniscus) had slightly higher points<br />
(Hengherr et al. 2009). Much higher supercooling<br />
points of )6.7 °C and )7.4 °C were originally observed<br />
in respectively Richtersius coronifer and Bertolanius<br />
nebulosus (Westh & Kristensen 1992). These high<br />
supercooling points are likely the result of the presence<br />
of ice-nucleating agents initiating the freezing process<br />
(Westh et al. 1991). Figure 5 shows thermograms of H.<br />
crispae kept in seawater with a salinity of 20 ppt<br />
(Fig. 5a) and distilled water (Fig. 5b) prior to differential<br />
scanning calorimetry (DSC). Water content in the<br />
animals kept at 20 ppt and in distilled water was<br />
respectively 73% and 81%. The latter group of tardigrades<br />
had a higher supercooling point as compared to<br />
the group kept in 20 ppt saltwater, illustrating the<br />
expected correlation between water content and crystallization<br />
temperature in species without ice-nucleating<br />
agents (see e.g. Hengherr et al. 2009). Thus, the extent<br />
of supercooling and thereby the ability to avoid freezing,<br />
is coupled to osmoregulation.<br />
Halobiotus crispae has a large capacity to tolerate<br />
perturbations in ambient salinity making it an ideal<br />
model for the study of osmoregulation and volume<br />
regulation in tardigrades. Experiments on this species<br />
revealed tolerance to a wide range of salinities, with<br />
specimens of the Greenlandic population remaining<br />
active in solutions ranging from distilled water to<br />
saltwater with osmolalities up to 2000 mOsm kg )1<br />
(<strong>Halberg</strong> et al. 2009b). During experiments with transfers<br />
to strong hypotonic solutions, active stage H.<br />
crispae swell with up to 60% before regulating back to<br />
416<br />
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Acta Physiol 2011, 202, 409–420<br />
N. Møbjerg et al. Æ<br />
Adaptation to extreme environments in tardigrades<br />
(a) 15<br />
(a) 1400<br />
Differential heat flow (mW)<br />
Differential heat flow (mW)<br />
7.5<br />
0<br />
(b) 15<br />
7.5<br />
0<br />
Exotherm Exotherm<br />
Cooling<br />
Heating<br />
near control values (Fig. 4e). Similarly, specimens<br />
transferred into hypertonic solutions shrink and thereafter<br />
respond by regulating body volume. In a series of<br />
experiments with transfers into saltwater solutions with<br />
a salinity between 2 ppt (62 mOsm kg )1 ) and 40 ppt<br />
(1245 mOsm kg )1 ) the active stage tardigrades hyperregulated<br />
at all times (Fig. 6a) (<strong>Halberg</strong> et al. 2009b).<br />
Hyperregulation is likely a general feature of at least the<br />
eutardigrades, as our data on R. coronifer reveal that<br />
this species also keeps its body fluids hyperosmotic as<br />
compared to the surroundings (Fig. 6b). Our data<br />
indicate that this limno-terrestrial cryptobiont is less<br />
tolerant of high salinity solutions than the littoral H.<br />
crispae. When exposed to water with increasing salt<br />
Endotherm<br />
20 ppt.<br />
–40 –35 –30 –25 –20 –15 –10 –5 0 5<br />
Cooling<br />
Heating<br />
Endotherm<br />
0 ppt.<br />
–40 –35 –30 –25 –20 –15 –10 –5 0 5<br />
Temperature (°C)<br />
Figure 5 Supercooling in Halobiotus crispae. (a) Supercooling<br />
in H. crispae kept in seawater with a salinity of 20 ppt.<br />
Thermogram modified from <strong>Halberg</strong> et al. (2009b). (b) Halobiotus<br />
crispae kept in distilled water. Cooling and subsequent<br />
reheating of a sample containing 46 specimens of H. crispae<br />
from Vellerup Vig, Denmark kept in distilled water for 45 min<br />
prior to the transfer into pans for differential scanning calorimetry<br />
(DSC). Freezing ()12.2 °C) and melting ()1.2 °C)<br />
temperatures were estimated as onsets of peaks of respectively<br />
exothermic and endothermic events using DSC7 software<br />
(cooling rate 2 °C min )1 ) (DSC7; Perkin Elmer Inc., Wellesley,<br />
MA, USA).<br />
Hemolymph osmolality (mOsm kg –1 ) Hemolymph osmolality (mOsm kg –1 )<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
(b) 1400<br />
1200<br />
1000<br />
H. crispae<br />
Osmotic performance<br />
Isoosmotic line<br />
0<br />
0 200 400 600 800 1000 1200 1400<br />
800<br />
600<br />
400<br />
200<br />
R. coronifer<br />
Osmotic performance<br />
Isoosmotic line<br />
0<br />
0 200 400 600 800 1000 1200 1400<br />
External Osmolality (mOsm kg –1 )<br />
Figure 6 Osmoregulation in eutardigrades. (a) Measured hemolymph<br />
osmolality of active stage Halobiotus crispae from<br />
Vellerup Vig, Denmark as a function of external osmolality.<br />
Figure modified from <strong>Halberg</strong> et al. (2009b). (b) Hemolymph<br />
osmolality of Richtersius coronifer from Öland, Sweden as a<br />
function of external osmolality. External solutions with osmotic<br />
concentrations of 100, 200, 300 and 500 mOsm kg )1 were<br />
prepared from artificial seawater salt (Tropic Marin, Dr Biener<br />
GmbH, Germany). Each point on the graph represents<br />
mean SD of hemolymph osmolality measurements (nanolitre<br />
osmometry; Clifton Technical Physics, Hartford, NY, USA)<br />
made in tardigrades kept in demineralized water and the different<br />
salt water solution for 30 min. Five animals were used for<br />
hemolymph determination at each of the experimental solutions.<br />
Arrow indicates the upper lethal line for R. coronifer.<br />
concentrations, R. coronifer will become inactive and<br />
eventually die, exhibiting an upper lethal limit of<br />
around 500 mOsm kg )1 (Fig. 6b). In solutions ranging<br />
from demineralized water to salt water with an osmolality<br />
of 500 mOsm kg )1 , the tardigrade, however,<br />
maintains a consistent osmotic gradient of around<br />
170 mOsm kg )1 above that of the external environment<br />
(Fig. 6b). In comparison, H. crispae exposed to the<br />
same salinity range maintains an osmotic gradient of<br />
around 300 mOsm kg )1 above that of the surroundings<br />
(Fig. 6a). These data on two different species of<br />
tardigrades indicate that eutardigrades have a relatively<br />
Ó 2011 The Authors<br />
Acta Physiologica Ó 2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 417
Adaptation to extreme environments in tardigrades Æ<br />
N. Møbjerg et al. Acta Physiol 2011, 202, 409–420<br />
high water turnover, and that they excrete a hypoosmotic<br />
fluid – the likely organs involved in this<br />
excretion being Malpighian tubules and the gut system<br />
(Fig. 4) (Møbjerg & Dahl 1996, <strong>Halberg</strong> et al. 2009b).<br />
At present the composition of tardigrade extracellular<br />
fluids (as well as intracellular fluids) are unknown.<br />
Obviously, information on the composition of the<br />
hemolymph is much needed for our understanding of<br />
tardigrade physiology and future discussions on how<br />
tardigrades regulate their body fluids and especially<br />
which osmolytes they are regulating. It is likely that the<br />
osmotic pressure obtained through hyperregulation is<br />
partly built by organic solutes, which in turn may act as<br />
bioprotectants or may be important for e.g. the above<br />
mentioned ability to supercool.<br />
In summary, recent research has shown that tardigrades<br />
in their active state may tolerate large fluctuations<br />
in abiotic factors and cope with environmental<br />
alterations by staying active and maintaining high<br />
metabolic rates. It would seem that these extraordinary<br />
organisms have two ways of handling environmental<br />
extremes; one way is entering a dormant state, i.e.<br />
cryptobiosis or diapause/encystment and the other,<br />
likely requiring a high metabolic rate, is relaying on<br />
e.g. osmoregulation and DNA repair, while staying<br />
active. The mechanisms enabling tardigrades to withstand<br />
environmental extremes in their active state may<br />
provide clues to the much debated mechanism underlying<br />
the phenomenon of cryptobiosis. Intriguingly,<br />
adaptations to some of the harshest environments on<br />
Earth have given tardigrades the ability to survive<br />
conditions that by far exceed the extremes presented by<br />
the environment (e.g. extreme levels of ionizing radiation<br />
and extremely low temperatures), presenting an<br />
unresolved puzzle for contemporary biology.<br />
Conflict of interest<br />
There are no conflicts of interest.<br />
We would like to thank Peter Westh for help in generating the<br />
DSC data presented in Figure 5. Special thanks are due to the<br />
organizers of the August Krogh Symposium 2010: Erik Hviid<br />
Larsen, Ylva Hellsten and Jørgen Wojtaszewski. Funding came<br />
from the 2008 Faculty of Science, University of Copenhagen<br />
Freja-Programme and from the Carlsberg Foundation.<br />
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420<br />
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Paper IV
Haugan et al. BMC Developmental Biology 2010, 10:56<br />
http://www.biomedcentral.com/1471-213X/10/56<br />
RESEARCH ARTICLE<br />
Research article<br />
Functional characterization of the vertebrate<br />
primary ureter: Structure and ion transport<br />
mechanisms of the pronephric duct in axolotl<br />
larvae (Amphibia)<br />
Birgitte M Haugan, <strong>Kenneth</strong> A <strong>Halberg</strong>, Åse Jespersen, Lea R Prehn and Nadja Møbjerg*<br />
Open Access<br />
Abstract<br />
Background: Three kidney systems appear during vertebrate development: the pronephroi, mesonephroi and<br />
metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the<br />
induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine<br />
modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model.<br />
Results: We investigated structural as well as physiological properties of the pronephric duct. The key elements of our<br />
