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A new interstitial arthrotardigrade, Renaudarctus psammocryptus n. gen., n. sp., is described from high-energy marine beaches of Fort Pierce and Boca Raton, Florida. It is found in sediment consisting of stratified layers of coarse quartz sand and shell hash. The tardigrade is distinguished particularly by its dorsal cuticular plates, toes, and claw structure. The Renaudarctidae is established as a new family in the order Arthrotardigrada. A recently described tardigrade, Neostygarctus acanthophorus from a marine cave in Italy, originally placed in the family Stygarctidae, is included in the Renaudarctidae based on toe and claw structure. The phylogenetic relationships of Renaudarctidae to other families in Heterotardigrada are discussed. Seven other species of tardigrades found with the new species included: Parastygarctus sterreri, Stygarctus gourbaultae, Halechiniscus remanei, Raiarctus colurus, Raiarctus sp., Tanarctus tauricus, and Batillipes bullacaudatus. Only B. bullacaudatus and H. remanei have been reported from the U.S.A. previously. The vertical and horizontal distribution of the tardigrades in the beaches appears to be related to granulometry more than to other physical or chemical factors, including depth in the beach. Since the discovery of the first marine tardigrade, Microlyda Dujardin, 1851, most marine biologists have considered these microscopic metazoans to be a rare component of the marine ecosystem. The first monograph on this phylum (Marcus, 1929) recorded only six marine species. Schulz (1935, 1951, 1955) described several new species. Since 1955, the most significant taxonomic contribution has been that of Renaud-Mornant, who has produced 32 publications and described 9 genera and 27 species. In a recent review of the marine Tardigrada (see Renaud-Mornant, 1982), 22 genera of Heterotardigrada were noted. These animals, a total of 56 species, have been found in a wide variety of marine habitats from the deep sea to tropical beaches. The material was obtained through the kind cooperation of the Smithsonian Marine Station at Link Port, Florida. We especially acknowledge the technical assistance of William Lee, of the Smithsonian Marine Station at Link Port, who assisted with sampling and processing; the cooperation of Carolyn Gast, of the Department of Invertebrate Zoology, Smithsonian Institution, who prepared the illustrations; and the aid of Marie Wallace, also of the Department of Invertebrate Zoology, who assisted with sample processing and in the preparation of the manuscript. Our appreciation is also expressed to Horton H. Hobbs, Jr., who critically reviewed this manuscript and made many valuable suggestions. This research was supported by the Danish Natural Science Research Council (Grant No. 11-3558), a NATO Science Fellowship and a Smithsonian Postdoctoral Fellowship to the senior author, and funds made available to the junior author through the Smithsonian Marine Station. 2 Institute of Cell Biology and Anatomy, University of Copenhagen, Universitetsparken 15, DK2100 Copenhagen 0, Denmark. TRANS. AM. MICROSC. Soc., 103(3): 295-311. 1984. This content downloaded from 157.55.39.177 on Wed, 16 Nov 2016 04:21:04 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. In the United States, the majority of marine tardigrade species have been reported from intertidal sandy beaches of the East Coast (Hallas & Kristensen, 1982; Lindgren, 1971; McGinty & Higgins, 1968; McKirdy, 1975; Pollock, 1970a,c, 1979). Although the marine tardigrades from intertidal, mostly highenergy, marine beaches of North America are relatively well known, little is known about these animals from other marine habitats. Renaud-Mornant (1982) predicted a remarkable increase in the number of species once deep-sea, coral sand, and other substrates were sampled. Indeed, five species of arthrotardigrades were described recently from the subtidal sediment of marine caves along the Italian coast (Grimaldi de Zio et al., 1982a,b), and the first semipelagic tardigrade, Tholoarctus natans, was found in subtidal coarse sand and shell gravel collected along the northern coast of France (Kristensen & RenaudMornant, 1983). In this investigation, a relatively new technique of momentarily subjecting large quantities of coarse sediment to freshwater (Kristensen & Higgins, 1984) is used. Furthermore, we have taken samples deeper than usual, to 1.8 m below the beach surface. Our research was directed toward substrate choice of the different species in a stratified coarse quartz sand habitat with layers of shell hash. In this paper, the first of two on tardigrades from coarse sediment, a new