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The tardigrade Hypsibius exemplaris has low tolerance to direct exposure to 50% relative humidity (RH) without preconditioning in a high RH environment. However, tardigrades pre‐treated with D942, a furancarboxylic acid derivative, partially acquired desiccation tolerance against 50% or even lower RH without preconditioning. Proteomic analysis of tardigrades treated with D942 indicated that responses to oxidative stress may be one of the underlying molecular mechanisms., The tardigrade Hypsibius exemplaris can undergo anhydrobiosis. Several chemicals that inhibit successful anhydrobiosis in H. exemplaris have been identified, and these chemicals inhibit the activity of signaling molecules. In the present study, we investigated whether upregulation of the activity of these signaling molecules could improve desiccation tolerance of H. exemplaris. Pre‐treatment with an indirect activator of AMP‐activated protein kinase [AMPK; which directly inhibits mammalian NAD(P)H dehydrogenase [quinone] 1 [NQO1] of mitochondrial complex I (D942)] significantly improved desiccation tolerance of H. exemplaris, whereas a direct activator of AMPK did not. To elucidate the underlying molecular mechanisms, we examined the proteome of tardigrades treated with D942. Two proteins, putative glutathione S‐transferase and pirin‐like protein, were upregulated by treatment. Both of these proteins are known to be associated with the response to oxidative stress. One of the downregulated proteins was serine/threonine‐proteinphosphatase 2A (PP2A) 65‐kDa regulatory subunit A alpha isoform, and it is interesting to note that PP2A activity was previously suggested to be required for successful anhydrobiosis in H. exemplaris. Taken together, our results suggest that D942 treatment may partially induce responses common to those of desiccation stress. The identification of a chemical that improves desiccation tolerance of H. exemplaris may facilitate further investigation into desiccation tolerance mechanisms.

Cytochrome b5 is an essential electron transfer protein, which is ubiquitously found in living systems and involved in wide variety of biological processes. Tardigrades (also known as water bears), some of which are famous for desiccation resistance, have many proteins unique to them. Here, we report spectroscopic and structural characterization of a cytochrome b5 like protein from one of the desiccation-tolerant tardigrades, Ramazzottius varieornatus strain YOKOZUNA-1 (RvCytb5 ). A 1.4 A resolution crystal structure revealed that RvCytb5 is a new cytochrome b5 protein specific to tardigrades.

Pigment-dispersing factor (PDF) denotes a conserved family of homologous neuropeptides present in several invertebrate groups, including mollusks, nematodes, insects, and crustaceans (referred to here as pigment-dispersing hormone [PDH]). With regard to their encoding genes (pdf, pdh), insects possess only one, nematodes two, and decapod crustaceans up to three, but their phylogenetic relationship is unknown. To shed light on the origin and diversification of pdf/pdh homologs in Panarthropoda (Onychophora + Tardigrada + Arthropoda) and other molting animals (Ecdysozoa), we analyzed the transcriptomes of five distantly related onychophorans and a representative tardigrade and searched for putative pdf homologs in publically available genomes of other protostomes. This revealed only one pdf homolog in several mollusk and annelid species; two in Onychophora, Priapulida, and Nematoda; and three in Tardigrada. Phylogenetic analyses suggest that the last common ancestor of Panarthropoda possessed two pdf homologs, one of which was lost in the arthropod or arthropod/tardigrade lineage, followed by subsequent duplications of the remaining homolog in some taxa. Immunolocalization of PDF-like peptides in six onychophoran species, by using a broadly reactive antibody that recognizes PDF/PDH peptides in numerous species, revealed an elaborate system of neurons and fibers in their central and peripheral nervous systems. Large varicose projections in the heart suggest that the PDF neuropeptides functioned as both circulating hormones and locally released transmitters in the last common ancestor of Onychophora and Arthropoda. The lack of PDF-like-immunoreactive somata associated with the onychophoran optic ganglion conforms to the hypothesis that onychophoran eyes are homologous to the arthropod median ocelli. J. Comp. Neurol. 523:1865–1885, 2015 © 2015 Wiley Periodicals, Inc.