methodology were: using histology, light and transmission electron microscopy as well as confocal laser scanning<br />
microscopy on fixed tissue and applying the microperfusion technique on isolated pronephric ducts in combination<br />
with single cell microelectrode impalements. Our data show that the fully differentiated pronephric duct is composed<br />
of a single layered epithelium consisting of one cell type comparable to the principal cell of the renal collecting duct<br />
system. The cells are characterized by a prominent basolateral labyrinth and a relatively smooth apical surface with one<br />
central cilium. Cellular impalements demonstrate the presence of apical Na + and K + conductances, as well as a large K +<br />
conductance in the basolateral cell membrane. Immunolabeling experiments indicate heavy expression of Na + /K + -<br />
ATPase in the basolateral labyrinth.<br />
Conclusions: We propose that the pronephric duct is important for the subsequent modification of urine produced by<br />
the pronephros. Our results indicate that it reabsorbs sodium and secretes potassium via channels present in the apical<br />
cell membrane with the driving force for ion movement provided by the Na + /K + pump. This is to our knowledge the<br />
first characterization of the pronephric duct, the precursor of the collecting duct system, which provides a model of<br />
cell structure and basic mechanisms for ion transport. Such information may be important in understanding the<br />
evolution of vertebrate kidney systems and human diseases associated with congenital malformations.<br />
Background<br />
During the development from embryo to adult life vertebrates<br />
use a succession of kidney forms to maintain extracellular<br />
fluid homeostasis and simultaneously rid the<br />
body of nitrogenous wastes [1,2]. Three spatially and<br />
temporally different kidney generations form from the<br />
intermediate mesoderm in an anterior to posterior direction<br />
i.e. the pronephroi, mesonephroi and metanephroi<br />
[3]. The functional unit in these paired kidneys is the<br />
* Correspondence: nmobjerg@bio.ku.dk<br />
1 Department of Biology, University of Copenhagen, Universitetsparken, DK-<br />
2100 Copenhagen, Denmark<br />
Full list of author information is available at the end of the article<br />
nephron, and it is composed of a filtration unit and a<br />
renal tubule. Urine is produced by the filtration of blood<br />
in the filtration unit, followed by the selective reabsorption<br />
and secretion of ions, organic molecules and water<br />
across highly specialized epithelia of the renal tubule [4].<br />
Nephrons open into a ureter, and in the meso- and metanephros<br />
they do so via a collecting duct system [5,6]. This<br />
system is the site for the important final adjustment of<br />
the urine. Vertebrate kidneys may produce urine, which is<br />
either hypoosmotic (diluted), isoosmotic or hyperosmotic<br />
(concentrated) relative to the body fluids [7-10].<br />
This ability is a function of i) the evolutionary state of the<br />
© 2010 Haugan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons<br />
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in<br />
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nephrons and ii) the regulation of filtration and of the<br />
transport of inorganic ions, organic molecules and water<br />
across the renal epithelia.<br />
The first kidneys to form - the embryonic pronephric<br />
kidneys - are the functional kidneys of fish and amphibian<br />
larvae [11-16]. These are very simple kidneys composed<br />
of a single nephron. A characteristic of vertebrate<br />
kidney organogenesis is the development of a pronephric<br />
duct in association with each pronephros [3]. These ducts<br />
are the first or primary ureters of vertebrate kidney systems.<br />
They form the collecting duct system of the mesoand<br />
metanephroi, and they, and their derivates, are the<br />
key players in the induction of the nephrogenic mesenchyme,<br />
which forms these latter kidney generations. Few<br />
functional studies exist on the pronephros and functional<br />
studies of the duct are virtually lacking. Molecular studies<br />
have been directed at mapping genes expressed in different<br />
segments of the pronephric nephron, and several<br />
recent reviews have highlighted the potential use of this<br />
embryonic kidney in drug and human kidney disease<br />
assessment [17-23]. To date, there has been little focus on<br />
the role of the pronephric duct in urine modification and<br />
it remains to be shown whether transepithelial transport<br />
processes are present in this structure. Ultrastructural<br />
investigations have shown duct cells with the characteristics<br />
of an epithelium involved in active transport e.g.<br />
many mitochondria and surface expansions of the basolateral<br />
cell membranes [14,24]. In addition, gene expression<br />
assays have indicated high expression of transporters<br />
known to be involved in ion transport, such as the Na + /<br />
K + -ATPase and the ROMK channel [17,18,25-27]. Collectively,<br />
these data suggest that the pronephric duct may<br />
play an important role in regulation of extracellular fluid<br />
homeostasis. Therefore we ask the question: Is the duct<br />
involved in urine modification<br />
In amphibians the pronephros is a large organ, which is<br />
functional for a considerable time, before it degenerates.<br />
We investigate structural and functional characteristics of<br />
the pronephric duct in the freshwater amphibian Ambystoma<br />
mexicanum (axolotl). Members of the Ambystoma<br />
genus have been used as models for the study of pronephric<br />
structure, function, development and evolution<br />
for more than a century [24,28,29] and the formation and<br />
caudal migration of the pronephric duct in the axolotl has<br />
been thoroughly investigated [30-32]. Numerous functional<br />
studies exist on the mesonephric collecting duct<br />
system of both urodele and anuran amphibians, which<br />
provide detailed information on the transport characteristics<br />
of these segments [Reviewed in [13]]. Our histological<br />
examinations and dissections of axolotl larvae<br />
indicate that the pronephros is functional from the time<br />
of hatching to larval stage 54. We investigate duct morphology<br />
and cellular transport mechanisms present in<br />
larvae with functional pronephroi, and show that the primary<br />
ureter is important for urine dilution in the axolotl.<br />
The single cell type found in the ureter shows the characteristics<br />
of the vertebrate collecting duct system principal<br />
cell and our data indicate that it reabsorbs Na + and<br />
secretes K + .<br />
Results and Discussion<br />
Identification of functional pronephroi and pronephric<br />
ducts<br />
We determined the interval in which the axolotl pronephros<br />
and pronephric duct is functional by investigating<br />
kidney structure in freshly dissected larvae and in<br />
larvae prepared for histology (Figure 1 and 2). The pronephroi<br />
of axolotl larvae are paired organs located on<br />
each side of the dorsal aorta in the most anterior part of<br />
the body cavity. They are visible from the outside on the<br />
dorsal side of the larva as two small bulges behind the<br />
gills (Figure 1A). Each of the two kidneys are composed of<br />
a filtrating unit - a glomus originating from the dorsal<br />
aorta, and a single convoluted renal tubule opening into<br />
the coelom via two ciliated nephrostomes (Figure 1B).<br />
The pronephros is fully functional when the axolotl larva<br />
Figure 1 Axolotl larva stage 45 and 52. A. Dorsal view of recently<br />
hatched axolotl larva (stage 45; forelimb present as limb buds). The<br />
pronephroi can be seen as two small bulges behind the gills. B. Schematic<br />
representation of the pronephric nephron in the stage 52 larva<br />
as revealed from light- and transmission electron microscopy on serial<br />
section of plastic embedded tissue. The pronephros consists of an external<br />
glomus (gl) and a single renal tubule, which opens into the<br />
coelom (co) via two ciliated nephrostomes (ne). In this late larval stage<br />
the tubule is divided into two ciliated tubules (ci), two proximal tubule<br />
branches (pt), a common proximal tubule, a ciliated intermediate segment<br />
(is) and a distal tubule (dt). The distal tubule continues as the pronephric<br />
duct (pd), which leaves the confines of the pronephros and<br />
empties into the cloaca.
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A<br />
pt<br />
dt<br />
nc<br />
ne<br />
gl<br />
is<br />
25 m<br />
co<br />
B<br />
50 m<br />
C<br />
pt<br />
mr<br />
wd<br />
mv<br />
pd<br />
co<br />
bl<br />
nu<br />
mu<br />
15m<br />
5 m<br />
Figure 2 Histology of pronephros and pronephric duct. A. Cross section of a stage 54 larva (forelimb completely developed) revealing the filtration<br />
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<br />
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 characterized<br />
by possessing a ciliated intermediate segment (is). nc, notochord; dt, distal tubule; pt, proximal tubule. B. Longitudinal section of pronephric duct<br />
(stage 52 larva). Araldite section, 2 μm, stained with toluidine blue. The pronephric duct (pd) leaves the confines of the pronephros. co, coelom; mu,<br />
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<br />
consists of two cell types: principal cells and intercalated, mitochondria-rich cells (mr). C. Transmission electron microscopy of pronephric duct shown<br />
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<br />
basal labyrinth (bl) formed by the highly invaginated basal and to some extent lateral cell membranes. nu, nucleus.
