New insights into osmobiosis and chemobiosis in tardigrades.

Tardigrades are renowned for their ability to enter the extremotolerant state of latent life known as cryptobiosis. While it is widely accepted that cryptobiosis can be induced by freezing (cryobiosis) and by desiccation (anhydrobiosis), the latter involving formation of a so-called tun, the exact mechanisms underlying the state-as well as the significance of other cryptobiosis inducing factors-remain ambiguous. Here, we focus on osmotic and chemical stress tolerance in the marine tidal tardigrade Echiniscoides sigismundi. We show that E. sigismundi enters the tun state following exposure to saturated seawater and upon exposure to locality seawater containing the mitochondrial uncoupler DNP. The latter experiments provide evidence of osmobiosis and chemobiosis, i.e., cryptobiosis induced by high levels of osmolytes and toxicants, respectively. A small decrease in survival was observed following simultaneous exposure to DNP and saturated seawater indicating that the tardigrades may not be entirely ametabolic while in the osmobiotic tun. The tardigrades easily handle exposure to ultrapure water, but hypo-osmotic shock impairs tun formation and when exposed to ultrapure water the tardigrades do not tolerate DNP, indicating that tolerance towards dilute solutions involves energy-consuming processes. We discuss our data in relation to earlier and more contemporary studies on cryptobiosis and we argue that osmobiosis should be defined as a state of cryptobiosis induced by high external osmotic pressure. Our investigation supports the hypothesis that the mechanisms underlying osmobiosis and anhydrobiosis are overlapping and that osmobiosis likely represents the evolutionary forerunner of cryptobiosis forms that involve body water deprivation.

Actinarctus doryphorus (Tanarctidae) DNA barcodes and phylogenetic reinvestigation of Arthrotardigrada with new A. doryphorus and Echiniscoididae sequences.

Little is still known about the diversity and evolution of marine arthrotardigrades, as they are generally difficult to sample, resulting in a limited amount of molecular data for barcoding and phylogenetic studies. With the current study, we provide the first investigation into COI haplotype diversity in a marine tanarctid and at the same time readdress arthrotardigrade phylogeny. Specifically, we provide COI mtDNA, 18S and 28S rDNA sequences from a population of Actinarctus doryphorus (Tanarctidae) sampled off the coast of Roscoff, France and further provide new 18S sequences from two marine echiniscoidids. A. doryphorus COI sequences confirmed the presence of a single species and further revealed five haplotypes shared among nine sequenced individuals. Our 18S and 28S rDNA datasets were individually and combined analysed with Bayesian inference and Maximum Likelihood. Actinarctus doryphorus was placed together with Tanarctus sequences within a maximally supported Tanarctidae, confirming previous interpretations that the clade is distinct from Halechiniscidae. Although several studies in recent decades have concluded that the marine arthrotardigrades are paraphyletic, recent studies have argued that the clade may not be paraphyletic. Our phylogenetic analyses consistently inferred Arthrotardigrada as paraphyletic, as the clade includes the monophyletic Echiniscoidea. Accordingly, we propose that it is time to suppress the order Arthrotardigrada as it clearly does not reflect tardigrade phylogeny.

Extreme freeze-tolerance in cryophilic tardigrades relies on controlled ice formation but does not involve significant change in transcription.

Subzero temperatures are among the most significant factors defining the distribution of organisms, yet, certain taxa have evolved to overcome this barrier. The microscopic tardigrades are among the most freeze-tolerant animals, with selected species reported to survive milli-Kelvin temperatures. Here, we estimate survival of fully hydrated eutardigrades of the species Ramazzottius varieornatus following exposures to -20 °C and  -80 °C as well as -196 °C with or without initial cooling to -80 °C. The tardigrades easily survive these temperatures, yet with a significant decrease in viability following rapid cooling by direct exposure to -196 °C. Hence, post-freeze recovery of R. varieornatus seems to rely on cooling rate and thus controlled ice formation. Cryophilic organisms are renowned for having cold-active enzymes that secure appropriate reaction rates at low temperatures. Hence, extreme freeze-tolerance in R. varieornatus could potentially involve syntheses of cryoprotectants and de novo transcription. We therefore generated a reference transcriptome for this cryophilic R. varieornatus population and explored for differential gene expression patterns following cooling to -80 °C as compared to active 5 °C controls. Specifically, we tested for fast transcription potentially occurring within 25 min of cooling from room temperature to a supercooling point of ca. -20 °C, at which the tardigrades presumably freeze and enter into the ametabolic state of cryobiosis. Our analyses revealed no evidence for differential gene expression. We, therefore, conclude that extreme freeze-tolerance in R. varieornatus relies on controlled extracellular freezing with any freeze-tolerance related genes being constitutively expressed.