Summary The use of epifluorescence microscopy coupled with ultraviolet (UV) autofluorescence is suggested as a means to view and interpret tardigrade structures. Endogenous fluorochromes are a known component of tardigrade cuticle, claws and bucco-pharyngeal apparatus. By imaging the autofluorescence from tardigrades, it is possible to document these structures in detail, including the subdivisions and boundaries of echiniscid (heterotardigrade) plates and the nature and spatial relationships of the texture (pores, granules, papillae and tubercles) on the various plates. This allows the determination of taxonomic features not easily seen with other microscopic techniques. Lay Description Here we describe the advantages of using UV autofluorescence to image the cuticle, claws and buccopharynx of water bears or tardigrades. Tardigrades are identified by their cuticle's surface, the shape of their mouth and buccopharynx, and the size and shape of their claws. Because the cuticle autofluoresces in proportion to its thickness, this method can tell pores and bumps apart when other light microscope techniques cannot. Some tardigrades, known as echiniscids, are armor-plated on their backs. These echiniscids are separated by the texture and shape of these plates and cannot be identified unless we know if a pore or a bump is present. Thus UV autofluorescence is a very useful tool for anyone wishing to look at tardigrades.

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.

Aim To analyse the importance of climatic and geographical isolation in determining the patterns of speciation and distribution of species within the tardigrade genus Echiniscoides. Location Marine intertidal zone, globally. Methods DNA was extracted from 465 individual tardigrades from 48 localities world-wide. The tardigrades were divided into clusters using several distance-based criteria. The phylogeny of these clusters was estimated with Bayesian analyses. The relationships between genetic distance and substrate, climate, and geographical distance were tested with a new improved Mantel test which incorporates phylogenetic uncertainties by analysing the raw tree data instead of the averaged tree. Results Approximately 40 clusters, each probably corresponding to species, were recovered from the genetic analysis; the number of clusters fluctuated depending on the criterion used for cluster delimitation. Each cluster had a limited temperature range and all clusters were confined to single oceans under all realistic criteria for cluster delimitation. Apart from a tropical cluster, each cluster occurred only in one hemisphere. Occurrence on different substrata was not correlated with genetic distance between clusters. Both geographical distance and climate were correlated with genetic distance; however, the correlation between geographical and genetic distance disappeared when the non-independence of climatic and geographical distance were controlled for. Main conclusions The distribution of individual species of Echiniscoides is limited by climate and geographical distance. Distance does not appear to be a major factor influencing phylogeny in this genus, but ecological speciation along a temperature gradient appears to be important.

Little is known about egg dormancy in tardigrades, except for their ability to survive desiccated for a long time. Our previous analyses of the life-history traits of a reared strain of the leaf litter-dwelling eutardigrade Paramacrobiotus richtersi revealed a variation in hatching phenology, suggesting the presence of diapause (resting) eggs in tardigrades. To study adaptive strategies in an unpredictable environment subject to stochastic variability, such as that colonized by tardigrades, we have analysed the hatching phenology of an apomictic triploid cytotype of P. richtersi. The first lab oviposition of mature females collected in the field in the spring and fall as well as cohorts of eggs laid by females born in the laboratory were used. The eggs of all samples, maintained under the same constant experimental conditions, had a high hatching percentage (from 75 to 93%) but a high variability occurred in the hatching time. Four patterns were identified. First, subitaneous eggs hatched within 30–40 days from oviposition. Second, delayed-hatching eggs hatched gradually over 41–62 days. Some eggs did not hatch within 90 days from oviposition when water was maintained in the culture. Within this group, 13% of eggs (diapause resting eggs; third category) do not hatch until they are subjected to desiccation, followed by rehydration, while 87% never complete their development (abortive eggs; fourth category). The four categories of eggs had no morphological differences. The high variability in the hatching time of tardigrade eggs might be considered a form of bet-hedging.

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.

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani & Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.

Phylum Tardigrada consists of ∼1000 tiny, hardy metazoan species distributed throughout terrestrial, limno-terrestrial and oceanic habitats. Their phylogenetic status has been debated, with current evidence placing them in the Ecdysozoa. Although there have been efforts to explore tardigrade phylogeny using both morphological and molecular data, limitations such as their few morphological characters and low genomic DNA concentrations have resulted in restricted taxonomic coverage. Using a protocol that allows us to identify and extract DNA from individuals, we have sequenced 18S rDNA from 343 tardigrades from across the globe. Using maximum parsimony and Bayesian analyses we have found support for dividing Order Parachela into three super-families and further evidence that indicates the traditional taxonomic perspective of families in the class Eutardigrada are nonmonophyletic and require re-working. It appears that conserved morphology within Tardigrada has resulted in conservative taxonomy as we have found cases of several discrete lineages grouped into single genera. Although this work substantially adds to the understanding of the evolution and taxonomy of the phylum, we highlight that inferences gained from this work are likely to be refined with the inclusion of further taxa-specifically representatives of the nine families yet to be sampled. © The Willi Hennig Society 2008.