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hatches from the egg at stage 44. This was confirmed by<br />
the appearance of blood cells in the capillaries of the glomus,<br />
which from embryonic stage 36 had a fully developed<br />
endothelium and a visceral layer with podocytes.<br />
From stage 44 the cilia of the nephrostomes points<br />
toward the lumen of the renal tubule, indicating a passage<br />
of fluid from the coelom. At this stage the kidney consists<br />
of an external glomus and a renal tubule with the following<br />
morphologically determined segments: two nephrostomes,<br />
each connected to a branch of proximal tubule, a<br />
common proximal tubule and a distal tubule. The pronephric<br />
duct runs caudally as an extension of the distal<br />
tubule opening into the cloaca. From the time of hatching<br />
the kidney was observed to increase in overall size due to<br />
further segmentation of the renal tubule i.e. the development<br />
of a ciliated intermediary segment; present from<br />
stage 52 (Figure 1B and 2A). The fully segmented pronephric<br />
tubule consists of the following morphological<br />
defined segments: two nephrostomial tubules, two proximal<br />
tubule branches, a common proximal tubule, a ciliated<br />
intermediary segment and a distal tubule (Figure<br />
2A). At stage 52 the mesonephros was clearly visible and<br />
functional as judged by glomerular maturation and presence<br />
of blood cells in the mesonephric glomerular capillaries.<br />
Hence, comparable to the situation found in<br />
anuran amphibians [14,33] the two kidney generations<br />
functionally overlap in axolotl larvae. At stage 54 the pronephros<br />
reaches its maximal size. Gonadal primordia<br />
were observed medioventral to the mesonephros (not<br />
shown). These were undifferentiated and sex determination<br />
was not possible. During the transition from stage 54<br />
to latter stages, characterized by fully developed hind<br />
limbs, overall pronephric tubule and glomus volume<br />
decreased, marking pronephric degeneration. Pronephric<br />
structure in stage 52-54 larvae of Ambystoma mexicanum<br />
resembled the description by Christensen (1964) of the<br />
functional pronephros in A. punctatum [24].<br />
Pronephric duct structure<br />
Structural examination of the pronephric duct in larvae<br />
with functional pronephroi revealed that the duct consists<br />
of a single cell type (Figure 2B and 2C). Thus, the<br />
heterocellularity with intercalated mitochondria-rich<br />
cells interposed between principal cells, characteristic of<br />
the collecting duct system of latter kidney generations, is<br />
not seen at this point (Figure 2B and 2C). This is comparable<br />
to the situation in the green toad, Bufo viridis - a<br />
terrestrial anuran amphibian [14]. The pronephric duct<br />
cells in A. mexicanum are approximately 20 μm high in<br />
early larval stages, but decrease in height to 10-15 μm in<br />
stage 52 and 54 larvae (Figure 2C). They have a relatively<br />
smooth apical surface. A single central cilium is present<br />
(not shown) in addition to small and sparse microvilli,<br />
which are more numerous at the point of the apical cell<br />
junctions. The nucleus is regularly shaped, centrally<br />
placed and contains a nucleolus and patches of heterochromatin.<br />
There is a conspicuous basal labyrinth, and<br />
lateral infoldings are seen as well (Figure 2C). The cytoplasm<br />
contains many mitochondria in addition to a Golgi<br />
complex and endoplasmatic reticulum. The morphology<br />
of the duct changes at the level of the mesonephros in late<br />
larval stages 52-54, revealing the presence of intercalated,<br />
mitochondria-rich cells (Figure 2B, insert).<br />
Ion transport mechanisms in the pronephric duct of larvae<br />
stage 46-54<br />
We examined if the pronephric duct participates in final<br />
urine modification with the aid of glass microelectrodes<br />
and ion substitution experiments in isolated and perfused<br />
ducts dissected from 22 larvae. Figure 3A is a frequency<br />
distribution of the membrane potential (V m ) of 64<br />
impaled cells. The data show a broad distribution with an<br />
Number of cells<br />
V m (mV)<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
-20<br />
-40<br />
-60<br />
-80<br />
-100<br />
A<br />
0<br />
-110 -90 -70 -50 -30<br />
V m (mV)<br />
B<br />
high K<br />
2 min<br />
V m (mV)<br />
-40<br />
-60<br />
-80<br />
-100<br />
high K<br />
Figure 3 Voltage recordings from single cells in isolated and perfused<br />
ducts. A. Frequency distribution of the membrane potential<br />
(V m ) in 64 cells from pronephric ducts dissected from axolotl larvae in<br />
the stage 46-54. B. Original voltage trace from single cell. Effect of raising<br />
bath [K + ] from 3 to 20 mmol/l. V m depolarized indicating the presence<br />
of a basolateral K + conductance. C. Summary data illustrating the<br />
effect on V m of the bath K + concentration step (n = 29).<br />
0<br />
-20<br />
C<br />
-10
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average V m between -75 and -80 mV. Transport characteristics<br />
of the duct did not seem to differ between early<br />
and late larval stages. As shown in figure 3B and 3C, raising<br />
the K + concentration in the basal solution from 3 to<br />
20 mmol/l resulted in a large, reversible depolarization of<br />
V m , revealing the presence of a large basolateral K + conductance.<br />
This indicates the presence of K + channels in<br />
the basolateral cell membrane. We examined whether the<br />
duct has luminal electrogenic transporters or channels in<br />
experiments with luminal K + and Na + steps. Figure 4<br />
V m (mV)<br />
0<br />
-20<br />
-40<br />
-60<br />
-80<br />
A<br />
low Na<br />
high K<br />
low Na<br />
shows the result of these substitution experiments. V m<br />
hyperpolarized upon a decrease in luminal Na + concentration<br />
from 102 to 7 mmol/l and depolarized upon an<br />
increase in luminal K + concentration from 3 to 20 mmol/<br />
l. This indicates that the luminal (apical) cell membrane<br />
possesses Na + as well as K + channels. In order to identify<br />
an ion pump, which can provide the driving force for<br />
luminal uptake of Na + as well as K + secretion, we isolated<br />
pronephric ducts and performed immunolabeling with<br />
an antibody directed against the Na + -K + -ATPase α-subunit.<br />
As shown in figure 5, the Na + -K + -ATPase is highly<br />
expressed in the pronephric ducts from these larvae, and<br />
is entirely localized to the lateral and highly invaginated<br />
basolateral cell membranes.<br />
Electrophysiological studies performed on the mesonephric<br />
collecting duct system of amphibians, have indicated<br />
that principal cells in aquatic urodeles, have a large<br />
apical Na + conductance and no, or very small, K + conductance.<br />
However, in terrestrial anurans, K + secretion<br />
through apical K + channels seems a major task of the<br />
principal cells [34-39]. In the current study we provide<br />
evidence for a principal cell, which has the characteristics<br />
of the mammalian collecting duct principal cell, i.e. with<br />
luminal Na + as well as K + conductances.<br />
-100<br />
5 min<br />
A<br />
0<br />
B<br />
-20 low Na -20 high K<br />
0<br />
C<br />
V m (mV)<br />
-40<br />
-60<br />
V m (mV)<br />
-40<br />
-60<br />
B<br />
25ìm<br />
-80<br />
-80<br />
-100<br />
-100<br />
Figure 4 Electrophysiological response to luminal fluid exchange.<br />
A. Original voltage trace from single cell. Effect of changing<br />
luminal [Na + ] from 102 to 7 mmol/l and luminal [K + ] from 3 to 20<br />
mmol/l. V m hyperpolarized upon the decrease in luminal [Na + ] and depolarized<br />
upon an increase in luminal [K + ]. This indicates that the luminal<br />
(apical) cell membrane possesses Na + as well as K + conductances.<br />
B. Summary data illustrating the effect on V m of concentration steps in<br />
luminal Na + (n = 6) and K + (n = 5).<br />
DAPI<br />
75ìm + +<br />
Na -K -ATPase<br />
Figure 5 Na + -K + -ATPase expression. A. The Na + -K + -ATPase is highly<br />
expressed in the pronephric ducts and entirely localized to the lateral<br />
and highly invaginated basolateral cell membranes. B. Na + -K + -ATPase<br />
expression in duct counterstained with DAPI. Images are three-dimensional<br />
reconstructions of the original CLSM z-series, showing a median<br />
longitudinal section of the pronephric duct.
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Conclusions<br />
We show that the pronephric duct, which is the first or<br />
primary ureter in all vertebrates, participates in urine<br />
adjustment in the axolotl. The cells constituting the duct<br />
are on the ultrastructural as well as cell physiological level<br />
comparable to principal cells found in the first segments<br />
of the mammalian collecting duct system [5,6,14,40-42].<br />
Notably, the pronephric duct lacks intercalated, mitochondria-rich<br />
cells. We propose that the duct is important<br />
for urine dilution through NaCl reabsorbtion, and<br />
that it in addition participates in the regulation of K +<br />
homeostasis. Figure 6 provides a model of the ion transport<br />
mechanisms, which we suggest are present in the<br />
duct cell. In this model a Na + -K + -ATPase in the basolateral<br />
cell membrane pumps Na + out of the cell and thereby<br />
provides the driving force for apical uptake of Na +<br />
through channels. The epithelial sodium channel (ENaC)<br />
is a likely candidate mediating this apical Na + uptake [43-<br />
45]. Na + exits the cell through the pump. K + is secreted<br />
through apical channels, and is recycled for the pump<br />
across the basolateral cell membrane. It is highly probable<br />
that ROMK channels, known to mediate K + secretion in<br />
the mammalian collecting duct system [42], and shown to<br />
be expressed in amphibian pronephric ducts [18], mediate<br />
K + secretion across the apical cell membrane. The<br />
active transport of Na + would create a lumen-negative<br />
transepithelial potential, and Cl - would presumably follow<br />
passively through the paracellular pathway of this epithelium<br />
[45]. This is to our knowledge the first characterization<br />
of the pronephric duct-the precursor of the<br />
+<br />
Na<br />
+<br />
Na<br />
+<br />
+<br />
K K<br />
ATP<br />
Figure 6 Suggested model for ion transport mechanisms in the<br />
cells of the vertebrate primary ureter. A Na + -K + -ATPase in the basolateral<br />
cell membrane pumps Na + out of the cell and thereby provides<br />
the driving force for apical uptake of Na + through channels. K + is secreted<br />
across the apical cell membrane through channels and recycled for<br />
the pump across the basolateral cell membrane.<br />
collecting duct system- which provides detailed information<br />
on cell structure and the basic mechanisms for ion<br />
transport.<br />
Methods<br />
Animals<br />
Specimens of the Mexican axolotl Ambystoma mexicanum<br />
(Shaw and Nodder, 1798) came from the animal stable<br />
of the August Krogh Building, part of the Campus<br />
Animal Research Facility at University of Copenhagen.<br />
Staging were performed according to [46] for embryos<br />
