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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.

The 14th International Symposium on Tardigrada took place in Copenhagen, Denmark from 30 July to 3 August 2018. Approximately 140 participants, representing 28 countries from five continents attended the meeting, and there were 58 talks and 74 posters of which 20 were selected for the Symposium Proceedings published in this special issue. The studies span phylogenomics, systematics, anatomy, morphology, reproductive biology, cryobiology, ecology, diet, microbial interactions and biogeography, taking the next step forward in broadening and deepening our understanding of tardigrade biology.

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.

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.

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.

Marine tidal heterotardigrades (Echiniscoididae) have gained increasing interest owing to their unique adaptations and evolutionary position, bridging marine and limnoterrestrial taxa. Echiniscoididae was established to accommodate the marine genera Anisonyches and Echiniscoides. However, it has become apparent that Anisonyches, with its claw configuration, median cirrus and seminal receptacles, clearly has little or no affinity to tidal echiniscoidids with supernumerary claws. Consequently, we establish Anisonychidae fam. nov. to accommodate Anisonyches in a paraphyletic Arthrotardigrada and discuss its affinity to other heterotardigrade taxa. We recently split Echiniscoides into Isoechiniscoides and Echiniscoides s.l. The latter remains a miscellany of species complexes and undescribed genera, and it has become evident that a larger number of echiniscoidids belonging to Echiniscoides, Isoechiniscoides and undescribed genera coexist in intertidal sediments. Here, we erect Neoechiniscoides aski gen. nov., sp. nov. from Roscoff, France, which has a unique anal system, characterized by prominent lateral lobes with a set of wing-like structures. Phylogenetic analyses based on COI sequences infer a close relationship between N. aski, an undescribed species from Roscoff and unidentified species from Maine, USA. We propose that the new genus includes the former Echiniscoides species Echiniscoides pollocki and Echiniscoides horningi, which we hereby transfer.

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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5912-x) contains supplementary material, which is available to authorized users.

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.

The so-called ‘tun’ state is best known from limno-terrestrial tardigrades and rotifers that rely on this compact body shape for anhydrobiotic survival. Little is known of tun formation in marine species and the evolutionary origin of the state is presently unknown. Here, we investigate desiccation tolerance and tun formation in the marine tidal echiniscoidean tardigrade, Echiniscoides sigismundi (M. Schultze, 1865). Groups of approximately 20 E. sigismundi sampled from Lynaes (Denmark) were dehydrated on filter paper from seawater as well as ultrapurified water and kept for 48 h at 5 °C, after which they were rehydrated in seawater. The activity and behaviour of the tardigrades was examined under a light microscope, whereas scanning electron microscopy was used for high-resolution three-dimensional imaging. When dehydrated from seawater, E. sigismundi enters a tun, however, when exposed to ultrapurified water, the tardigrade swells and becomes incapable of movement, and thus incapable of tun formation. Nonetheless, E. sigismundi tolerates being dehydrated from ultrapurified water, revealing an exceptional and unparalleled resilience towards losing structural integrity. Our results confirm previous investigations, which suggest that tun formation relies on a functional musculature. They further suggest that tun formation may have evolved as a response to elevated external pressure rather than desiccation per se.

Little is known about ionic and osmotic stress tolerance in tardigrades. Here, we examine salt stress tolerance in Ramazzottius oberhaeuseri and Echiniscus testudo from Nivå (Denmark) and address whether limno-terrestrial tardigrades can enter a state of quiescence (osmobiosis) in the face of high external osmolyte concentrations. Direct transfers into NaCl solutions showed an upper tolerance level of around 600 mOsm kg−1 in R. oberhaeuseri and 200 mOsm kg−1 in E. testudo. During salt exposures, R. oberhaeuseri contracted into a ‘tun’, whereas E. testudo remained active leaving it more susceptible to acute effects of the ions. Further experiments focused on the more resilient R. oberhaeuseri, which entered a tun and readily regained activity when directly exposed to polyethylene glycol and sucrose of up to 872 ± 0 and 813 ± 3 mOsm kg−1, respectively, revealing a higher tolerance towards non-ionic osmolytes as compared to NaCl. Ramazzottius oberhaeuseri furthermore readily regained activity following gradual increases in non-ionic osmolytes and NaCl of up to 2434 ± 28 and 1905 ± 3 mOsm kg−1, respectively, showing that short-term acclimation promoted salt stress tolerance. Our results suggest that the limno-terrestrial R. oberhaeuseri enters a state of quiescence in the face of high external osmotic pressure and that it, in this state, is highly tolerant of ionic and osmotic stress.

