Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa, as do the two major invertebrate model organisms: Caenorhabditis elegans and Drosophila melanogaster. We present a brief description of the tardigrades and highlight species that are currently used as models for physiological and molecular investigations. Tardigrades are uniquely adapted to a range of environmental extremes. Cryptobiosis, currently referred to as a reversible ametabolic state induced by e.g. desiccation, is common especially among limno-terrestrial species. It has been shown that the entry and exit of cryptobiosis may involve synthesis of bioprotectants in the form of selective carbohydrates and proteins as well as high levels of antioxidant enzymes and other free radical scavengers. However, at present a general scheme of mechanisms explaining this phenomenon is lacking. Importantly, recent research has shown that tardigrades even in their active states may be extremely tolerant to environmental stress, handling extreme levels of ionizing radiation, large fluctuation in external salinity and avoiding freezing by supercooling to below -20 °C, presumably relying on efficient DNA repair mechanisms and osmoregulation. This review summarizes the current knowledge on adaptations found among tardigrades, and presents new data on tardigrade cell numbers and osmoregulation.

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.

Halobiotus crispae is a marine eutardigrade belonging to Hypsibiidae. A characteristic of this species is the appearance of seasonal cyclic changes in morphology and physiology, i.e. cyclomorphosis. Halobiotus crispae was originally described from Nipisat Bay, Disko Island, Greenland. The present study investigates the distribution of this species and describes the seasonal appearance of cyclomorphic stages at the southernmost locality, Vellerup Vig in the Isefjord, Denmark. Our sampling data indicate that the distribution of H. crispae is restricted to the Northern Hemisphere where we now have found this species at seven localities. At Vellerup Vig data from sampling cover all seasons of the year and all of the originally described cyclomorphic stages have been found at this locality. However, when comparing the lifecycles of H. crispae at Nipisat Bay and Vellerup Vig, profound differences are found in the time of year, as well as the period in which these stages appear. Noticeably, at Nipisat Bay the pseudosimplex 1 stage is a hibernating stage occurring during the long Arctic winter. In contrast, at Vellerup Vig, this stage appears during the summer. Thus, while pseudosimplex 1 seems to be an adaptation to withstand low temperatures in Greenland, this stage possibly enables the animal to tolerate periods of oxygen depletion and heat stress during the Danish summer. Moreover, a characteristic of the Danish population is the presence of a prolonged pseudosimplex 2 stage. The environmental or endogenous signals underlying the transition between different stages remain unknown. In addition, we report the genetic diversity and phylogenetic position of H. crispae based on the first molecular data obtained from this species. Our molecular data confirm that H. crispae from Greenland and Denmark are in fact the same species. Thus, the observed life cycle changes occur within a species and do not represent life cycle variation between different species. In addition, our molecular data suggest that Halobiotus has evolved within Isohypsibius. Further investigations on the lifecycle of members of the Halobiotus genus as well as other members of the Hypsibiidae is needed in order to establish whether cyclomorphosis is i) a general theme among members of Hypsibiidae or ii) an autapomorphy for Halobiotus

Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state termed anhydrobiosis in response to desiccation. In the anhydrobiotic state, tardigrades display an incredible capacity to tolerate extreme environmental stress, not necessarily encountered in their natural habitat. In this study, we determine the effect of different extreme stresses on initial survival, long-term survival and fecundity of selected species 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 microgravity environment. Experiments on Whatman-3 filters show an effect of cosmic radiation on the survival of the eutardigrade Richtersius coronifer just after returning to Earth; however, after 2 years of desiccation on Whatman-3 filters, none of the tardigrades previously exposed to cosmic radiation could be revived. In a microcosmos experiment, the tardigrades R. coronifer, Ramazzottius oberhauseri and Echiniscus testudo were desiccated on a moss substrate together with rotifers and nematodes. Very low survival rates were observed in this experiment, likely due to the applied desiccation protocol. Embryos of the tardigrade Milnesium tardigradum were also exposed to cosmic radiation; they all hatched in the laboratory after the flight. In addition, experiments testing extreme cold and vacuum tolerance in R. coronifer show that tardigrades in anhydrobiosis are unaffected by these conditions.

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.

SUMMARY. Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.

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.

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.