and the larvae were designated according to the degree of<br />
limb development as defined by the Ambystoma Genetic<br />
Stock Center, University of Kentucky; http://www.ambystoma.org.<br />
Larvae used for experiments, were in the stage<br />
44 to 54. They were euthanized by decapitation, followed<br />
by brain destruction, and were subsequently either prepared<br />
for histology or pronephric ducts (o.d. 50-70 μm,<br />
dissected length 300-1000 μm) were free hand dissected<br />
at 6°C and prepared for microperfusion experiments or<br />
immunolabeling. The pronephric ducts were dissected<br />
from the region in front of the mesonephros. Dissections<br />
were performed in media containing (in mmol/l): 75<br />
NaCl, 20 NaHCO 3 , 3.0 KCl, 1.8 CaCl 2 , 1.0 MgSO 4 , 0.8<br />
Na 2 HPO 4 , 0.2 NaH 2 PO 4 , 5.5 glucose, 3.3 glycine, 0.4 PVP,<br />
5.0 HEPES, titrated to pH 7.8 with NaOH.<br />
Histology, Light and Transmission Electron Microscopy<br />
Dissections were performed for every developmental<br />
stage of post-hatched axolotls from stage 44 to 57. In<br />
addition, we examined pronephric development in<br />
embryos. Light microscopic imaging of live and dissected<br />
larvae was performed using Zeiss Stemi 2000-CS and<br />
Leica MZ 16 microscopes equipped with an Infinity X<br />
Digital Camera (DeltaPix, Denmark). For histology, a<br />
total of 31 larvae in the stage 44-54 and 19 embryos from<br />
stage 21 to 44 were used. Specimens were fixed for 12<br />
hours at room temperature in an aldehyde fixative containing:<br />
1.2% glutaraldehyde, 1% paraformaldehyde, 0.05<br />
mol/l sucrose and 0.05 mol/l sodium cacodylate buffer<br />
(pH 7.4) and subsequently rinsed and stored in 0.05 mol/l<br />
sodium cacodylate buffer with 0.05 mol/l sucrose. Following<br />
1 hour's post fixation in 2% OsO 4 with 0.1 mol/l<br />
sodium cacodylate, specimens were dehydrated through a<br />
graded series of ethanol and propylenoxide and embedded<br />
in Araldite. For light microscopy, 1.5 μm sections<br />
were cut with glass knives on a Leica ultramicrotome EM<br />
UC6 and stained with toluidine blue. Ultrathin sections<br />
for transmission electron microscopy were cut on the<br />
same microtome with a Diatome diamond knife and subsequently<br />
stained with uranyl acetate and lead citrate.<br />
Transmission electron microscopic images were acquired<br />
using JEOL 100SX and JEOL JEM 1011 transmission electron<br />
microscopes. Kodak negatives obtained from the
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JEOL 100SX were digitized using an Epson Perfection<br />
4990 Photo scanner. The JEOL JEM-1011 was equipped<br />
with a GATAN digital camera. Digital images were optimized<br />
for contrast and color using CorelDraw X4.<br />
Microperfusion and cellular impalements<br />
Pronephric ducts were transferred to a bath chamber<br />
mounted on an inverted microscope and perfused in vitro<br />
with a set of pipettes made to fit the diameter of the<br />
tubules [37-39]. The tubule perfusion system used (Luigs<br />
& Neumann, Germany) was designed for accurate adjustment<br />
and movement of concentric pipettes [47]. Holding<br />
and perfusion pipettes were hand made from glass tubing<br />
(Drummond Scientific Company, PA, USA; holding<br />
pipettes: o.d. 2.1 mm, i.d. 1.6 mm; perfusion pipettes: o.d.<br />
1.2 mm, i.d. 1.0 mm) in a microforge equipped with a<br />
microscope (SM II/1 Puller from Luigs & Neumann, Germany).<br />
The tubules were perfused with a single-barrelled<br />
perfusion pipette containing a small glass capillary (o.d.<br />
0.3 mm, i.d. 0.2 mm, Drummond Scientific Company, PA,<br />
USA) connected to a manual Hamilton valve (Hamilton<br />
Co., NV, USA) with a four-way distribution system. Fluid<br />
exchange during luminal perfusion experiments was<br />
made through this capillary, the tip of the capillary being<br />
placed close to the opening of the perfusion pipette,<br />
ensuring fast fluid exchange in the tubule. Luminal perfusion<br />
was performed either by hand through a syringe<br />
connected to one of the ports in the valve or it was gravity-driven<br />
through a port connected to a fluid filled reservoir.<br />
The pressure applied was adjusted by monitoring<br />
tubule diameter, ensuring that the tubule neither collapsed<br />
nor over expanded. The bath was perfused at 8 ml/<br />
min and fluid exchange was performed through beakers<br />
attached to the outside of the Faraday cage, which surrounds<br />
the microperfusion set-up.<br />
Perfusions of tubule bath and luminal fluid were carried<br />
out at room temperature with a perfusion solution containing<br />
(in mmol/l): 75 NaCl, 25 NaHCO 3 , 3.0 KCl, 1.8<br />
CaCl 2 , 1.0 MgSO 4 , 0.8 Na 2 HPO 4 , 0.2 NaH 2 PO 4 . The perfusion<br />
solution was equilibrated with 1.8% CO 2 in O 2 and<br />
had a measured pH of 7.8. Experimental solutions with<br />
different sodium and potassium concentrations were prepared<br />
from this control solution. In the high K + solution,<br />
the K + concentration was raised to 20 mmol/l by equimolar<br />
substitution with Na + . In the low Na + solution with a<br />
[Na + ] of 6.8 mmol/l, Na + was replaced by choline or N-<br />
methyl-D-glucamine (NMDG + ) titrated with HCl.<br />
Pronephric duct cells were impaled across the basal cell<br />
membrane with KCl (1-3 mol/l) filled glass microelectrodes<br />
(R electrode ≈ 100 MΩ) and the membrane potential<br />
(V m ) was recorded with respect to the grounded bath.<br />
The microelectrodes were pulled from borosilicate glass<br />
with filament (Clark Electromedical, UK) on a vertical<br />
electrode puller (Narishige, Japan). Impalements were<br />
achieved by placing the microelectrode tip against the<br />
basal surface of the cell and gently taping the micromanipulator<br />
(Leitz, Germany) on which the electrodes were<br />
mounted. Voltage recordings were made by a WPI Duo<br />
773 electrometer (World Precision Instruments, USA)<br />
and digitized by a PowerLab/4S data acquisition system<br />
(ADInstruments, Australia). The recording of V m was<br />
accepted if the impalement was achieved by a sudden<br />
change in the potential read by the electrode and if the<br />
impalement was stable.<br />
The results are based on 64 cell impalements made in<br />
22 pronephric ducts dissected from 22 axolotl larvae in<br />
the stage 46-54. Figures were made in Origin 7.5 (Microcal,<br />
USA) and CorelDRAW X4.<br />
Immunolabeling, Confocal Laser Scanning Microscopy and<br />
3D reconstruction<br />
For identification and localization of the Na + -K + -ATPase,<br />
three separate immunolabeling experiments were conducted<br />
with equal results covering larvae in stages 46-54.<br />
In each experiment, pronephric ducts were isolated from<br />
four to five specimens and subsequently fixed on ice for<br />
approximately 60 minutes in 3% paraformaldehyde buffered<br />
to pH 7.4 with 0.1 mol/l sodium cacodylate. After<br />
being rinsed in PBS (perfusion solution), the tissue was<br />
incubated overnight at 4°C in PBS containing 10% goat<br />
serum (Invitrogen, CA, USA), 1% triton-X and the Na + -<br />
K + ATPase monoclonal mouse antibody α5-IgG (10 μg/<br />
ml). The α5 antibody developed by D.M. Fambrough was<br />
obtained from the Developmental Studies Hybridoma<br />
Bank developed under the auspices of the NICHD and<br />
maintained by The University of Iowa, Department of<br />
Biology (Iowa City, IA 52242). Following an extensive<br />
wash in PBS, the pronephric ducts were incubated with<br />
Alexa-488-conjugated goat anti-mouse IgG (1:100, Invitrogen,<br />
CA, USA) overnight at 4°C. Following rinses in<br />
PBS, the tissue was mounted on glass coverslips in Vecta<br />
shield (Vector Laboratories Inc., CA, USA). In some<br />
preparations, the renal tubules were incubated in DAPI<br />
(1:250, Invitrogen, CA, USA) for approximately 5 min<br />
and washed in PBS, prior to mounting. Image acquisition<br />
was performed on a Leica DM RXE 6 TL inverted microscope<br />
equipped with a Leica TCS SP2 AOBS confocal<br />
laser scanning unit, using the 488 nm line of an argon/<br />
crypton laser. The image series were processed and<br />
edited in the 3-D reconstruction software IMARIS (Bitplane<br />
AG, Zürich, Switzerland). The confocal images are<br />
based on 150-170 optical sections of a Z-series performed<br />
at intervals of 0.3-0.5 μm. All control preparations<br />
were negative for immunostaining.<br />
Abbreviations<br />
bl: basal labyrinth; ci: ciliated tubule; co: coelom; dt: distal tubule; gl: glomus; is:<br />
ciliated intermediate segment; nc: notochord; ne: nephrostome; nu: nucleus;
Haugan et al. BMC Developmental Biology 2010, 10:56<br />
http://www.biomedcentral.com/1471-213X/10/56<br />
Page 8 of 9<br />
mr: intercalated mitochondria-rich cell; mu: muscle; mv: microvilli; pd: pronephric<br />
duct; pt: proximal tubule; V m : membrane potential; wd: Wolffian duct.<br />
Authors' contributions<br />
NM conceived and designed the study. BMH and NM fixed larvae and performed<br />
the dissections for the structural investigation. BMH sectioned specimens,<br />
and made LM and TEM investigations with help from ÅJ and NM. NM<br />
made the microperfusion experiments and microelectrode impalements. KAH,<br />
LRP and NM carried out immunostaining experiments and KAH performed<br />
CLSM and prepared the 3D images. All authors participated in discussions and<br />
interpretation of the data. NM wrote the paper with inputs from the other<br />
authors. All authors read and approved the final version of the manuscript.<br />
Acknowledgements<br />
We sincerely thank Mrs. Jette Lyby Michelsen and Mrs. Kristine J.K. Sørensen for<br />
technical assistance. Funding came from the 2008 Faculty of Science, University<br />
of Copenhagen Freja-Programme and from the Carlsberg Foundation<br />
(grant numbers: 2004_04_0572; 2006_01_0534; 2008_01_0466). The funders<br />
had no role in study design, data collection and analysis, decision to publish, or<br />
preparation of the manuscript.<br />
Author Details<br />
Department of Biology, University of Copenhagen, Universitetsparken, DK-<br />
2100 Copenhagen, Denmark<br />
Received: 18 September 2009 Accepted: 27 May 2010<br />
Published: 27 May 2010<br />
© This BMC 2010 is article Developmental an Haugan Open is available Access et al; Biology licensee from: article 2010, http://www.biomedcentral.com/1471-213X/10/56<br />
distributed BioMed 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.<br />
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doi: 10.1186/1471-213X-10-56<br />
Cite this article as: Haugan et al., Functional characterization of the vertebrate<br />
primary ureter: Structure and ion transport mechanisms of the pronephric<br />
duct in axolotl larvae (Amphibia) BMC Developmental Biology 2010,<br />
10:56
Paper V
2803<br />
The Journal of Experimental Biology 212, 2803-2811<br />
Published by The Company of Biologists 2009<br />
doi:10.1242/jeb.029413<br />
Cyclomorphosis in Tardigrada: adaptation to environmental constraints<br />
<strong>Kenneth</strong> Agerlin <strong>Halberg</strong> 1 , Dennis Persson 1,2 , Hans Ramløv 3 , Peter Westh 3 , Reinhardt Møbjerg Kristensen 2<br />
and Nadja Møbjerg 1, *<br />