Differential expression profiling of heat stressed tardigrades reveals major shift in the transcriptome.

Tardigrades are renowned for their extreme stress tolerance, which includes the ability to endure complete desiccation, high levels of radiation and very low sub-zero temperatures. Nevertheless, tardigrades appear to be vulnerable to high temperatures and thus the potential effects of global warming. Here, we provide the first analysis of transcriptome data obtained from heat stressed specimens of the eutardigrade Ramazzottius varieornatus, with the aim of providing new insights into the molecular processes affected by high temperatures. Specifically, we compare RNA-seq datasets obtained from active, heat-exposed (35 °C) tardigrades to that of active controls kept at 5 °C. Our data reveal a surprising shift in transcription, involving 9634 differentially expressed transcripts, corresponding to >35% of the transcriptome. The latter data are in striking contrast to the hitherto observed constitutive expression underlying tardigrade extreme stress tolerance and entrance into the latent state of life, known as cryptobiosis. Thus, when examining the molecular response, heat-stress appears to be more stressful for R. varieornatus than extreme conditions, such as desiccation or freezing. A gene ontology analysis reveals that the heat stress response involves a change in transcription and presumably translation, including an adjustment of metabolism, and, putatively, preparation for encystment and subsequent diapause. Among the differentially expressed transcripts we find heat-shock proteins as well as the eutardigrade specific proteins (CAHS, SAHS, MAHS, RvLEAM, and Dsup). The latter proteins thus seem to contribute to a general stress response, and may not be directly related to cryptobiosis.

New records on the rich loriciferan fauna of Trezen ar Skoden (Roscoff, France): Description of two new species of Nanaloricus and the new genus Scutiloricus.

Loricifera is a phylum of microscopic animals that inhabit marine environments worldwide. Named after their conspicuous and protective lorica, the phylum was first described from Roscoff (France) in 1983 and, hitherto, it contains only 40 species. Based on data collected from Roscoff during the past four decades, we here describe two new species of Nanaloricus, namely Nanaloricus valdemari sp. nov. and Nanaloricus mathildeae sp. nov., as well as a new genus and species, Scutiloricus hugoi gen. et sp. nov. Adults of N. valdemari sp. nov. are distinguished by a pair of unique cuticular ridges, here referred to as longitudinal stripes, spanning laterally along the anterior two thirds of the dorsal lorical plate. N. mathildeae sp. nov. is characterized by strong sexual dimorphism. Specifically, the branches composing the multiform male clavoscalids are much broader as compared to other Nanaloricus species. The two new Nanaloricus species are both characterized by unique sensory organs associated with the double trichoscalids. The size and exact position of these organs differ between the two species. Adults of Scutiloricus hugoi gen. et sp. nov. are characterized by, among other features, a square lorica composed of six cuticular plates with a total of 14 anterior spikes, of which 12 have transverse cuticular ridges and thus appear fenestrated; laterodorsal flosculi arranged linearly; a posterior lorical region characterized by an anal field with a small anal cone flanked by a pair of spurs. Notably, mature females are characterized by a pair of seminal receptacles, a character not previously reported in Loricifera. We discuss the new findings and compare N. valdemari sp. nov. and N. mathildeae sp.nov. with other species assigned to genus Nanaloricus. The distinguishing features of Scutiloricus hugoi gen. et sp. nov. are discussed from a comparative perspective with the other genera of family Nanaloricidae.

New insights into survival strategies of tardigrades.