family of Arthrotardigrada is described. A future paper will include descriptions of tardigrades found in the same kind of sediment located subtidally, about 10 km off the coast of Fort Pierce, Florida. MATERIALS AND METHODS Samples were collected from two different beaches located on the East Coast of Florida, Pepper State Park Beach at Fort Pierce and Red Reef Park Beach at Boca Raton. During the period of investigation, from 5 October 1982 to 9 April 1983, almost monthly samples were taken within 1 h of low tide along a transect of four stations, extending from midto low-tide regions, perpendicular to the shore. A single hole was dug at each station to ground-water level. The ground-water salinity, measured with an American Optical refractometer, and the temperature were recorded. From each hole, about 30 kg of sediment were placed in a plastic bucket and saturated with seawater. Extraction of organisms from the sediment was accomplished by transferring small amounts (a few handfuls) of sediment to a bucket containing about 10 liters of freshwater. The osmotic shock causes the tardigrades (and other interstitial organisms) to release their otherwise tenacious hold on the sand grains. The sediment was agitated gently and the water decanted through a 62-,m mesh net. After repeating this process once with the same sediment, the sediment was discarded and a unit of untreated sediment was used to repeat the process. After the second unit had been processed, the contents of the net were washed into a container with 2-3% formalin buffered with Borax, or fresh seawater was used to wash the animals into a watch glass for live observation. (The reintroduction of seawater allowed most of the meiofauna to recover from the osmotic shock.) The animals were observed with the aid of 50x magnification of a stereomicroscope and by 1,250x oil immersion phase-con296 This content downloaded from 157.55.39.177 on Wed, 16 Nov 2016 04:21:04 UTC All use subject to http://about.jstor.org/terms VOL. 103, NO. 3, JULY 1984 trast microscopy. Sorted animals later were transferred with a drop of 2% formalin to microslides and covered with coverslips. The formalin preparation was infused with a 10% solution of glycerin in 96% ethyl alcohol and allowed to evaporate to glycerin over a period of several days; the resulting wholemount preparation was sealed by Murrayite. The holotype, allotype, and one paratype were illustrated by aid of a camera lucida. One specimen was pressed tightly between the coverslip and slide in order to make certain critical observations. Zeiss differential interference-contrast optics (Nomarski) were used to make photomicrographs while whole-mount specimens were still in 2% formalin. Measurements are given in micrometers (ium) and the resulting data are expressed in a terminology used by Kristensen & Renaud-Mornant (1983) and Kristensen & Higgins (1984). The holotype and allotype of the new family are deposited at the National Museum of Natural History, Smithsonian Institution (USNM), Washington, D.C. (U.S.A.). The squashed preparation and two other paratypes are deposited at the Zoological Museum of Copenhagen, Denmark, and bear the senior author's (RMK) specimen numbers only.

No tardigrades were reported from Oklahoma prior to the study, and very little was known concerning the tardigrade fauna of the surrounding states. Of 16 species found, two (Pseudechiniscus cor- nutus lobatus and Macrobiotus hibiscus) had not been previously re- ported from North America.

Differences in the species of bryophytes (mosses) and their tardigrade (water-bear) inhabitants in tundra adjacent to the trans-Alaska Pipeline haul road (now the Dalton Highway) are associated with environmental perturbations induced by the presence of the road, primarily road dust. Vehicular traffic on this gravel road generates a calcium-rich mineral dust that is windblown onto the tundra and is considered among the road's most significant environmental impacts. Sphagnum mosses rarely occur within 10 m of the road, where dustfall is greatest. Sphagnum specimens (S. lenense, S. balticum, S. rubellum, and S. aongstroemii) were collected in transects paralleling the road at 50 and 500 m. Roadside moss samples are dominated by species tolerant of high calcium and drier habitats (e.g., Aulacomnium turgidum, Dicranum angustum). Tardigrade species dwelling within these mosses are typical of more xeric environments (Diphascon scoticum and Hypsibius dujardini). While these fungivorous and algal feeding species numerically dominate roadside mosses, areas farther from the road are dominated by omnivores and carnivores (Macrobiotus hufelandi and M. harmsworthi, respectively).