Desiccation tolerance commonly found among tardigrades allows them to cope with temporal variation of available water. Although the long-term survival of adults has been demonstrated in several species, desiccation tolerance of eggs and embryos is less well studied, however it is an important aspect from an ecological and evolutionary point of view. For the first time we evaluated the desiccation tolerance and subsequent hatching success of five different developmental stages of the tardigrade species Milnesium tardigradum, when rehydrated following drying at eight different humidity levels (10, 20, 31, 40, 54, 59, 72, 81%). Humidity level and developmental stage are significant factors in determining successful hatch rates. The results showed that the less developed stages were quite sensitive to desiccation. Low humidity levels during the first 3 days of development lead to a decrease in hatch rates following rehydration. Later developmental stages showed higher hatch rates than embryos dried at earlier stages. However, fast drying at low humidity levels resulted in delayed development and lower hatch rates following rehydration. In general, further developed embryos exhibit a better survival capacity compared with younger stages.

Living in harsh and variable environments that are prone to periodic desiccation, tardigrades exhibit remarkable tolerance against physical extremes through a state known as anhydrobiosis. To study the effect of this state on the longevity and hence the lifecycle in the taxon Tardigrada for the first time, we exposed a tardigrade species, Milnesium tardigradum, to alternating periods of drying and active feeding periods in a hydrated state. Compared with a hydrated control, the periodically dried animals showed a similar longevity, indicating that the time spent in anhydrobiosis was ignored by the internal clock. Thus, desiccation can produce a time shift in the age of tardigrades similar to the model described for rotifers that has been termed ‘Sleeping Beauty’.

To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non-reducing disaccharide trehalose to protect against dehydration damage. The trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.

Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).

Allozymes were assessed by starch gel electrophoresis in 3 populations of a eutardigrade, Richtersius coronifer, with different reproductive modes. One population from Italy (with 2 sub-populations) was amphimictic and 2 populations (1 from Italy and 1 from Sweden) were parthenogenetic. All populations, irrespective of their reproductive mode, were diploid with the same chromosome number (2n=12) and had bivalents in the oocytes. Of the 14 loci analyzed, only 3 were polymorphic. The amphimictic population had a higher degree of genetic variability (mean heterozygosity >0.25) than the parthenogenetic populations (mean heterozygosity of the 2 populations

We describe a terrestrial faunal community including only Tardigrada and Rotifera, present on inland nunataks of Ellsworth Land, Antarctica (∼75°–77° S, 70°–73° W). The fauna is exceptional in its simplicity, including five tardigrade species (three new to science) and at least two rotifer species, which comprise two consumer trophic levels. Nematode worms, the most important element of the simplest faunal communities previously reported worldwide (from the Ross Sea Dry Valley region of continental Antarctica), and microarthropods, otherwise represented in all known Antarctic terrestrial communities, are absent. The tardigrade community composition shows affinity with the continental Antarctic fauna, with which it shares three species. The remaining two species are unique to Ellsworth Land and may suggest a prolonged existence as a distinct biogeographical unit.

For anhydrobiotic metazoans the rate of desiccation is an important factor influencing the probability of survival in a dry anhydrobiotic state. Formation of animal aggregations, in which the exposed body surface area of individual animals is reduced, represents one way to reduce the rate of evaporation. Such aggregations have earlier been documented in e.g., nematodes. We experimentally evaluate the effect of aggregation size (number of animals in a group of desiccating animals) on anhydrobiotic survival in the eutardigrade Richtersius coronifer. The experiment shows that aggregation provides a clear improvement on anhydrobiotic survival. The most likely explanation for this is that aggregated animals were exposed to a lower rate of desiccation. Although the empirical evidence of aggregation in tardigrades is scarce, our study suggests that aggregation could potentially be an important survival factor for tardigrades living in environments characterized by periods of rapid desiccation.