The marine heterotardigrades belonging to Echiniscoididae inhabit a wide range of substrates and represent the dominant tardigrade group in intertidal zones worldwide. Notably, the evolution of this family is interesting as it holds key evidence as to how heterotardigrades colonized terrestrial and freshwater environments. We inferred the phylogeny of Echiniscoididae using 28S and COI sequences from GenBank and from new specimens collected at Roscoff, France. Morphological characterizations of several major clades, inferred from the molecular data, are currently lacking. However, one of the clades is erected as a new taxon, Isoechiniscoides gen. nov., for the former Echiniscoides higginsi-group, which comprises species with isonych claws, pillars in the epicuticle and exceptionally long sensory appendages. The type species for the new genus is described here as Isoechiniscoides sifae nov. gen., sp. nov. Isoechiniscoides comprises six clawed interstitial species, whereas Echiniscoides taxa possess a higher claw number and primarily inhabit algae, barnacles and lichens in tidal zones. The new genus, Isoechiniscoides, together with morphologically undetermined specimens from British Columbia (Canada), is sister-group to all other Echiniscoididae taxa. Consequently, we propose to establish Isoechiniscoidinae subfam. nov. to accommodate the new genus. Isoechiniscoidinae provides an evolutionary link between Echiniscoidea and Arthrotardigrada. The latter comprises exclusively marine and mostly interstitial species.

Tardigrade phylogeny is currently the object of intense investigations driven by an increasing amount of molecular data from a broad taxonomic range of tardigrades. New information obtained from these investigations plays a crucial role in establishing a reliable systematic framework of Tardigrada. Importantly, contemporary phylogenetic investigations readily incorporate and reinvestigate morphological characters and character systems. Most of these newer investigations are, however, primarily based on a few conservative nuclear genes (in addition to the mitochondrial COI). Emerging transcriptomic and genomic data sets provide promising new information for future phylogenetic studies.

Tardigrades are microscopic aquatic bilaterians that despite their size have a complex morphology and anatomy. These minute animals need a surrounding film of water to be active, and tardigrades residing in terrestrial environments live in moist microhabitats. The latter species are referred to as semiterrestrial, and they endure rapid dehydration by losing water over highly permeable integuments while forming a quiescent tun. Species of the class Eutardigrada are predominantly semiterrestrial and limnic with a relatively uniform morphology and anatomy. Heterotardigrades (class Heterotardigrada) comprising echiniscoideans and arthrotardigrades are much more diverse with highly variable external morphologies, ranging from clearly segmented species, over species with extremely large external sense organs and cuticular wings to species with a more “bear”-like eutardigrade body outline. In this chapter, we review the current understanding of tardigrade morphology and functional anatomy and put focus on tardigrade diversity emphasizing the often neglected marine arthrotardigrades. We give an introduction to the structure and function of major tardigrade organ systems, including the integument, body cavity, and digestive, muscular, nervous, and reproductive systems, and we highlight the diversity of tardigrade sensory appendages and overall external morphology.

Stressful environmental conditions generally limit animal survival, growth, and reproduction and may induce dormancy in the form of various resting stages. Tardigrades represent one of a few animal phyla in which different forms of dormancy are frequently encountered. One of these forms, cryptobiosis, a quick response to sudden changes in the environment, has gained a great deal of attention, whereas much less is known of the slower emerging form of dormancy, diapause. In this review we present the current knowledge of diapause in tardigrades.