Eight intertidal beach localities along the Pacific coast of the United States were surveyed: two in Washington, one in Oregon, and five in California. Seven species of marine Heterotardigrada were encountered including Stygarctus bradypus, S. spinifer, Halechiniscus remanei, Batillipes gilmartini, B. mirus, and Echiniscoides sp. A new species, B. tridentatus n. sp., from Washington and California is distinctive in possessing two pairs of lateral projections between legs III and IV, a prominent spike on the shank of legs IV, and a two-pointed caudal apparatus. Despite interest in marine Tardigrada worldwide (see Renaud-Mornant, 1982) and along the east coast of the United States (e.g., Kristensen & Higgins, 1984b; Lindgren, 1971; McGinty & Higgins, 1968; McKirdy, 1975; Pollock, 1970a,b), the west coast of North America has received little attention. Only four species, all from California, are reported from the entire coast. Echiniscoides sigismundi (Schultze, 1865) is known from Dillon Beach (Schuster & Grigarick, 1965) and from Santa Cruz Island (Schuster & Grigarick, 1970). (These animals were tentatively referred to the subspecies, E. sigismundi groenlandicus, by Kristensen & Hallas, 1980.) Styraconyx sargassi Thulin, 1942 was first reported from California by Mathews (1938, as Bathyechiniscus tetronyx) and later by Schuster & Grigarick (1965) from Dillon Beach. The genus Batillipes is represented by the description of B. gilmartini from Pacific Grove (McGinty, 1969), where it was found with Halechiniscus remanei. Finally, Meyer (personal communication) reported Batillipes mirus in the San Diego area. The specific objective of this survey was to expand our knowledge of the marine tardigrade fauna of the Pacific coast of the United States. In a larger context, added information regarding this poorly understood segment of the Pacific tardigrade fauna should contribute to future discussions of global tardigrade biogeography. MATERIALS AND METHODS During summer 1987, collections were made from selected beaches along the northeastern Pacific coast from Seattle, Washington to southern California; these included two beaches in Washington state, one from Oregon, and four from California. Collections were made in 1983 from a fifth California site. Although this study was not exhaustive, collections were made to survey the full intertidal range of each beach comparably at each locale. Publication costs, in part, are being met by a grant from the Spencer-Tolles Fund of the American Microscopical Society. TRANS. AM. MICROSC. SOC., 108(2): 169-189. 1989. ? Copyright, 1989, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.235 on Fri, 07 Oct 2016 04:56:52 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. A semi-quantitative sampling procedure was used. Sample sites were located at the low tidal line and at /4, /2, and 3/4 tidal heights along the beach. At most sites, samples were taken from the sand surface to the depth of the low tidal water table (or close thereto) at 15-cm intervals (1-15, 15-30, 30-45, 45-60 cm, etc.). Each sample consisted of approximately 250 cm3 of sediment representing equal quantities taken from each 15-cm depth interval. Each sample was soaked in approximately 3 liters of fresh water, following a procedure modified from Kristensen & Higgins (1984b). After 10 min, including periodic swirling of the bucket and its contents, sand and meiofauna (presumed to be osmotically incapacitated) were thoroughly suspended in the water medium. Heavier material, mostly sand, was allowed to settle briefly and lighter objects, including meiofauna, were decanted through a filter of 62-gm mesh. Organisms and debris retained by the net were preserved in 10% formalin, lightly tinted with rose bengal stain. During subsequent storage, preserved organisms absorbed pink coloring from the stain making them easier to discern from surrounding debris during sorting, manipulation, and identification. In the laboratory, subsamples of each resuspended sample were diluted with tap water and examined using a dissecting microscope at 40-60 x. Specimens were examined as uncovered wet mounts using an inverted phase-contrast microscope or as temporarily covered wet mounts with differential interference microscopy. The former technique has the advantage of avoiding distortion from cover glass pressure and of permitting reorientation of specimens during observation. Permanent mounts were made by adding some specimens to a 10% solution of glycerine in ethanol which was allowed to evaporate to pure glycerine. Tardigrades were mounted in glycerine on Cobb slides using a double cover glass technique. Cover glasses were sealed by Cytomount? and ringed with epoxy paint. Drawings were made using a video-camera lucida technique. Specimens were projected by video camera onto a high resolution (700-line) television monitor. A plate of one-way mirror glass was mounted at a 45? slope (low side toward the monitor) on a light table placed immediately in front of the monitor. When observed from above this sloping mirror, a clear and undistorted reflection of the televised image was observable coincident with a faint image of a drawing pencil placed on paper on the light table surface beneath. The televised image could then be traced accurately. Measurements were made from drawings using a digitizing tablet attached to a microcomputer. Granulometric surveys followed procedures of Hulings & Gray (1971). Single samples of sand representative of each beach were screened and weighed. The graphic mean and sorting were calculated. Dried, weighed samples were repeatedly flooded with excess 10% HC1 until no further reaction was observed and then redried to calculate the percentage calcium carbonate in samples. Collection sites. Pertinent data regarding study sites are summarized in Table I. Granulometric analyses for five of these sites are shown in Fig. 1. Site 1: Golden Gardens Park, Meadow Point, on Shilshole Bay, Puget Sound north of Seattle, Washington. This beach includes a wide assortment of grain sizes, including gravel and cobble components. Collection was limited to me170 This content downloaded from 157.55.39.235 on Fri, 07 Oct 2016 04:56:52 UTC All use subject to http://about.jstor.org/terms VOL. 108, NO. 2, APRIL 1989