The ability of some animal taxa (e.g., nematodes, rotifers, and tardigrades) to enter an ametabolic (cryptobiotic) state is well known. Nevertheless, the phenotypic factors affecting successful anhydrobiosis have rarely been investigated. We report a laboratory study on the effects of body size, reproductive condition, and energetic condition on anhydrobiotic survival in a population of the eutardigrade Richtersius coronifer. Body size and energetic condition interacted in affecting the probability of survival, while reproductive condition had no effect. Large tardigrades had a lower probability of survival than medium-sized tardigrades and showed a positive response in survival to energetic condition. This suggests that energy constrained the possibility for large tardigrades to enter and to leave anhydrobiosis. As a possible alternative explanation for low survival in the largest specimens we discuss the expression of senescence. In line with the view that processes related to ahhydrobiosis are connected with energetic costs we documented a decrease in the size of storage cells over a period of anhydrobiosis, showing for the first time that energy is consumed in the process of anhydrobiosis in tardigrades. (C) 2002 Wiley-Liss, Inc. (Less)

This study represents the first systematic investigation of long-term anhydrobiotic survival in tardigrades, rotifers and nematodes inhabiting mosses and lichens. Sixty-three different samples from public and private collections, kept dry for 9-138 years, were examined. Rotifers of the genus Mniohia and the eutardigrade Ramazzottius oberhaeuseri (hatched from eggs) were found alive from one of the samples (9 years old). These observations represent the longest record for rotifers in the anhydrobiotic state. For tardigrades, our results confirm previous reports on the upper limit of anhydrobiotic survival under atmospheric oxygen conditions. This study suggests the possibility that tardigrade eggs are able to withstand longer periods in anhydrobiosis than animals. Some problems related to the evaluation of long-term anhydrobiotic survival, such as contamination and chemical treatments of samples, are reported. The possible role of the microenvironment in which the anhydrobiotic animals are kept is discussed.

Summary 1Tardigrades comprise a micrometazoan phylum that is a sister group of the arthropods. 2They are components of the meiobenthos in lotic habitats, and ≈ 50–70 species have been reported in such habitats world-wide. Approximately 800 species have been identified from all marine, freshwater, and terrestrial habitats. 3Taxonomy is based primarily on the morphology of the claws, buccal-pharyngeal apparatus, cuticle and eggs. 4Reproductive modes include sexual reproduction (amphimixis) and parthenogenesis. The sexual condition of individuals may be either gonochorism, unisexuality, or hermaphroditism. Moulting occurs throughout the life of the tardigrade. 5Latent states (cryptobiosis, including encystment, anoxybiosis, cryobiosis, osmobiosis and anhydrobiosis) enable tardigrades to withstand unfavourable environmental conditions. 6Population densities, life histories, dissemination and biogeography of freshwater species are poorly known.

The Tardigrada is a cosmopolitan phylum of pre-Pangaean origin, yet tardigrade families and genera show distinct biogeographic components isolated by two major geological events. Separate Laurasian and Gondwanan familial clusters correlate with the Triassic disintegration of Pangaea, while discrete Antarctic, Australian and New Zealand familial/generic clusters relate to the subsequent Jurassic/Cretaceous disintegration of Gondwana

The ultrastructure of the adult pharynx of the eutardigrade Halobiotus crispaeis described for the active, sexually mature summer form as well as for the winter form known as pseudosimplex 1. The total number of cells is 54: 27 muscle cells, 12 apical cells, nine nerve cells and six buccal tube cells. The development of the pharynx is described on the basis of five ontogenetic stages. Three of them are reconstructed three-dimensionally with the help of computer techniques. The number of cells composing the pharynx remains constant from an early embryological stage and each cell can be traced and recognized in the adult pharynx. The findings regarding the ultrastructure of the pharynx are compared to previous descriptions. A hypothesis regarding the coordinated function of the organ and a preliminary attempt to define the plesiomorphic condition of the tardigrade pharynx are presented. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.

This study considers specimens of Macrobiotus hufelandi C. A. S. Schultze, 1834 collected from Italy and Germany, including the type locality. In addition to a qualitative analysis of the animals and eggs, statistics were performed on the measurements of the sclerified parts of the animals. The data illustrate low intra-species variability in the morphology of both the animals and the eggs. Differences in egg shell morphology, previously attributed to the variability of M. hufelandi, were found to fall into distinct types, related to different animal morphotypes. The data also indicate that M. hufelandi contains several new species: M. macrocalix sp.n., M. sandrae sp.n. and M. terminalis sp.n.; M. hufelandi is redescribed, and a neotype is assigned.