Tardigrades are microscopic aquatic animals renowned for their tolerance towards extreme environmental conditions. The current study is the first to investigate their tolerance towards heavy metals and we present a novel tardigrade toxicant tolerance assay based on activity assessments as a measure of survival. Specifically, we compare tolerance towards 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 hours of exposure, tolerance was high with no apparent link to lineage or habitat. EC50s (95% CI), 24 hours 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 hours after removal from approximately 3 mg l-1 copper, suggesting that tardigrade cysts have increased tolerance towards toxicants. In order to identify putative tolerance related genes, an E. sigismundi transcriptome was generated and 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 towards 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.

Tardigrades are well known for their ability to survive extreme conditions such as desiccation and freezing by entering cryptobiosis, a state in which metabolism becomes immeasurable. Within tardigrades, cryptobiosis has been investigated almost entirely in eutardigrades and echiniscoideans living in semi-terrestrial habitats (predominantly mosses); information regarding cryptobiosis in the neglected marine arthrotardigrades remains sporadic, with the current understanding that arthrotardigrades are unable to undergo cryptobiosis. In the present study we have investigated two marine arthrotardigrade species, Styraconyx haploceros (Halechiniscidae) and Batillipes pennaki (Batillipedidae), inhabiting the tidal zone of Roscoff, France. These two species are likely candidates for cryptobiotic arthrotardigrades as they may experience large variations in environmental parameters. Our results show that S. haploceros, which live on the lichen Lichina, is able to withstand complete desiccation. Upon exposu...

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.

Tardigrades form an important component of meiofaunal communities across the globe. However, our knowledge on tardigrade ecology is very limited. Here, we report the results of 21 field samplings of the marine tardigrade Halobiotus crispae collected over a period of 74 months at the locality of Vellerup Vig, Denmark, with the aim of providing novel insights into its ecology. Uniquely, H. crispae is characterized by the presence of seasonal cyclic changes in the phenotype of the animal, i.e. cyclomorphosis. Our sampling data include (i) total number of animals extracted, (ii) dominant cyclomorphic stage found, and (iii) important environmental parameters such as temperature, salinity and pH. Our accumulated data constitute a tentative model for the annual fluctuations in animal density, which reveals an annual peak in abundance during the months of February and March. In contrast, tardigrade density appears to decrease in response to increasing temperatures during late spring/early summer. The the...

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 position of Tardigrada in the animal tree of life is a subject that has received much attention, but still remains controversial. Whereas some think tardigrades should be categorized as cycloneuralians, most authors argue in favor of a phylogenetic position within Panarthropoda as a sister group to Arthropoda or Arthropoda + Onychophora. Thus far, neither molecular nor morphological investigations have provided conclusive results as to the tardigrade sister group relationships. In this article, we present a detailed description of the nervous system of the eutardigrade Halobiotus crispae, using immunostainings, confocal laser scanning microscopy, and computer-aided three-dimensional reconstructions supported by transmission electron microscopy. We report details regarding the structure of the brain as well as the ganglia of the ventral nerve cord. In contrast to the newest investigation, we find transverse commissures in the ventral ganglia, and our data suggest that the brain is partitioned into at least three lobes. Additionally, we can confirm the existence of a subpharyngeal ganglion previously called subesophagal ganglion. According to our results, the original suggestion of a brain comprised of at least three parts cannot be rejected, and the data presented supports a sister group relationship of Tardigrada to 1) Arthropoda or 2) Onychophora or 3) Arthropoda + Onychophora. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.

Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state termed anhydrobiosis in response to desiccation. In theanhydrobiotic state, tardigrades display an incredible capacity to tolerate extreme environmental stress, not necessarily encountered in theirnatural habitat. In this study, we determine the effect of different extreme stresses on initial survival, long-term survival and fecundity of selectedspecies of limno-terrestrial tardigrades. The primary focus was to assess the effect of cosmic radiation. This was achieved through the RoTaRad(Rotifers, Tardigrades and Radiation) project on the BIOPAN 6 mission, funded by Agenzia Spaziale Italiana under the European Space Agency.To test their tolerance of space environment, tardigrades were sent into low earth orbit, and exposed to cosmic radiation and a microgravityenvironment. Experiments on Whatman-3 filters show an effect of cosmic radiation on the survival of the eutardigrade Richtersius coronifer justafter returning to Earth; however, after 2 years of desiccation on Whatman-3 filters, none of the tardigrades previously exposed to cosmicradiation could be revived. In a microcosmos experiment, the tardigrades R. coronifer, Ramazzottius oberhauseri and Echiniscus testudo weredesiccated on a moss substrate together with rotifers and nematodes. Very low survival rates were observed in this experiment, likely due to theapplied desiccation protocol. Embryos of the tardigrade Milnesium tardigradum were also exposed to cosmic radiation; they all hatched in thelaboratory after the flight. In addition, experiments testing extreme cold and vacuum tolerance in R. coronifer show that tardigrades inanhydrobiosis are unaffected by these conditions.Key words: Tardigrada – anhydrobiosis – cosmic radiation – extreme freezing – vacuum