1 Department of Biology, University of Copenhagen, August Krogh Building, Universitetsparken 13, DK-2100 Copenhagen Ø,<br />
Denmark, 2 Natural History Museum of Denmark, Zoological Museum, Invertebrate Department, Universitetsparken 15, DK-2100<br />
Copenhagen Ø, Denmark and 3 Department of Nature, Systems and Models, University of Roskilde, Universitetsvej 1, DK-4000<br />
Roskilde, Denmark<br />
*Author for correspondence (e-mail: nmobjerg@bio.ku.dk)<br />
Accepted 9 June 2009<br />
SUMMARY<br />
Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of<br />
survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found<br />
cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called<br />
pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice<br />
formation proceeds rapidly at a crystallization temperature of around –20°C, revealing extensive supercooling in both stages,<br />
while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show<br />
that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage<br />
specimens (350–500 μm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was<br />
approximately 950 mOsm kg –1 . Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory<br />
volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a<br />
stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction,<br />
followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in<br />
hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water<br />
turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.<br />
Key words: cyclomorphosis, environmental stress, freeze tolerance, Halobiotus crispae, invertebrate, osmoregulation, tardigrade, volume<br />
regulation.<br />
INTRODUCTION<br />
The phylum Tardigrada comprises a group of hydrophilous micrometazoans,<br />
exhibiting close affinities to the euarthropod complex<br />
(Garey et al., 1996; Giribet et al., 1996; Mallatt et al., 2004). They<br />
occupy a range of niches in terrestrial, freshwater and marine<br />
environments from continental Antarctica (Convey and McInnes,<br />
2005) to the icecap of Greenland (Grøngaard et al., 1999) yet are<br />
especially abundant in mosses and lichens, where they constitute a<br />
major component of the cryptic fauna. Along with nematodes and<br />
rotifers, selected species of tardigrades exhibit a remarkable<br />
resilience against physical extremes, including low and high<br />
temperatures (–253°C to +151°C), ionizing radiation (up to<br />
6000Gy), vacuum, high pressure (up to 600MPa) and extreme<br />
desiccation (Ramløv and Westh, 1992; Westh and Kristensen, 1992;<br />
Ramløv and Westh, 2001; Schill et al., 2004; Horikawa et al., 2006;<br />
Jönson and Schill, 2007; Hengherr et al., 2008; Hengherr et al.,<br />
2009). However, the underlying physiological and biochemical<br />
mechanisms mediating these unique tolerances are still largely<br />
unidentified and represent an exciting challenge to contemporary<br />
biology.<br />
The marine eutardigrade Halobiotus crispae Kristensen 1982<br />
(Fig.1) colonizes tidal and subtidal habitats at numerous localities<br />
throughout the northern hemisphere (Møbjerg et al., 2007). This<br />
species is characterized by the appearance of seasonal cyclic<br />
changes in morphology, i.e. cyclomorphosis (Kristensen, 1982).<br />
Three distinct cyclomorphic stages have been recognized: (1) the<br />
active stage, (2) the pseudosimplex 1 (P1) stage and (3) the<br />
pseudosimplex 2 (P2) stage (Møbjerg et al., 2007). The defining<br />
physiological and biochemical characteristics of the individual stages<br />
are largely unknown but most likely correlate with dominant abiotic<br />
factors. A ubiquitous factor in all tidal and subtidal habitats is the<br />
large temporal and spatial fluctuations in external salinity. Yet,<br />
additional adaptations are necessary at high latitudes to ensure winter<br />
survival due to prolonged exposure to subzero temperatures. In the<br />
present study, we focus on the adaptive significance of the two main<br />
cyclomorphic stages in H. crispae, i.e. the active stage corresponding<br />
to the reproductive stage of other tardigrades and the P1 stage, a<br />
hibernation stage, which is comparable to the cysts found in other<br />
tardigrades (e.g. Guidetti et al., 2008). We do not deal with the P2<br />
stage, which is a sexual maturation stage that has not yet been<br />
reported from other tardigrades. Our preliminary data, however,<br />
suggest that this stage has a unique osmoregulatory profile. We show<br />
that the transition between the active and P1 stages is associated<br />
with profound changes in the physiology of the animal. The P1 stage<br />
is the only stage at which H. crispae survives internal ice formation.<br />
The active stage tolerates large shifts in ambient salinity and we<br />
investigate in detail the volume and osmoregulatory capacity of this<br />
stage. Our study presents the first detailed analysis of osmoregulation<br />
in tardigrades. The data show that active-stage H. crispae hyperregulate<br />
at any investigated external salinity, which would indicate<br />
excretion of dilute urine. This is likely to be a general feature of<br />
eutardigrades, which all possess Malpighian tubules.<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2804<br />
K. A. <strong>Halberg</strong> and others<br />
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<br />
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<br />
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).<br />
(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<br />
of the posterior area of the active stage, revealing the open tri-lobed cloaca (scale bar=10 μm).<br />
MATERIALS AND METHODS<br />
Tardigrade sampling<br />
Specimens of Halobiotus crispae were collected at regular intervals<br />
in the period 2005 to 2008 at Vellerup Vig, Isefjord, Denmark<br />
(55°44.209N, 11°51.365E) and at Nipisat Bay, Disko Island, West<br />
Greenland (69°25.934N, 54°10.768E) in August 2006. At Vellerup<br />
Vig, bottom samples were collected at an approximate depth of<br />
1.0–2.5m, while samples from Nipisat Bay were taken in the subtidal<br />
zone 3–4cm below low tide. With the exception of the data on<br />
osmotic stress tolerance presented in Fig.3, the obtained results are<br />
based entirely on animals collected at Vellerup Vig. Detailed<br />
descriptions of the two localities can be found elsewhere (Kristensen,<br />
1982; Møbjerg et al., 2007). Collected samples were freshwatershocked,<br />
decanted into a conical net (mesh size 62 μm) and<br />
transferred to Petri dishes. These dishes were supplied with fresh<br />
seawater (SW; 18–20ppt; pH8–9) and substrate from the locality.<br />
Tardigrades were localized using a Leica MZ16 stereomicroscope<br />
(Leica Microsystems, Wetzlar, Germany), and primarily found on<br />
the haptera of various filamentous algae present in the substrate.<br />
Isolated tardigrades from Vellerup Vig were kept at 4°C in SW for<br />
periods of up to 6months by regularly supplying fresh substrate<br />
from the locality. Different cyclomorphic stages (see Fig.1) were<br />
identified using an Olympus BX 51 interference-contrast microscope<br />
(Olympus, Tokyo, Japan).<br />
Scanning electron microscopy<br />
For scanning electron microscopy, specimens were fixed in 2.5%<br />
glutaraldehyde in 0.1moll –1 sodium cacodylate buffer (pH7.4),<br />
rinsed in the buffer and subsequently postfixed in 1% OsO 4 in<br />
0.1moll –1 sodium cacodylate buffer (pH7.4). Following fixation,<br />
the specimens were dehydrated through a graded series of ethanol<br />
and acetone. They were critical point dried (Bal-Tec CPD 030 critical<br />
point dryer, Bal-Tec Union, Balzers, Liechtenstein), mounted on<br />
aluminum stubs, sputter-coated with platinum–palladium (thickness<br />
~12nm) using a JEOL JFC-2300HR (JEOL, Tokyo, Japan) and<br />
examined in a JEOL JSM-6335F Field Emission scanning electron<br />
microscope (JEOL, Japan).<br />
Cold hardiness<br />
Six groups of 10 animals in both the active and P1 stage were<br />
transferred to Eppendorf tubes containing 1.5ml of SW from<br />
Vellerup Vig (20ppt). The samples were cooled to a constant<br />
temperature of –20°C at a cooling rate of approximately 1°Cmin –1<br />
(Block, 1991) and held at the target temperature for a period of 24h.<br />
The animals were thawed at room temperature and the survival<br />
assessed successively over the course of 96h. Animals retaining<br />
locomotory function or responsive to tactile stimuli following this<br />
period were considered alive.<br />
As subzero temperatures may be experienced for longer periods<br />
of time in Arctic habitats, the long-term survival at subzero<br />
temperatures was investigated. An additional six groups of 10<br />
specimens in each cyclomorphic stage were frozen to –20°C at a<br />
cooling rate of approximately 1°Cmin –1 and kept frozen for a total<br />
of 36days. Survival was assessed as described above.<br />
Differential scanning calorimetry<br />
The quantity and kinetics of ice formation associated with cooling<br />
of H. crispae from Vellerup Vig (20ppt) in active and P1 stages<br />
were studied by differential scanning calorimetry (DSC). Groups<br />
of 40–75 animals in each respective stage were transferred to 30μl<br />
aluminum DSC pans. In order to avoid dehydrating the animals<br />
during the removal of external water, the tardigrades were clumped<br />
in the central part of the DSC pan and excess water was subsequently<br />
removed with small pieces of delicate task wipes. Sample mass was<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Cyclomorphosis in tardigrades<br />
2805<br />
determined gravimetrically to the nearest 0.01mg using a fine-scale<br />
AT261 Deltarange (Mettler-Toledo, Columbus, OH, USA), yielding<br />
a total mass of 0.16–0.39mg (wet mass). The pans were sealed and<br />
transferred to a calorimeter (Perkin Elmer DSC 7 equipped with an<br />
Intercooler II mechanical cooling device), with an empty pan as<br />
reference. The calorimeter was calibrated with gallium [melting<br />
point, T m =29.78°C; melting enthalpy, H m =80.1 J g –1 ], water<br />
(T m =0°C) and n-decane (T m =–29.66°C). All scans involved cooling<br />
from 5°C to –40°C and subsequent reheating to 5°C at a cooling<br />
rate of 5°Cmin –1 . Samples were reweighed following the DSC run<br />
to ensure that no water loss had occurred. In order to determine the<br />
water content of samples following the freeze/thaw cycle, pans were<br />
punctured and dried at 80°C to a constant mass (dry mass). A<br />
minimum of three groups of animals in each stage was used (see<br />
Table1). The obtained thermograms (heat flows vs temperature)<br />
were analyzed with respect to crystallization temperature (T c ),<br />
amount of ice formed during the freezing exotherm (assuming that<br />
the latent heat of crystallization is the same as for pure water), T m<br />
and the osmotic pressure of the extracellular fluids as calculated by<br />
the standard DSC 7 software. Ice contents were calculated using<br />
the water content and the enthalpy of the freeze exotherm. The<br />
temperature dependence of the enthalpy of crystallization of water<br />
was taken into account as previously described (Kristiansen and<br />