Life is set within a narrow frame of physicochemical factors, yet, some species have adapted to conditions far beyond these constraints. Nature appears to have evolved two principal strategies for living organisms to cope with hostile conditions. One way is to remain active, retaining metabolism through adaptations that enable the organism to match the physiological requirements of environmental change. The other is to enter a state of dormancy with metabolic suppression. One form of metabolic suppression, known as cryptobiosis, is a widespread state across life kingdoms, in which metabolism comes to a reversible standstill. Among animals, nematodes, rotifers and tardigrades, comprise species that have the ability to enter cryptobiosis at all stages of their life cycle. Tardigrades are microscopic cosmopolitan metazoans found in permanent and temporal aquatic environments. They are renowned for their ability to tolerate extreme stress and are particularly resistant after having entered a cryptobiotic state known as a "tun". As new molecular tools allow for a more detailed investigation into their enigmatic adaptations, tardigrades are gaining increasing attention. In this graphical review, we provide an outline of survival strategies found among tardigrades and we summarize current knowledge of the adaptive mechanisms that underlie their unique tolerance to extreme or changing environments.

New insights into the limited thermotolerance of anhydrobiotic tardigrades.

The recent discovery of an upper limit in the tolerance of an extremotolerant tardigrade to high temperatures is astounding. Although these microinvertebrates are able to endure severe environmental conditions, including desiccation, freezing and high levels of radiation, high temperatures seem to be an Achilles' heel for active tardigrades. Moreover, exposure-time appears to be a limiting factor for the heat stress tolerance of the otherwise highly resilient desiccated (anhydrobiotic) tardigrades. Indeed, the survival rate of desiccated tardigrades exposed to high temperatures for 24 hours is significantly lower than for exposures of only 1 hour. Here, we investigate the effect of 1 week of high temperature exposures on desiccated tardigrades with the aim of elucidating whether exposure-times longer than 24 hours decrease survival even further. From our analyses we estimate a significant decrease in the 50% mortality temperature from 63ºC to 56ºC for Ramazzottius varieornatus exposed to high temperatures in the desiccated tun state for 24 hours and 1 week, respectively. This negative correlation between exposure-time and tolerance to high temperatures probably results from the interference of intracellular temperature with the homeostasis of macromolecules. We hypothesize that high temperatures denature molecules that play a vital role in sustaining and protecting the anhydrobiotic state.

Thermotolerance experiments on active and desiccated states of Ramazzottius varieornatus emphasize that tardigrades are sensitive to high temperatures.

Global warming is already having harmful effects on habitats worldwide and it is therefore important to gain an understanding of how rising temperatures may affect extant animals. Here, we investigate the tolerance to high temperatures of Ramazzottius varieornatus, a tardigrade frequently found in transient freshwater habitats. Using logistic modelling on activity we evaluate the effect of 24 hour temperature exposures on active tardigrades, with or without a short acclimation period, compared to exposures of desiccated tardigrades. We estimate that the 50% mortality temperature for non-acclimated active tardigrades is 37.1 °C, with a small but significant increase to 37.6 °C following acclimation. Desiccated specimens tolerate much higher temperatures, with an estimated 50% mortality temperature of 82.7 °C following 1 hour exposures, but with a significant decrease to 63.1 °C following 24 hour exposures. Our results show that metabolically active tardigrades are vulnerable to high temperatures, yet acclimatization could provide a tolerance increase. Desiccated specimens show a much higher resilience-exposure-time is, however, a limiting factor giving tardigrades a restricted window of high temperature tolerance. Tardigrades are renowned for their ability to tolerate extreme conditions, but their endurance towards high temperatures clearly has an upper limit-high temperatures thus seem to be their Achilles heel.

Comparative myoanatomy of Tardigrada: new insights from the heterotardigrades Actinarctus doryphorus (Tanarctidae) and Echiniscoides sigismundi (Echiniscoididae).