The heterotardigrade family Echiniscidae consists of 12 currently recognized genera that are generally represented by few species with the exception of the genera Echiniscus and Pseudechiniscus, which have numerous species. The echiniscids inhabit a wide variety of different moss and lichen substrates. Phylogenetic relationships within the Echiniscidae have previously been investigated using morphological characters. This study includes molecular data from the nuclear 18S and 28S genes and the mitochondrial COI gene, as well as the morphological data to investigate the incongruence between the studies by Kristensen (1987) and Jorgensen (2000) and between morphology and molecules. The arthrotardigrades Batillipes and Florarctus and the echiniscoideans Echiniscoides and Oreella have been included as out-groups and ten genera of echiniscids as the in-group in the phylogenetic analyses. The data sets were analysed individually and combined with Bayesian inference, maximum likelihood and maximum parsimony. In this study, Echiniscidae was always inferred to be monophyletic in analyses of the combined data set (18S, 28S and morphology) excluding COI. The analyses inferred a ‘Pseudechiniscus lineage’ not including Mopsechiniscus. Furthermore, the inferred ‘Pseudechiniscus lineage’ only included part of the Pseudechiniscus species making the genus polyphyletic. The five Echiniscus species, which were included in the analysis, were monophyletic and sister-group to Testechiniscus (COI had Echiniscus paraphyletic). The clade [Hypechiniscus, (Echiniscus + Testechinsicus)] was also well supported. Parechiniscus was inferred to be the sister-group to all other echiniscid taxa, a phylogenetic position that corresponds well with its weakly sclerotized dorsal plates. Echiniscoidea was not inferred to be monophyletic in contradiction to recently phylogenetic analyses focused on Arthrotardigrada.

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

The muscular architecture of Halobiotus crispae (Eutardigrada: Hypsibiidae) was examined by means of fluorescent-coupled phalloidin in combination with confocal laser scanning microscopy and computer-aided three-dimensional reconstruction, in addition to light microscopy (Nomarski), scanning electron microscopy, and transmission electron microscopy (TEM). The somatic musculature of H. crispae is composed of structurally independent muscle fibers, which can be divided into a dorsal, ventral, dorsoventral, and a lateral musculature. Moreover, a distinct leg musculature is found. The number and arrangement of muscles differ in each leg. Noticeably, the fourth leg contains much fewer muscles when compared with the other legs. Buccopharyngeal musculature (myoepithelial muscles), intestinal musculature, and cloacal musculature comprise the animal's visceral musculature. TEM of stylet and leg musculature revealed ultrastructural similarities between these two muscle groups. Furthermore, microtubules are found in the epidermal cells of both leg and stylet muscle attachments. This would indicate that the stylet and stylet glands are homologues to the claw and claw glands, respectively. When comparing with previously published data on both heterotardigrade and eutardigrade species, it becomes obvious that eutardigrades possess very similar numbers and arrangement of muscles, yet differ in a number of significant details of their myoanatomy. This study establishes a morphological framework for the use of muscular architecture in elucidating tardigrade phylogeny. J. Morphol. 2009. © 2009 Wiley-Liss, Inc.

Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascites cells elicited the formation of long microvillus-like protrusions, rapid translocation of endogenous ERM proteins and green fluorescent protein-tagged ezrin to the cortical region including these protrusions, and Thr567/564/558(ezrin/radixin/moesin) phosphorylation of cortical ERM proteins. Reduced cell volume appeared to be the critical parameter in hypertonicity-induced ERM protein activation, whereas alterations in extracellular ionic strength or intracellular pH were not involved. A shrinkage-induced increase in the level of membrane-associated phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] appeared to play an important role in ERM protein activation, which was prevented after PtdIns(4,5)P2depletion by expression of the synaptojanin-2 phosphatase domain. While expression of constitutively active RhoA increased basal ERM phosphorylation, the Rho-Rho kinase pathway did not appear to be involved in shrinkage-induced ERM protein phosphorylation, which was also unaffected by the inhibition or absence of Na+/H+exchanger isoform (NHE1). Ezrin knockdown by small interfering RNA increased shrinkage-induced NHE1 activity, reduced basal and shrinkage-induced Rho activity, and attenuated the shrinkage-induced formation of microvillus-like protrusions. Hyperosmolarity-induced cell death was unaltered by ezrin knockdown or after phosphatidylinositol 3-kinase (PI3K) inhibition. In conclusion, ERM proteins are activated by osmotic shrinkage in a PtdIns(4,5)P2-dependent, NHE1-independent manner. This in turn mitigates the shrinkage-induced activation of NHE1, augments Rho activity, and may also contribute to F-actin rearrangement. In contrast, no evidence was found for the involvement of an NHE1-ezrin-PI3K-PKB pathway in counteracting shrinkage-induced cell death.

In the present study we investigate the genetic diversity within the asexually reproducing tardigrade Echiniscus testudo. The present study is the first to sample a tardigrade species for comparison of DNA sequence diversity between widely separated samples. Echiniscus testudo was sampled at 13 localities spanning three continents. DNA sequences of the mitochondrial COI gene and the nuclear ITS2 sequence were used to investigate the genetic diversity and phylogeographic structure of the various asexual lineages. Terrestrial tardigrades with the capability of entering a cryptobiotic state are assumed to have a high passive dispersal potential through airborne transport. Our results show moderate (ITS2) to high (COI) haplotype diversity and low sequence diversity that indicate evolution of haplotypes within distinct asexual lineages and a high dispersal potential. No isolation by distance was detected by Mantel tests. Different phylogeny inference methods (neighbor-joining, maximum parsimony, maximum likelihood and Bayesian inference) revealed little topological resolution, but minimum spanning networks showed some phylogeographic patterns. The COI and ITS2 minimum spanning networks show patterns that indicate dispersal of several haplotypes from founding populations. In conclusion our data show a low genetic diversity and a relatively high haplotype diversity indicating that E. testudo is a young species with a high dispersal potential.

The aim of this study was to elucidate mechanisms of P(i) handling in toads (Bufo bufo). We introduced toads to experimental solutions of various [P(i)] and high P(i) diets and measured urine and lymph [P(i)]. Both lymph and urine [P(i)] increased with increasing P(i) loads, indicating P(i) absorption across skin and intestine. An initial fragment of a NaPi-II type transporter was amplified from kidney, and the full-length sequence was obtained. The protein showed the molecular hallmarks of NaPi-IIb transporters. When expressed in Xenopus oocytes the clone showed unusual pH dependence, but apparent affinity constants for P(i) and Na(+) were in the range of other NaPi-II transporters. Expression profiling showed that the transporter was present in skin, intestine and kidney. Reverse transcription-polymerase chain reaction assays on dissected renal tubules indicated expression in the collecting duct system. Collecting tubules and ducts were isolated, perfused and microelectrode recordings showed electrogenic P(i) transport in apical and basolateral membranes. Taken together, our results show that P(i) is handled by intestine, kidney and skin. The presently cloned NaPi-IIb is a likely candidate involved in P(i) absorption across these epithelia. In addition, electrophysiological experiments suggest that the collecting duct system plays an important role in P(i) homeostasis.

The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by DiamondBossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine andor = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.