Westh, 1991). For hemolymph osmolality calculations, the onset<br />
melting point measured by the DSC 7 software was determined<br />
according to the approach of Nicholajsen and Hvidt (Nicholajsen<br />
and Hvidt, 1994), in which the established melting point of the body<br />
fluid was derived from a standard curve made from predetermined<br />
NaCl solutions.<br />
Preparation of experimental solutions<br />
Salt water solutions of different osmotic pressure were made by<br />
successive dilution with distilled water or by evaporative reduction<br />
of 100% SW from the locality. Measurements of osmotic pressure<br />
were made in parallel on a Vapro 5520 vapor pressure osmometer<br />
(Wescor, Logan, UT, USA) and on a refractometer (S-1 Shibuya<br />
Land, Tokyo, Japan).<br />
Osmotic stress tolerance<br />
Animals collected from the Danish as well as the Greenlandic<br />
population were used for the current experiment. Groups of 20<br />
specimens were transferred to small glass vials containing 4ml of<br />
100% SW at 4°C, and specimens were either exposed to a gradual<br />
increase or decrease in salinity. The gradual changes in salinity were<br />
performed over the course of 4–5h by periodically replacing small<br />
volumes of SW with prefixed solutions of either a higher or lower<br />
salinity. Animal activity was concomitantly assessed. Animals were<br />
allowed a period of 20–40min of acclimatization following a salinity<br />
change prior to assessment. Individuals responsive to tactile stimuli<br />
were considered active. Five groups of specimens in active and P1<br />
stages were assessed at both hypo- and hyperosmotic salinities.<br />
Volume measurements<br />
Individual adult active-stage specimens of H. crispae (size<br />
300–500μm) from Vellerup Vig (20ppt) were visualized in an<br />
Olympus BX 51 microscope (Olympus), photographed using a<br />
digital camera (C-5050, Olympus) and subsequently exposed for<br />
set time periods of 30min, 1, 2, 4, 24 and 48h to saltwater solutions<br />
with salinities of 2ppt, 10ppt and 40ppt. The osmotic treatments<br />
were conducted in small glass vials containing 4ml of SW at 4°C.<br />
At the end of each time interval, individuals were transferred, in a<br />
drop of the appropriate solution, to glass microscope slides and<br />
photographed under cover slips for subsequent estimations of body<br />
volume. During photography, great care was taken to minimize the<br />
time spent by the animals under the cover slips, in order to avoid<br />
evaporative water loss, which would alter the osmotic pressure of<br />
the solution. The animals were ensured total freedom of movement.<br />
Following photography, individuals were returned to the respective<br />
salinities until the end of the next set time period, when the process<br />
was repeated. At each of the time intervals, 10–14 individuals were<br />
photographed at each of the SW treatments. Images were analyzed<br />
using DP-soft TM (Olympus), and total body volume was calculated<br />
according to the equation: V total =π(r 2 bodyh body +2r 2 legh leg ), where V<br />
is the volume of the specimen, r is the measured radius, and h is<br />
the measured length of the body and hind legs, respectively.<br />
In order to assess the behavioral response of H. crispae during<br />
osmotic shock and to quantify potential mortality related to the<br />
respective treatments, a separate experiment was performed.<br />
Specimens (N=10) were transferred directly to 2ppt, 10ppt, 20ppt<br />
(control) and 40 ppt, respectively, and animal activity was<br />
subsequently monitored over the course of 48h at 4°C. Individuals<br />
responsive to tactile stimuli at the above-mentioned set time periods<br />
were considered active and alive (see Fig.7). Three groups exposed<br />
to each treatment were assessed.<br />
Measurement of hemolymph osmolality<br />
Hemolymph osmolality was measured in individual tardigrades<br />
following exposure for 30min, 4 and 48h to the experimental<br />
solutions of 2ppt (62mOsmkg –1 ), 10ppt (311mOsmkg –1 ) and 40ppt<br />
(1245 mOsm kg –1 ). Six animals were used for osmolality<br />
determination in each of the experimental solutions. Six animals<br />
kept at 20ppt (623mOsmkg –1 ) served as a control. Hemolymph<br />
Table 1. Post-freeze survival and data obtained from differential scanning calorimetry on H. crispae from Vellerup Vig, Denmark (20 ppt)<br />
in the active and P1 stages<br />
Sample<br />
Post-freeze<br />
survival (%)<br />
(frozen for 24 h)<br />
Post-freeze<br />
survival (%)<br />
(frozen for<br />
36 days)<br />
Crystallization<br />
temp. (°C)<br />
Melting temp.<br />
(°C)<br />
Water content<br />
(%)<br />
Body-water<br />
frozen during<br />
freezing<br />
exotherm (%)<br />
Osmolality of<br />
extracellular<br />
–1<br />
fluids (mOsm kg )<br />
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)<br />
Active 0 (6) 0 (6) –21.6±2.1 (3) –4.48±0.15 (3) 68±4 (3) 69±5 (3) 975±36 (3)<br />
t-test (P0.05 (NS, not significant), P
2806<br />
K. A. <strong>Halberg</strong> and others<br />
samples (2–3nl) were collected by piercing individual specimens<br />
under immersion oil (type A; 150 centistoke; Cargille Laboratories,<br />
Cedar Grove, NJ, USA) using hand-pulled glass capillary tubes<br />
(capacity 1 μl; Micro-caps, Drummond Scientific Company,<br />
Broomall, PA, USA). Hemolymph samples were acquired through<br />
capillary action and subsequently ejected into immersion oil. Care<br />
was taken to ensure that the measurements were made on fluid<br />
originating from hemolymph alone and samples containing gut<br />
contents were discarded. Prior to sample collection, immersion oil<br />
was collected into the capillary tube in order to avoid any evaporative<br />
water loss. Using an Irvin loop, samples were immediately<br />
transferred in a drop of immersion oil into sample oil wells (type<br />
B; 1250 centistoke; Cargille Laboratories) of a calibrated nanoliter<br />
osmometer (Clifton Technical Physics, Hartford, NY, USA), and<br />
the osmolality (mOsmkg –1 ) was determined by melting point<br />
depression (MDP=1.858°COsm –1 ).<br />
Statistics<br />
Significant differences between experimental and control conditions<br />
were tested using unpaired, two-tailed t-tests, and a significance level<br />
of P≤0.05.<br />
RESULTS<br />
Survival at sub-zero temperatures<br />
The external morphology of H. crispae in the active and the P1<br />
stage is shown in Fig.1. Notably, the P1 stage is characterized by<br />
a conspicuous double cuticle, in which both the mouth and cloaca<br />
are closed by cuticular thickenings (Fig.1A–C). Post-freeze survival<br />
following both short- and long-term exposure to subzero<br />
temperatures is listed in Table1. An example of the quantitative<br />
kinetics of ice formation associated with the freeze/thaw cycle of<br />
P1-stage H. crispae kept at 20ppt is illustrated in Fig.2. The<br />
collective DSC analysis of the onsets and areas of peaks after the<br />
freeze/thaw cycles, together with the water content of the samples,<br />
provide the remaining results listed in Table1.<br />
Whereas animals in the active stage and P2 stage (data not shown)<br />
were intolerant of freezing, animals in the P1 stage were<br />
demonstrated to be freeze-tolerant. Post-freeze survival following<br />
24h exposure to –20°C was 53.3±15%; however, when prolonging<br />
the period spent at subzero temperatures to 36days, the survival of<br />
animals in the P1 stage decreased to 12.7±7% (Table1). Animal<br />
recovery was monitored over a period of 96 h following the<br />
freeze/thaw cycle; however, the majority of animals had resumed<br />
activity after a period of 48h. No additional recovery was monitored<br />
beyond 96h following any of the investigated treatments.<br />
Substantial ice formation proceeded rapidly following the first<br />
ice nucleation in the freeze-tolerant P1 stage with a mean of<br />
–19.6±3.1°C; as indicated by the large exotherm in Fig.2. At the<br />
given salinity, the amount of ice formed during the freezing<br />
exotherm amounted to approximately 60% of the body water<br />
(Table1). The absence of additional small exothermic peaks during<br />
the subsequent cooling to –40°C indicated that no additional ice<br />
formation occurred following the initial large freezing exotherm.<br />
The freeze exotherm lasted less than one minute. The initial<br />
separation of ice (T c ) occurred in the temperature range of –15.4 to<br />
–23.2°C.<br />
Only marginal differences in T c of animals in the P1 and active<br />
stage were observed (Table1), suggesting an absence of seasonal<br />
variations in ice-nucleating activity in H. crispae. In spite of invariant<br />
water contents between the two stages, the amount of water<br />
crystallizing during cooling in animals in the active stage was<br />
significantly higher than in animals in the P1 stage; however, the<br />
melting points of the two stages remained largely unaltered. The<br />
latter indicates that body fluid osmolality at a given external salinity<br />
is unaffected by the animal’s transition from the active to the P1<br />
stage.<br />
Volume- and osmoregulatory capacity<br />
Fig.3 shows the percentage of active animals of H. crispae following<br />
the exposure to gradual changes in the external salinity. When<br />
comparing Danish P1 and active-stage H. crispae, the active stage<br />
displayed a larger tolerance towards the more concentrated SW<br />
solutions and were slightly more tolerant of the very dilute solutions.<br />
Indeed, a significantly higher percentage of active-stage specimens<br />
Differential heat flow (mW)<br />
15<br />
7.5<br />
Exotherm<br />
Cooling<br />
Heating<br />
Endotherm<br />
0<br />
–40 –35 –30 –25 –20 –15 –10 –5 0 5<br />
Temperature (°C)<br />
Fig. 2. Representative thermogram displaying the exothermic and<br />
endothermic events associated with the cooling and heating of a sample<br />
containing P1-stage Halobiotus crispae from Vellerup Vig, Denmark<br />
(20 ppt). The crystallization and melting temperatures were estimated as<br />
the onsets of peaks (–17.5°C and –4.5°C, respectively) using the DSC 7<br />
software (see also Table 1). The very low crystallization temperatures<br />
measured exclude the presence of physiologically relevant ice-nucleating<br />
agents.<br />
Activity (%)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Active, Vellerup<br />
P1, Vellerup<br />
Active, Nipisat<br />
Osmolality (mOsm kg –1 )<br />
0 500 1000 1500 2000 2500<br />
*<br />
*<br />
0 10 20 30 40 50 60 70 80 90<br />
Salinity (ppt)<br />
100<br />
Fig. 3. Osmotic stress tolerance of Halobiotus crispae in the P1 and active<br />
stages. Active-stage () and P1-stage () specimens from the Danish<br />
population at Vellerup Vig. Active-stage () specimens from the<br />
Greenlandic population at Nipisat Bay. Data are means ± s.e.m. from five<br />
independent experiments. *, significantly different from Vellerup Vig P1<br />
stage (P
Cyclomorphosis in tardigrades<br />
2807<br />
remained active in the salinity spectrum of 0–3ppt and 45–60ppt,<br />
as compared with the animals in the P1 stage, and in general seemed<br />
less affected by the impositions of osmotic stress. Our preliminary<br />