Changes in the trehalose level in the tardigrade Adorybiotus coronifer were ob- served during induction of, and arousal from, anhydrobiosis. A trehalose accumulation surpassing 1.6% dry weight (d.w.) in anhydrobiotic animals collected dry on their biotope was rapidly reduced on rehydration, reaching the level of active animals (0.1-0.2% d.w.) after 6 hours. Tardigrades dried while embedded in sand in the laboratory accumulated trehalose from 0.1% to 2.3% d.w. within 5-7 hours. Induction of aerobic acidosis in arousing tardigrades by CO, perfusion reversibly arrested re- activation for at least 36 hours and induced a reduction in anabolic and catabolic activities, mea- sured as a significant reduction in trehalose degradation, and a sevenfold reduction in the rate of protein synthesis. These data support the hypotheis that trehalose generally serves a protective role in desiccation- tolerant Metazoa, but indicate that tardigrades require only a moderate level for efficient pro- tection. Trehalose accumulation A positive correlation between the survival of the cryptobiotic state and the accumulation of the disaccharide trehalose has been demonstrated for a number of anhydrobiotic organisms, and con- centrations as high as 13-18% dry weight have been reported in anhydrobiotic cysts of the crusta- cean Artemia salina' (Clegg, '64; '65) and in the nematode Aphelenchus avenae (Madin and Crowe, '75; Crowe et al., '77). Moreover, trehalose reduces damage, induced by freeze-drying, to iso- lated liposomes (Crowe et al., '861, microsomes from sarcoplasmatic reticulum (Crowe et al., '83), and the enzyme phosphofructokinase (Carpenter et al., '87; '88). Other data such as the physical properties of nearly dry Artemia salina cysts (Clegg et al., '82; Clegg, '86) and computerized modeling of molecular interactions between tre- halose and a phospholipid bilayer (Gaber et al., '86) indicate that trehalose is a likely candidate for the polyhydroxy compound that, according to the water replacement hypothesis (Webb, '65; Crowe, '711, replaces the structural water of the cellular compounds during desiccation. Anhydrobiosis is widespread in the phylum Tardigrada, but quantification of trehalose accu- mulation during the induction of anhydrobiosis has not yet been made in this phylum. Crowe ('75) reported that paper chromatography analysis of ethanol extracts of the tardigrade Macrobiotus

Tardigrades were sampled from diverse biotypes and classified according to four xeric variables to assess susceptibility to desiccation: exposure to insolation, elevation, and standardized desiccation rate and hydration capacity of the habitat plant. Fifteen tardigrade species were recorded including Hypsibius cataphractus Maucci, a new addition to the British fauna. Xeric associations with seven tardigrade species were analysed by multiple regression, using incidence and population density as independent abundance estimates. Species show considerable variation in ecotype. The hygrophilic Macrobiotus hufelandi and Hypsibius dujar dini are excluded from rapidly desiccating habitats. Xerophiles, Milnesium tardigradum and H. oberhaeuseri, do not favour high insolation or high desiccation rate but apparently shun poorly drained sites and/ or prolonged hydration. Significant interspecific associations are identified between Milnesium tardigradum and two Hypsibius species which it may exploit for food. Negative associations between three other species—M. hufelandi, M. richtersi and H. prosostomus—suggest competitive exclusion. The interstitial meiofauna of a dehydrating moss cushion migrates vertically to the proximal C-zone, although this behaviour is not observed in Echiniscus testudo; implications of this for the species' ecotype are discussed.

The hindgut of the semi-terrestrial tardigrade, Milnesium tardigradum was examined with light and electron microscopy. The hindgut consists of a cloaca and an anterior hindgut. It is delineated anteriorly by the pylorus into which four Malpighian tubules empty and posteriorly, by a broad cloacal slit. A single oviduct enters the hindgut at the junction between the cloaca and the anterior hindgut. Two pairs of muscles insert on the cloaca and anterior hindgut respectively. Electron microscopic observations demonstrate that the anterior hindgut is a specialized transporting epithelium. The luminal surface is covered by a thin layer of cuticle which penetrates into channel-like invaginations. Numerous mitochondria are concentrated apically. The basal and lateral surfaces are also folded. The cells are joined apically by deep tight junctions and a simple basal lamina lines the entire hindgut. The cloaca which receives the contents of the gut and Malpighian tubules as well as gametes of the reproductive tract is a transitional organ that exhibits several characteristics of the hypodermis and anterior hindgut. The cuticle of the cloaca changes sequentially from the complex structure of the integument to a simple layer of the anterior hindgut. The function of the hindgut is discussed with emphasis on the possible response of the anterior hindgut to a hypoosmotic habitat, evaporative water loss during the induction of anhydrobiosis and low oxygen tension.