By mathematical modelling, we analyse conditions for near-isotonic and isotonic transport by mammalian kidney proximal tubule.The model comprises compliant lateral intercellular space (lis) and cells, and infinitely large luminal and peritubular compartments with diffusible species: Na+, K+, Cl- and an intracellular non-diffusible anion. Unknown model variables are solute concentrations, electrical potentials, volumes and hydrostatic pressures in cell and lis, and transepithelial potential. We used data mainly from rat proximal tubule to model epithelial cells and interspace with luminal and peritubular baths of identical composition.The model of the tubular epithelium with physiological water permeability and paracellular electrical resistance generates solute coupled water uptake with an approx. 3% hypertonic absorbate. This function remains unperturbed following 'blocking' of apical water channels and in 'aquaporin-null' simulation. Reduced rate of volume reabsorption in AQP(-/-) mice would also require decreased apical sodium permeability. Paracellular convection accounts for approx. 36% of the net Na+ absorption, and the model epithelium accomplishes uphill water transport similar to rat proximal tubule. Na+ recirculation is required for truly isotonic transport. The tonicity of the absorbate and the recirculation flux depend critically on ion permeabilities of interspace basement membrane.Our model based on solute-solvent coupling in lateral space simulates major physiological features of proximal tubule, including significantly lower water permeability of the AQP1-null preparation, and a ratio of net sodium uptake and oxygen consumption exceeding that predicted from stoichiometry of the Na+/K+-pump. Physical properties of interspace basement membrane are critical for obtaining near-isotonic and truly isotonic transport.

This study deals with the morphology and ultrastructure of the mesonephros in adult caecilians of the species Geotrypetes seraphini. Based on serial sections in paraffin and araldite, nephrons are reconstructed and the cellular characteristics of different nephron segments described. The long and slender mesonephric kidneys of G. seraphini are broadest caudally and taper toward the front, where the organs are divided into smaller segmental divisions. Two nephron types can be distinguished on the basis of their connections to the coelom and their position within the nephric tissue: ventral nephrons connect to the coelom via a ciliated peritoneal funnel, whereas medial nephrons lack this connection. Both nephron types are composed of a filtration unit, the Malpighian corpuscle, and a renal tubule, which can be divided into six morphologically distinct segments: neck segment, proximal tubule, intermediate segment, early distal tubule, late distal tubule, and collecting tubule. Collecting tubules merge and form a branch system that opens into collecting ducts. Collecting ducts empty into the Wolffian duct. Proximal tubules of nephrons in the frontal divisions are morphologically different from the proximal tubules of more caudal kidney regions. Distal tubule subdivision is only clearly recognizable at the electron microscopic level. The length of each nephron segment is calculated from a ventral nephron with a total length of approximately 3.8 mm, and the course of the segments within the nephric tissue is reported. The number of nephrons was estimated at 1,700 units in each kidney. The segmentation and ultrastructure of the mesonephric nephrons in G. seraphini are discussed in relation to nephron descriptions from other caecilians and we further discuss the evolutionary origin of the amphibian nephron.

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.

SUMMARYWe studied the mechanisms of K+ transport in cells from isolated and perfused collecting tubules and ducts from the mesonephric kidney of the toad Bufo bufo. Cells were impaled with microelectrodes across the basal cell membrane. The basolateral membrane potential(Vbl) depolarized upon change of bath [K+] from 3 to 20 mmol l-1 demonstrating a large K+ conductance in this membrane. In collecting tubules and collecting ducts a Vbl of -66±2 mV and -74±4 mV depolarized by 30±2 mV and 36±3 mV, respectively (N=23; 15). The K+ channel inhibitor Ba2+ (1 mmol l-1)inhibited the basolateral K+ conductance and depolarized a Vbl of -64±4 mV by 30±6 mV (N=8). Luminal K+ steps (3 to 20 mmol l-1) demonstrated a K+ conductance in the apical cell membrane. In collecting tubules and collecting ducts a Vbl of -70±3 mV and-73±3 mV depolarized by 11±3 mV and 16±3 mV, respectively(N=11; 11). This conductance could also be inhibited by Ba2+, which depolarized a Vbl of -71±5 mV by 9±3 mV (N=5). The pump inhibitor ouabain (1 mmol l-1) depolarized Vbl, but addition of furosemide to bath solution did not affect Vbl. The[K+] in urine varied from 1.3 to 22.8 mmol l-1. In conclusion, we propose that the collecting duct system of B. bufosecretes K+ into the urine via luminal K+channels.

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.