The purpose of this study was to investigate some physical and biological parameters affecting the revival of the tardigrade, Macrobiotus areolatus Murray, 1907, from a cryptobiotic state. The animals were dried on filter paper under controlled conditions. Based on per cent survival, optimum recovery conditions were established within experimental ranges of pH 6-8, 5-27 C, and 5-9 mgm/L dissolved oxygen. Studies of revival time over the optimum range indicate significant temperature and oxygen effects, revival time appearing to vary inversely with temperature and dissolved oxygen concentration. The pH effect on revival time is not significant. Preliminary data indicate that nutriton may have an effect on revival time. Well-fed animals revive more rapidly than starved ones. Revival time appears to be directly propor- tional to the duration of the cryptobiotic state. Possible errors and suggestions for improvement of experimental techniques are cited. Numerous animals are known to exhibit latent stages, but the most interesting are those in which a nearly complete arrest of metabolic activities occurs. Such an arrest is a cryptobiotic state. Desiccation in tardigrades, with which this paper is concerned, represents a particular kind of cryptobiosis. In the desiccated state tardigrades appear to be lifeless. Upon the addition of water they rapidly swell and resume active life. In the active tardigrade homeostatic processes regulate an organism-environ- ment interaction which is at a point far removed from chemical equilibrium, just as in any other organism. In the desiccated tardigrade, however, material and energy exchange with the surrounding environment are greatly reduced and the metabolic rate approaches zero. The adaptive significance of such a phe- nomenon appears obvious. Under environmental stress the biotic integrity of the organism is preserved when the same stress might otherwise result in its destruction. It is doubtful that metabolism in the cryptobiotic tardigrade ever reaches zero. Baumann (1922) was of the opinion that tardigrades will not survive complete desiccation, and probably carry on some metabolic activities, though at an extremely low rate, in the cryptobiotic state. Schmidt (1948) found that rotifers revive from the cryptobiotic state more slowly, and fewer survive, with an increase in the time that they are kept dry. Pigon & Weglarska (1953, 1955) detected a measurable oxygen uptake in desiccated tardigrades at high humidities. At lower humidities the measurable rate of oxygen uptake was low and unreliable. They believed that metabolic activities were carried on even at the lowest humidities, however. It is possible that oxygen uptake measured at low humidities is due not to respiratory activities of the tardigrade cells which may lead to starvation, but, instead, to abnormal oxidation which may be more harmful than slow respiration.

Tardigrades have been reported from only 21 of the states in this country. This scarcity of published records is hardly indicative of the abundance of these animals. There are two good reasons for this lack of literature. First, unless one is working in rotifers, free-living nematodes, water-bears, or plankton and benthose, tradigrades probably won't be discovered. Secondly, even when found, they will be classified no further than "tardigrade" by the average biologist. To date no tardigrades have been reported from Kansas. Packard (1873) is usually given credit for first reporting tardigrades from the United States, and indeed, he is the first to publish drawings, description, and identification. However, tardigrades were known to occur in this country before this. Baily (1850), while traveling through the southern states during the winter of 1849 and the following spring, made microscopic examinations of moist and aquatic habitats. He reports observing "Tardigradi." His comment could well be that of most zoologists: "Of these I made no record, as I did not possess sufficient knowledge concerning them. They will well reward the attention of southern naturalists."

Background The phenomenon of desiccation tolerance, also called anhydrobiosis, involves the ability of an organism to survive the loss of almost all cellular water without sustaining irreversible damage. Although there are several physiological, morphological and ecological studies on tardigrades, only limited DNA sequence information is available. Therefore, we explored the transcriptome in the active and anhydrobiotic state of the tardigrade Milnesium tardigradum which has extraordinary tolerance to desiccation and freezing. In this study, we present the first overview of the transcriptome of M. tardigradum and its response to desiccation and discuss potential parallels to stress responses in other organisms. Results We sequenced a total of 9984 expressed sequence tags (ESTs) from two cDNA libraries from the eutardigrade M. tardigradum in its active and inactive, anhydrobiotic (tun) stage. Assembly of these ESTs resulted in 3283 putative unique transcripts, whereof ~50% showed significant sequence similarity to known genes. The resulting unigenes were functionally annotated using the Gene Ontology (GO) vocabulary. A GO term enrichment analysis revealed several GOs that were significantly underrepresented in the inactive stage. Furthermore we compared the putative unigenes of M. tardigradum with ESTs from two other eutardigrade species that are available from public sequence databases, namely Richtersius coronifer and Hypsibius dujardini. The processed sequences of the three tardigrade species revealed similar functional content and the M. tardigradum dataset contained additional sequences from tardigrades not present in the other two. Conclusions This study describes novel sequence data from the tardigrade M. tardigradum, which significantly contributes to the available tardigrade sequence data and will help to establish this extraordinary tardigrade as a model for studying anhydrobiosis. Functional comparison of active and anhydrobiotic tardigrades revealed a differential distribution of Gene Ontology terms associated with chromatin structure and the translation machinery, which are underrepresented in the inactive animals. These findings imply a widespread metabolic response of the animals on dehydration. The collective tardigrade transcriptome data will serve as a reference for further studies and support the identification and characterization of genes involved in the anhydrobiotic response.