BACKGROUND: Tardigrada is a group of microscopic invertebrates distributed worldwide in permanent and temporal aquatic habitats. Famous for their extreme stress tolerance, tardigrades are also of interest due to their close relationship with Arthropoda and Cycloneuralia. Despite recent efforts in analyzing the musculature of a number of tardigrade species, data on the class Heterotardigrada remain scarce. Aiming to expand the current morphological framework, and to promote the use of muscular body plans in elucidating tardigrade phylogeny, the myoanatomy of two heterotardigrades, Actinarctus doryphorus and Echiniscoides sigismundi, was analyzed by cytochemistry, scanning electron and confocal laser scanning microscopy and 3D imaging. We discuss our findings with reference to other tardigrades and internal phylogenetic relationships of the phylum. RESULTS: We focus our analyses on the somatic musculature, which in tardigrades includes muscle groups spanning dorsal, ventral, and lateral body regions, with the legs being musculated by fibers belonging to all three groups. A pronounced reduction of the trunk musculature is seen in the dorsoventrally compressed A. doryphorus, a species that generally has fewer cuticle attachment sites as compared to E. sigismundi and members of the class Eutardigrada. Interestingly, F-actin positive signals were found in the head appendages of A. doryphorus. Our analyses further indicate that cross-striation is a feature common to the somatic muscles of heterotardigrades and that E. sigismundi-as previously proposed for other echiniscoidean heterotardigrades-has relatively thick somatic muscle fibers. CONCLUSIONS: We provide new insights into the myoanatomical differences that characterize distinct evolutionary lineages within Tardigrada, highlighting characters that potentially can be informative in future phylogenetic analyses. We focus our current analyses on the ventral trunk musculature. Our observations suggest that seven paired ventromedian attachment sites anchoring a large number of muscles can be regarded as part of the ground pattern of Tardigrada and that fusion and reduction of cuticular attachment sites is a derived condition. Specifically, the pattern of these sites differs in particular details between tardigrade taxa. In the future, a deeper understanding of the tardigrade myoanatomical ground pattern will require more investigations in order to include all major tardigrade lineages.

Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages.

BACKGROUND: Tardigrades are renowned for their ability to enter cryptobiosis (latent life) and endure extreme stress, including desiccation and freezing. Increased focus is on revealing molecular mechanisms underlying this tolerance. Here, we provide the first transcriptomes from the heterotardigrade Echiniscoides cf. sigismundi and the eutardigrade Richtersius cf. coronifer, and compare these with data from other tardigrades and six eukaryote models. Investigating 107 genes/gene families, our study provides a thorough analysis of tardigrade gene content with focus on stress tolerance. RESULTS: E. cf. sigismundi, a strong cryptobiont, apparently lacks expression of a number of stress related genes. Most conspicuous is the lack of transcripts from genes involved in classical Non-Homologous End Joining. Our analyses suggest that post-cryptobiotic survival in tardigrades could rely on high fidelity transcription-coupled DNA repair. Tardigrades seem to lack many peroxins, but they all have a comprehensive number of genes encoding proteins involved in antioxidant defense. The "tardigrade unique proteins" (CAHS, SAHS, MAHS, RvLEAM), seem to be missing in the heterotardigrade lineage, revealing that cryptobiosis in general cannot be attributed solely to these proteins. Our investigation further reveals a unique and highly expressed cold shock domain. We hypothesize that the cold shock protein acts as a RNA-chaperone involved in regulation of translation following freezing. CONCLUSIONS: Our results show common gene family contractions and expansions within stress related gene pathways in tardigrades, but also indicate that evolutionary lineages have a high degree of divergence. Different taxa and lineages may exhibit unique physiological adaptations towards stress conditions involving possible unknown functional homologues and/or novel physiological and biochemical mechanisms. To further substantiate the current results genome assemblies coupled with transcriptome data and experimental investigations are needed from tardigrades belonging to different evolutionary lineages.

Modelling extreme desiccation tolerance in a marine tardigrade.

It has recently been argued that the enigmatic tardigrades (water bears) will endure until the sun dies, surviving any astrophysical calamities in Earth's oceans. Yet, our knowledge of stress tolerance among marine tardigrade species is very limited and most investigations revolve around species living in moist habitats on land. Here, we investigate desiccation tolerance in the cosmopolitan marine tidal tardigrade, Echiniscoides sigismundi, providing the first thorough analysis on recovery upon desiccation from seawater. We test the influence on survival of desiccation surface, time spent desiccated (up to 1 year) and initial water volume. We propose analysis methods for survival estimates, which can be used as a future platform for evaluating and analysing recovery rates in organisms subjected to extreme stress. Our data reveal that marine tidal tardigrades tolerate extremely rapid and extended periods of desiccation from seawater supporting the argument that these animals are among the toughest organisms on Earth.

Comparative Investigation of Copper Tolerance and Identification of Putative Tolerance Related Genes in Tardigrades.