data on P2 from Vellerup Vig show that this stage tolerates very<br />
dilute solutions better than the other stages, yet is the least tolerant<br />
of increases in salinity, becoming inactive at around 50ppt. As a<br />
comparison, the active stage from Greenland (Nipisat Bay), living<br />
in a more exposed habitat compared with the Vellerup Vig<br />
population, displayed an even higher tolerance to concentrated SW,<br />
with observed activity at 80ppt.<br />
In the following, we investigate in detail volume and<br />
osmoregulation in active-stage H. crispae from Vellerup Vig kept<br />
at a control salinity of 20ppt (Figs4–8). When exposed to a severe<br />
hypo-osmotic shock of 2 ppt (63 mOsm kg –1 ), the animals<br />
exhibited a large significant increase in body volume, resulting<br />
in a total body volume of 127±11% after merely 0.5h of exposure<br />
(Fig.4A,B). During this time period, animals became bloated and<br />
rigid and most specimens lost locomotory functions (Fig.7A).<br />
This passive uptake of water continued during the initial 2h of<br />
the exposure, culminating in a total body volume of 162±17%.<br />
However, after this period of time, a regulatory volume decrease<br />
(RVD) was observed. The total body volume of specimens was<br />
considerably reduced to 114±14% following 48h immersion and<br />
was not significantly different from the controls (Fig. 4B).<br />
Additionally, an increase in the number of active animals was<br />
observed, yet a considerable number of specimens remained<br />
passive throughout the treatment (Fig. 7A). Nevertheless,<br />
following a gradual return to 20 ppt, all animals regained<br />
locomotory functions. No mortality was observed in any of the<br />
treatments.<br />
Upon immersion of individual specimens into a less severe hypoosmotic<br />
media of 10ppt (311mOsmkg –1 ) a similarly significant<br />
increase in total body volume was observed; reaching a mean value<br />
of 132±11% after 0.5h exposure (Fig.5A,B). However, following<br />
this initial increase, total body volume stabilized, and a RVD was<br />
observed after 1–2h exposure. After 4h incubation, total body<br />
volume was 110±8%, which was not significantly different from<br />
the control situation. An effect on the locomotory functions was<br />
observed initially, as some animals displayed sluggish movements,<br />
yet only a limited number of animals were passive during this<br />
experiment (Fig.7B).<br />
When transferring H. crispae to a hyperosmotic solution of 40ppt<br />
(1245mOsmkg –1 ), a significant decrease in total body volume was<br />
observed (Fig.6A,B). Total body volume was significantly reduced<br />
to 66±9% following the first 0.5h of immersion and remained largely<br />
unaltered during the following hours of the treatment. After 24h,<br />
most specimens had displayed a regulatory volume increase,<br />
resulting in a mean total body volume of 82±9%, yet total body<br />
volume remained significantly different from the control situation<br />
even after 48h. Nevertheless, animal motility was little affected by<br />
the hypertonic shock, neither at the initial transfer nor throughout<br />
the rest of the experiment (Fig.7C).<br />
20 ppt,<br />
2 ppt,<br />
623 mOsm kg –1 62 mOsm kg –1<br />
A<br />
0h 0.5h 1h 2h 4h 24h 48h<br />
20 ppt,<br />
10 ppt,<br />
623 mOsm kg –1 311 mOsm kg –1<br />
A<br />
0h 0.5h 1h 2h 4h 24h 48h<br />
B<br />
Total body volume (%)<br />
200<br />
180<br />
160<br />
(12)<br />
(6)<br />
140<br />
(12)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
(6)<br />
(12)<br />
(12)<br />
(6)<br />
(10)<br />
0 1 2 3 4 20 30 40 50<br />
Exposure time (h)<br />
(10)<br />
(6)<br />
1400<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
Hemolymph osmotic pressure (mOsm kg –1 )<br />
B<br />
Total body volume (%)<br />
200<br />
180<br />
160<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
(6)(10)(12) (12)<br />
(10) (13)<br />
(6)<br />
(6)<br />
(11)<br />
0 1 2 3 4 20 30 40 50<br />
Exposure time (h)<br />
(6)<br />
1400<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
Hemolymph osmotic pressure (mOsm kg –1 )<br />
Fig. 4. (A) Halobiotus crispae in active stage from Vellerup Vig, Denmark<br />
(20 ppt). Light-microscopical images at different time points following<br />
exposure to 2 ppt (62 mOsm kg –1 ) of a single specimen (scale bar=100 μm).<br />
(B) Changes in total body volume () and measured internal osmolality ()<br />
over a period of 48 h following exposure to an external salinity of 2 ppt.<br />
Data are expressed as means ± s.d. Numbers in parentheses indicate the<br />
number of animals used for assessment of body volume and hemolymph<br />
osmolality at each time point.<br />
Fig. 5. (A) Halobiotus crispae in active stage from Vellerup Vig, Denmark<br />
(20 ppt). Light-microscopical images at different time points following<br />
exposure to 10 ppt (311 mOsm kg –1 ) of a single specimen (scale<br />
bar=100 μm). (B) Changes in total body volume () and measured internal<br />
osmolality () over a period of 48 h following an exposure to an external<br />
salinity of 10 ppt. Data are expressed as means ± s.d. Numbers in<br />
parentheses indicate the number of animals used for assessment of body<br />
volume and hemolymph osmolality at each time point.<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2808<br />
K. A. <strong>Halberg</strong> and others<br />
20 ppt,<br />
40 ppt,<br />
623 mOsm kg –1 1245 mOsm kg –1<br />
A<br />
B<br />
Total body volume (%)<br />
0h 0.5h 1h 2h 4h 24h 48h<br />
200<br />
180<br />
160<br />
140<br />
120<br />
100<br />
80<br />
60<br />
(6)<br />
(6)<br />
(12)<br />
(12) (14)<br />
(11)<br />
(13) (11)<br />
1400<br />
1200<br />
1000<br />
200<br />
40<br />
0 1 2 3 4 20 30 40 50<br />
Exposure time (h)<br />
Fig. 6 (A) Halobiotus crispae in active stage from Vellerup Vig, Denmark<br />
(20 ppt). Light-microscopical images at different time points following<br />
exposure to 40 ppt (1245 mOsm kg –1 ) of a single specimen (scale<br />
bar=100 μm). (B) Changes in total body volume () and measured internal<br />
osmolality () over a period of 48 h following an exposure to an external<br />
salinity of 40 ppt. Data are expressed as means ± s.d. Numbers in<br />
parentheses indicate the number of animals used for assessment of body<br />
volume and hemolymph osmolality at each time point.<br />
Notably, hemolymph osmotic pressure differed significantly<br />
from the control condition at all time points examined during the<br />
various salinity treatments (Figs4–6). Hemolymph osmolality of H.<br />
crispae varied in proportion to the gradient set up by the salinity<br />
transfer. The body fluids of animals kept under control conditions<br />
(20ppt, 623mOsmkg –1 ) had an osmolality of 926±29mOsmkg –1 ,<br />
(6)<br />
(6)<br />
800<br />
600<br />
400<br />
Hemolymph osmotic pressure (mOsm kg –1 )<br />
indicating that H. crispae hyper-regulates during steady-state<br />
conditions. This hyperosmotic regulation was independently<br />
confirmed by the DSC investigation in which the hemolymph<br />
osmolality was measured at 975±36mOsmkg –1 (Table1). The two<br />
measurements are not significantly different (t-test, P
Cyclomorphosis in tardigrades<br />
2809<br />
Hemolymph osmolality (mOsm kg –1 )<br />
1400<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
Active stage<br />
Osmotic performance<br />
Isoosmotic line<br />
0<br />
0 200 400 600 800 1000 1200 1400<br />
External osmolality (mOsm kg –1 )<br />
Fig. 8. Measured hemolymph osmolality of Halobiotus crispae (active stage<br />
from Vellerup Vig, Denmark) during steady-state conditions after 48 h<br />
acclimation to 2 ppt (63 mOsm kg –1 ), 10 ppt (311 mOsm kg –1 ), 20 ppt<br />
(623 mOsm kg –1 ) and 40 ppt (1245 mOsm kg –1 ), respectively. Each point<br />
represents the mean ± s.d. of the individual experiments. The broken line<br />
indicates the isoosmotic line at which no osmoregulation occurs.<br />
functionally characterized as a movable cyst (Kristensen, 1982;<br />
Møbjerg et al., 2007; Guidetti et al., 2008). In Denmark (Vellerup<br />
Vig), this stage is dominant during the summer months, presumably<br />
enabling H. crispae to withstand heat stress and oxygen depletion.<br />
The active stage, the only stage at which active feeding and sexual<br />
reproduction occur, is the dominant stage during the Greenlandic<br />
summer, whereas this stage is present during late winter and the<br />
spring months in Denmark (Møbjerg et al., 2007).<br />
Freeze avoidance and freeze tolerance<br />
Winter temperatures are frequently below the equilibrium freezing<br />
point of the surrounding seawater at least in some portions of the<br />
natural environments of H. crispae, and certain habitats may even<br />
become completely frozen for extended periods of the year<br />
(Kristensen, 1982). Enduring such hostile surroundings requires<br />
corresponding cold-tolerance strategies that enable long-term<br />
survival. Traditionally, two main options are exploited by<br />
ectothermic animals when faced with subzero temperatures, i.e.<br />
freeze avoidance and freeze tolerance (Lee, 1991). When exposing<br />
animals in the P1 stage of H. crispae to temperatures below the<br />
equilibrium freezing point (T c ) of their body fluids, freeze tolerance<br />
is demonstrated, indicating that winter survival could involve<br />
extracellular ice formation in this species. However, the finding that<br />
mortality increases with prolonged exposure to subzero temperatures<br />
suggests that the consequent damages accumulate in proportion to<br />
the time spent exposed to freezing conditions. This observation is<br />
likely explained by the depletion of essential metabolites and is of<br />
particular interest in Arctic habitats in which subfreezing<br />
temperatures have to be endured for long periods of time.<br />
In freeze-tolerant organisms, ice formation is usually promoted<br />
at relatively high subzero temperatures (–2 to –10°C) by icenucleating<br />
agents present in the extracellular fluid (Zachariassen,<br />
1985; Block, 1991; Westh and Kristensen, 1992). The adaptive<br />
advantage of such a strategy is that the process of ice formation<br />
proceeds relatively slowly at relatively high temperatures, enabling<br />
the organism to maintain the damage associated with freezing within<br />
tolerable boundaries. Indeed, both the localization and the amount<br />
of ice formed in freeze-tolerant organisms are usually under tight<br />
control. Surprisingly, our calorimetric investigation of the freezetolerant<br />
P1 stage reveals that ice crystallization occurs at<br />
approximately –20°C, excluding the presence of any physiologically<br />
relevant ice-nucleating agents in this stage. In fact, the very low<br />
crystallization temperatures measured in both stages suggest that<br />
the capacity for supercooling is maintained throughout the majority<br />
of the year, which is in general contrast to the pattern observed in<br />
most other freeze-tolerant invertebrates (Block, 1991; Westh and<br />
Kristensen, 1992; Ramløv et al., 1996). However, in spite of an<br />
invariant melting point and water content between the two stages,<br />
the amount of water crystallized during cooling to –40°C appears<br />