Tardigrades are microscopic aquatic animals renowned for their tolerance toward extreme environmental conditions. The current study is the first to investigate their tolerance toward heavy metals and we present a novel tardigrade toxicant tolerance assay based on activity assessments as a measure of survival. Specifically, we compare tolerance toward copper in four species representing different evolutionary lineages, habitats and adaptation strategies, i.e., a marine heterotardigrade, Echiniscoides sigismundi, a limno-terrestrial heterotardigrade, Echiniscus testudo, a limno-terrestrial eutardigrade, Ramazzottius oberhaeuseri, and a marine eutardigrade, Halobiotus crispae. The latter was sampled at a time of year, when the population is predominantly represented by aberrant P1 cysts, while the other species were in normal active states prior to exposure. Based on volume measurements and a general relation between body mass and copper tolerance, expected tardigrade EC50 values were estimated at 0.5-2 µg l-1. Following 24 h of exposure, tolerance was high with no apparent link to lineage or habitat. EC50s (95% CI), 24 h after exposure, were estimated at 178 (168-186) and 310 (295-328) µg l-1, respectively, for E. sigismundi and R. oberhaeuseri, whereas E. testudo and H. crispae were less affected. Highest tolerance was observed in H. crispae with a mean ± s.e.m. activity of 77 ± 2% (n = 3) 24 h after removal from ~3 mg l-1 copper, suggesting that tardigrade cysts have increased tolerance toward toxicants. In order to identify putative tolerance related genes, an E. sigismundi transcriptome was searched for key enzymes involved in osmoregulation, antioxidant defense and copper metabolism. We found high expression of Na/K ATPase and carbonic anhydrase, known targets for copper. Our transcriptome, furthermore, revealed high expression of antioxidant enzymes, copper transporters, ATOX1, and a Cu-ATPase. In summary, our results indicate that tardigrades express well-known key osmoregulatory enzymes, supporting the hypothesis that copper inhibits sodium turnover as demonstrated for other aquatic organisms. Tardigrades, nevertheless, have high tolerance toward the toxicant, which is likely linked to high expression of antioxidant enzymes and an ability to enter dormant states. Tardigrades, furthermore, seem to have a well-developed battery of cuproproteins involved in copper homeostasis, providing basis for active copper sequestering and excretion.

Tolerance to Gamma Radiation in the Marine Heterotardigrade, Echiniscoides sigismundi.

Tardigrades belong to the most radiation tolerant animals on Earth, as documented by a number of studies using both low-LET and high-LET ionizing radiation. Previous studies have focused on semi-terrestrial species, which are also very tolerant to desiccation. The predominant view on the reason for the high radiation tolerance among these semi-terrestrial species is that it relies on molecular mechanisms that evolved as adaptations for surviving dehydration. In this study we report the first study on radiation tolerance in a marine tardigrade, Echiniscoides sigismundi. Adult specimens in the hydrated active state were exposed to doses of gamma radiation from 100 to 5000 Gy. The results showed little effect of radiation at 100 and 500 Gy but a clear decline in activity at 1000 Gy and higher. The highest dose survived was 4000 Gy, at which ca. 8% of the tardigrades were active 7 days after irradiation. LD50 in the first 7 days after irradiation was in the range of 1100-1600 Gy. Compared to previous studies on radiation tolerance in semi-terrestrial and limnic tardigrades, Echiniscoides sigismundi seems to have a lower tolerance. However, the species still fits into the category of tardigrades that have high tolerance to both desiccation and radiation, supporting the hypothesis that radiation tolerance is a by-product of adaptive mechanisms to survive desiccation. More studies on radiation tolerance in tardigrade species adapted to permanently wet conditions, both marine and freshwater, are needed to obtain a more comprehensive picture of the patterns of radiation tolerance.

The tardigrade fauna of Australian marine caves: with descriptions of nine new species of Arthrotardigrada.

Marine caves are known to support a rich macrofauna; however, few studies have focused on meiofauna. Marine cave meiofaunal tardigrades have been reported from Japan and the Mediterranean Sea and a preliminary list of species including a redescription of Actinarctus neretinus Grimaldi de Zio, D'Addabbo Gallo, Morone De Lucia, Vaccarella and Grimaldi, 1982 was reported from Fish Rock Cave and Jim's Cave on the coast of Australia. This study is the fourth in a series describing the unique meiofauna in two Australian submarine caves located off the coast of New South Wales, describing nine new species.        Only 67 tardigrades were collected from the two caves, yet these contained a high diversity of at least 16 different species which are quite different in the two caves. The fauna includes nine arthrotardigrade genera: Actinarctus, Batillipes, Dipodarctus, Halechiniscus, Raiarctus, Styraconyx, Tanarctus, Tholoarctus, and Wingstrandarctus. This fauna is different from that reported for the high energy beaches along the East Coast of Australia.        We describe nine new species comprising a single batillipedid and eight halechiniscids: Batillipes solitarius nov. sp., Dipodarctus australiensis nov. sp., Dipodarctus susannae nov. sp., Raiarctus jesperi nov. sp., Raiarctus katrinae nov. sp., Tanarctus hirsutospinosus nov. sp., Tholoarctus oleseni nov. sp., Wingstrandarctus stinae nov. sp. and Wingstrandarctus unsculptus nov. sp.

Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada).

Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships of Tardigrada. Here, we present new insight into the morphology of the brain in a marine arthrotardigrade, Actinarctus doryphorus, based on transmission electron microscopy, supported by scanning electron microscopy, conventional light microscopy as well as confocal laser scanning microscopy. Arthrotardigrades contain a large number of plesiomorphic characters and likely represent ancestral tardigrades. They often have segmented body outlines and each trunk segment, with its paired set of legs, may have up to five sensory appendages. Noticeably, the head carries numerous cephalic appendages that are structurally equivalent to the sensory appendages of the trunk segments. Our data reveal that the brain of A. doryphorus is partitioned into three paired lobes, and that these lobes exhibit a more pronounced separation as compared to that of eutardigrades. The first brain lobe in A. doryphorus is located anteriodorsally, with the second lobe just below it in an anterioventral position. Both of these two paired lobes are located anterior to the buccal tube. The third pair of brain lobes are situated posterioventrally to the first two lobes, and flank the buccal tube. In addition, A. doryphorus possesses a subpharyngeal ganglion, which is connected with the first of the four ventral trunk ganglia. The first and second brain lobes in A. doryphorus innervate the clavae and cirri of the head. The innervations of these structures indicate a homology between, respectively, the clavae and cirri of A. doryphorus and the temporalia and papilla cephalica of eutardigrades. The third brain lobes innervate the buccal lamella and the stylets as described for eutardigrades. Collectively, these findings suggest that the head region of extant tardigrades is the result of cephalization of multiple segments. Our results on the brain anatomy of Actinarctus doryphorus support the monophyly of Panarthropoda.

Surface enhanced Raman scattering on Tardigrada--towards monitoring and imaging molecular structures in live cryptobiotic organisms.

Tardigrades are microscopic metazoans which are able to survive extreme physical and chemical conditions by entering a stress tolerant state called cryptobiosis. At present, the molecular mechanisms behind cryptobiosis are still poorly understood. We show that surface enhanced Raman scattering supported by plasmonic gold nanoparticles can measure molecular constituents and their local distribution in live tardigrades. Surface enhanced Raman signatures allow to differentiate between two species and indicate molecular structural differences between tardigrades in water and in a dry state. This opens new avenues for exploring cryptobiosis by studying molecular changes in live cryptobiotic organisms.

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

Many species of tardigrades are known to tolerate extreme environmental stress, yet detailed knowledge of the mechanisms underlying the remarkable adaptations of tardigrades is still lacking, as are answers to many questions regarding their basic biology. Here, we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) using high-performance liquid chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na(+) and Cl(-) are the principal inorganic ions in tardigrade fluids, albeit other ions, i.e. K(+), NH4(+), Ca(2+), Mg(2+), F(-), SO4(2-) and PO4(3-) were also detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared with that of the external medium (Na(+), ×70-800; K(+), ×20-90; Ca(2+) and Mg(2+), ×30-200; F(-), ×160-1040, Cl(-), ×20-50; PO4(3-), ×700-2800; SO4(2-), ×30-150). In contrast, in the marine species H. crispae, Na(+), Cl(-) and SO4(2-) are almost in ionic equilibrium with (brackish) salt water, while K(+), Ca(2+), Mg(2+) and F(-) are only slightly concentrated (×2-10). An anion deficit of ~120 mEq l(-1) in M. tardigradum and H. crispae indicates the presence of unidentified ionic components in these species. Body fluid osmolality ranges from 361±49 mOsm kg(-1) in R. coronifer to 961±43 mOsm kg(-1) in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not lose large quantities of inorganic ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicate that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.