to be about 60% for animals in the P1 stage and 70% for animals<br />
in the active stage (see Table 1). Consequently, the cellular<br />
dehydration induced by freezing is expected to be much higher in<br />
animals in the active stage, compared with that of specimens in the<br />
P1 stage. This reduction in ice accumulation in specimens in the<br />
P1 stage could potentially be explained by an increased production<br />
of macromolecules [which kinetically inhibits ice formation but has<br />
negligible effect on the melting temperature (see Westh and<br />
Kristensen, 1992)], as compared with the active stage. Whether this<br />
observation alone explains the observed freeze tolerance is difficult<br />
to determine. Selected cryptobiotic species of tardigrades, nematodes<br />
as well as some freeze-tolerant insects tolerate as much as 80% of<br />
the body water being converted into ice (Westh and Kristensen,<br />
1992; Ramløv and Westh, 1993; Wharton and Block, 1997;<br />
Hengherr et al., 2009).<br />
The apparent morphological difference between the two stages<br />
is similarly relevant in regard to the observed freeze tolerance. The<br />
P1 stage is formed from an incomplete molt in which both the mouth<br />
and cloaca become sealed by cuticular thickenings (see Fig.1), and<br />
the gut content is often shed prior to this transition. In nature, ice<br />
nucleation can be initiated by a wide range of exogenous substances<br />
(Wharton and Worland, 1998). Consequently, the additional layer<br />
of cuticle could increase the capacity to avoid inoculative freezing<br />
in animals in the P1 stage, as has been demonstrated for eggs of<br />
the nematode Panagrolaimus davidi Timm 1971 (see Wharton,<br />
1994). Indeed, the extensive capacity for supercooling in both the<br />
active and P1 stage, along with the additional layer of cuticle and<br />
the clearing of gut contents in P1, would indicate that H. crispae<br />
preferentially seek to avoid internal ice formation. Nevertheless,<br />
animals in the P1 stage tolerate internal ice formation for both shorter<br />
and longer periods of time.<br />
Volume and osmoregulation<br />
Our results indicate that active-stage H. crispae is the most tolerant<br />
of changes in external salinity. Specimens from the population from<br />
Nipisat Bay, Greenland exhibit an increased tolerance towards<br />
concentrated SW solutions as compared with animals from Vellerup<br />
Vig, Denmark, suggesting that the ability to tolerate large increases<br />
in salinity is potentiated by living in a more-exposed habitat. The<br />
observed volume regulatory response of active-stage H. crispae<br />
during hypo- and hypertonic treatments differs in a significant way.<br />
When exposed to the hypo-osmotic solutions, the initial increase in<br />
total body volume was regulated to a new steady state, which was<br />
not significantly different from the control condition. In fact, a new<br />
steady-state value was demonstrated after merely 4h immersion in<br />
the external medium of 10ppt. Conversely, during acute exposure<br />
to concentrated seawater (40ppt), a partial recovery to normal levels<br />
was demonstrated; however, total body volume remained<br />
significantly different from the control condition even after 48h<br />
immersion. These data suggest that the body volume of H. crispae<br />
is more tightly regulated during exposure to dilute as compared with<br />
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2810<br />
K. A. <strong>Halberg</strong> and others<br />
more concentrated saltwater solutions. Interestingly, this observation<br />
seems reflected in an evolutionary context. According to our<br />
previous study, Halobiotus has evolved within the freshwater genus<br />
Isohypsibius, thus potentially explaining the enhanced volume<br />
regulatory response during exposure to dilute media (Møbjerg et<br />
al., 2007).<br />
When submitted to osmotic shock of 10 ppt and 40 ppt,<br />
respectively, our data show that H. crispae experience few<br />
limitations in terms of motility. Active-stage specimens from the<br />
Nipisat population even retain activity when exposed to a gradual<br />
salinity increase to 60ppt (Fig.3). However, upon direct transfer to<br />
an extreme seawater dilution of 2ppt, animal activity is markedly<br />
reduced, probably due to the pronounced increase in hydrostatic<br />
pressure and concomitant reduction in hemolymph osmotic pressure.<br />
Proper locomotory function in tardigrades relies on the hydrostatic<br />
pressure of the body cavity (Kinchin, 1994); thus, maintaining an<br />
appropriate body volume is essential to normal coordination of<br />
movement. In addition, as the membrane potentials of animal cells<br />
are highly dependent upon extracellular ionic strength (Spyropoulos<br />
and Teorell, 1968), the concomitant changes in hemolymph osmotic<br />
pressure could influence animal motility due to inhibition of neuromuscular<br />
activity. Indeed, the fact that a significant number of<br />
animals were observed passive during exposure to 2ppt, while<br />
animals remained largely unaffected during exposure to 10ppt, in<br />
spite of experiencing comparable average changes in body volume<br />
(compare Fig.4B and Fig.5B after 0.5h immersion), suggests that<br />
not only total body volume but also hemolymph osmolality is an<br />
important factor in maintaining locomotory functions.<br />
Exposing H. crispae to severe osmotic stress reveals that this<br />
species is a euryhaline osmoconformer, in which the hemolymph<br />
osmotic pressure is largely governed by the external environment.<br />
However, when analyzing hemolymph osmotic pressure at steady<br />
state following 48h exposure to the various salinity treatments as<br />
a function of the external osmolality, an interesting pattern emerges.<br />
H. crispae maintains a large osmotic pressure gradient between the<br />
internal and external environment, thus distinctly hyper-regulating<br />
during all investigated salinity treatments – albeit markedly less in<br />
concentrated seawater. This would imply a large water turnover in<br />
this animal, with osmotic water uptake being balanced by the<br />
excretion of dilute urine.<br />
Hyperosmoregulation is known in other euryhaline invertebrates.<br />
The crayfish Procambarus clarkii Girard 1852 (Arthropoda)<br />
produces highly dilute urine and is a strong hyperosmoregulator in<br />
freshwater (Sarver et al., 1994). However, the excreted urine<br />
becomes progressively more concentrated in media of higher ionic<br />
strength and is nearly isoosmotic when exposed to an external<br />
concentration of 750mOsmkg –1 , at which P. clarkii cease to hyperregulate<br />
(Sarver et al., 1994). Moreover, similar osmoregulatory<br />
responses have been reported from nematodes (Fusé et al., 1993;<br />
Forster, 1998). Indeed, the internal osmolality of the parasitic<br />
nematode Pseudoterranova decipiens (Krabbe, 1878) was<br />
maintained 90mOsmkg –1 above that of the external environment<br />
during exposure to media of widely varying osmolality (Fusé et al.,<br />
1993).<br />
Three glands positioned at the transition zone between the<br />
midgut and rectum of eutardigrades are traditionally ascribed an<br />
osmoregulatory function. The term used for these structures, i.e.<br />
Malpighian tubules, was introduced more than a century ago (Plate,<br />
1889). The positional conformity of the Malpighian tubules in<br />
eutardigrades and in hexapods has been used as a strong argument<br />
in favor of a homology between these structures (Greven, 1982;<br />
Møbjerg and Dahl, 1996). However, at present, no functional data<br />
exist relating the Malpighian tubules of tardigrades to an<br />
osmoregulatory role. Nevertheless, several detailed morphological<br />
investigations of the tubules support the hypothesis. These studies<br />
have provided ultrastructural data, which are in agreement with an<br />
active transporting epithelium involved in solute and fluid transport<br />
(Greven, 1979; Weglarska, 1987a; Weglarska, 1987b; Møbjerg and<br />
Dahl, 1996; Peltzer et al., 2007). As holds for insects, the Malpighian<br />
tubules of tardigrades are considered secretion–reabsorption kidneys.<br />
In light of the ultrastructural data available on tardigrade Malpighian<br />
tubules, it seems reasonable to assume that the first steps in urine<br />
formation take place across initial segment cells, characterized by<br />
a conspicuous basal labyrinth, numerous mitochondria and an<br />
enlarged apical surface. There is, moreover, ultrastructural support<br />
for assigning the tardigrade rectum an osmoregulatory function<br />
(Dewel and Dewel, 1979). A possible mode of urine formation was<br />
outlined by Dewel and Dewel (Dewel and Dewel, 1979). They<br />
suggest that isoosmotic urine produced by the Malpighian tubules<br />
is modified in the rectum through the active reabsorption of solutes,<br />
leading to the excretion of hypo-osmotic urine. Our data seem in<br />
favor of such a mechanism of urine formation. Interestingly,<br />
preliminary and unpublished data (H.R. research group) on<br />
Richtersius coronifer (Richters 1903) indicate that this species also<br />
remains hyperosmotic during exposures to a range of external<br />
salinities and it is therefore likely that hyper-regulation, and possibly<br />
hypo-osmotic urine formation, is a general feature of eutardigrades.<br />
However, until functional studies at the cellular and molecular level<br />
are performed, the exact mechanisms involved in osmoregulation<br />
in tardigrades remain to be elucidated.<br />
In conclusion, we show that the transition between the individual<br />
cyclomorphic stages of H. crispae is associated with profound<br />
changes in the physiology of the animal. Our results show that<br />
animals in the active stage tolerate large changes in the external<br />
osmotic pressure by regulating their total body volume and by<br />
enduring large concomitant changes in hemolymph osmotic<br />
pressure. H. crispae remains hyperosmotic at any investigated<br />
external salinity, suggesting that this species is a strong hyperregulator.<br />
Our study is the first to provide evidence for the volume<br />
and osmoregulatory capacity in Tardigrada. Whereas animals in the<br />
active stage are intolerant of freezing, the P1 stage is demonstrated<br />
to be freeze tolerant. The relatively low crystallization temperature<br />
reveals that extensive supercooling of the body fluids takes place<br />
during cooling and that no physiologically relevant ice-nucleating<br />
agents are present.<br />
We would like to thank the crew onboard the research vessel R/W Porsild (Arctic<br />
Station, Qeqertarsuaq, Greenland) who made the collection at the type locality,<br />
Nipisat, possible. Funding came from the Carlsberg Foundation and from the<br />
2008 Faculty of Science, University of Copenhagen Freja-Programme.<br />
DSC<br />
MDP<br />
RVD<br />
SW<br />
T c<br />
T m<br />
LIST OF ABBREVIATIONS<br />
differential scanning calorimetry<br />
melting point depression<br />
regulatory volume decrease<br />
seawater<br />
crystallization temperature<br />
melting temperature<br />
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