Comparison of the transcriptomes of two tardigrades with different hatching coordination.

BACKGROUND: Tardigrades are microscopic organisms, famous for their tolerance against extreme environments. The establishment of rearing systems of multiple species has allowed for comparison of tardigrade physiology, in particular in embryogenesis. Interestingly, in-lab cultures of limnic species showed smaller variation in hatching timing than terrestrial species, suggesting a hatching regulation mechanism acquired by adaptation to their habitat. RESULTS: To this end, we screened for coordinated gene expression during the development of two species of tardigrades, Hypsibius exemplaris and Ramazzottius varieornatus, and observed induction of the arthropod molting pathway. Exposure of ecdysteroids and juvenile hormone analog affected egg hatching but not embryonic development in only the limnic H. exemplaris. CONCLUSION: These observations suggest a hatching regulation mechanism by the molting pathway in H. exemplaris.

Miniaturization of tardigrades (water bears): Morphological and genomic perspectives.

Tardigrades form a monophyletic group of microscopic ecdysozoans best known for surviving extreme environmental conditions. Due to their key phylogenetic position as a subgroup of the Panarthropoda, understanding tardigrade biology is important for comparative studies with related groups like Arthropoda. Panarthropods - and Ecdysozoa as a whole - likely evolved from macroscopic ancestors, with several taxa becoming secondarily miniaturized. Morphological and genomic evidence likewise points to a miniaturized tardigrade ancestor. The five-segmented tardigrade body typically measures less than 1 mm in length and consists of only about 1000 cells. Most organs comprise a relatively small number of cells, with the highest proportion belonging to the central nervous system, while muscles are reduced to a single cell each. Similarly, fully sequenced genomes of three tardigrade species - together with Hox gene expression data - point to extensive modifications, rearrangements, and major losses of genes and even a large body region. Parallels are evident with related ecdysozoans that may have also undergone genomic reductions, such as the nematode Caenorhabditis elegans. We interpret these data together as evidence of miniaturization in the tardigrade lineage, while cautioning that the effects of miniaturization may manifest in different ways depending on the organ or organ system under examination.

Carbonylation accumulation of the Hypsibius exemplaris anhydrobiote reveals age-associated marks.

Together with nematodes and rotifers, tardigrade belong to micrometazoans that can cope with environmental extremes such as UV and solar radiations, dehydration, supercooling or overheating. Tardigrade can resist the harshest conditions by turning to cryptobiosis, an anhydrobiotic state that results from almost complete dehydration and is characterized by an ametabolic status. Although reports have challenged the molecular basis of the mechanisms underlying genomic injury resistance, little is yet known regarding the possible involvement of other tardigrade macromolecules in injury during a stress experience. In this report, we show that the tardigrade Hypsibius exemplaris can accumulate molecular damages by means of in situ detection of carbonyls. Furthermore, we demonstrate that living tardigrade can accumulate carbonylation. Finally, we reveal that anhydrobiotic tardigrade can be constitutively affected by carbonylation that marks aging in other metazoans.

Laboratory Culture of Hypsibius exemplaris.

I have reared a culture of the tardigrade Hypsibius exemplaris for 30 years, since 1987. Here, I present my culture protocol.

Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus.

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.

Eremobiotus ginevrae sp. nov. and Paramacrobiotus pius sp. nov., two new species of Eutardigrada.

Two new eutardigrade species are described: Eremobiotus ginevrae sp. nov. and Paramacrobiotus pius sp. nov. The first is similar to Eremobiotus alicatai (Binda, 1969) but differs in the claw shape and dimensions. It has been found in Sicily, Israel and Russia. The second species, belonging to the richtersi group, is currently found exclusively in Sicily. It has a smooth cuticle, three macroplacoids and a microplacoid, eggs with reticulated trunco-conical processes with small terminal thorns; the egg-shell is areolated and the areolae are clearly sculptured.

Tardigrades from Nahuel Huapi National Park (Argentina, South America) with descriptions of two new Macrobiotidae species.

In 31 samples of mosses and lichens collected in the Argentinean province of Río Negro, 657 tardigrades, 53 exuviae and 219 free-laid eggs were found. In total, 20 species were identified: Diphascon chilenense, Dip. mitrense, Echiniscus bigranulatus, Ech. corrugicaudatus, Ech. merokensis merokensis, Ech. testudo, Hebesuncus mollispinus, Hypsibius convergens, Macrobiotus cf. anderssoni, Mac. andinus, Mac. kazmierskii, Mac. patagonicus, Mesobiotus szeptyckii, Mes. pseudoblocki sp. nov., Milnesium argentinum, Mil. beatae, Mil. brachyungue, Mil. granulatum, Mopsechiniscus granulosus, Minibiotus pseudostellarus sp. nov. Of the two new species, Mesobiotus pseudoblocki sp. nov. is most similar to Mes. blocki, but it differs mainly by the lack of dentate lunules, smaller eggs and presence of reticular design on egg processes. Minibiotus pseudostellarus sp. nov. is most similar to Min. constellatus, Min. eichhorni, Min. sidereus or Min. vinciguerrae, but it differs from them by the presence of 'pseudo-star'-shaped pores in the dorsal cuticle instead of fully developed 'stars' and by other morphometric characters.

Mesobiotus philippinicus sp. nov., the first limnoterrestrial tardigrade from the Philippines.

The limnoterrestrial tardigrade fauna of the Philippines is completely unknown. In this paper, we describe the first ever limnoterrestrial water bear species from this southeast Asian country, Mesobiotus philippinicus sp. nov., found in a moss sample collected in Quezon City. Apart from morphometrics and imaging in light microscopy, we also analysed the new species under scanning electron microscope and sequenced four DNA markers differing in mutation rates, three nuclear (18S rRNA, 28S rRNA, and ITS-2) and one mitochondrial (COI). This allowed not only a detailed description but also provided barcodes to aid future species identification. The new species belongs to the harmsworthi group and is most similar to M. diffusus (Binda & Pilato, 1987), M. pseudocoronatus (Pilato et al., 2006), M. montanus (Murray, 1910) and M. mottai (Binda & Pilato, 1994), but differs from these species by whorled egg processes and dimensions of some morphometric traits. The 28S rRNA, ITS-2 and COI sequences presented in this paper are the first published DNA sequences for the genus Mesobiotus.

Two new species of Milnesium (Tardigrada: Milnesiidae).

Two new species of Milnesium are described: Milnesium vorax sp. nov from Sicily and Milnesium tumanovi sp. nov. from Crimea. Milnesium vorax sp. nov. is uncoloured, with eye spots, smooth cuticle, wide buccal tube, stylet supports inserted on the buccal tube at almost 62 % of its length; claws with configuration [2-3]-[3-2]; accessory points present on the main branches. Milnesium tumanovi sp. nov. is also uncoloured, has eye spots, smooth cuticle, and wide buccal tube, but it has the stylet supports inserted on the buccal tube at about 52 % of its length, and claws with configuration [3-3]-[3-3]; the accessory points are present on the main branches.

Tardigrades of Sweden; an updated check-list.

Tardigrades occur worldwide and in a variety of ecosystems and habitats representing an important component of the micrometazoan biodiversity. Several studies documenting the occurrence of tardigrades in Sweden have been published since the first reports in early 1900, but no comprehensive summary of these studies have been published. We compiled the available information on recorded tardigrades from Sweden, using material from published studies and museum and university collections. In total, our review document 101 species of tardigrades that have been recorded from Sweden (an updated checklist of tardigrades from Sweden will be available online), of which 14 species are new records for the country. The highest number of species was recorded in the northernmost province of Lappland and the more southern provinces of Uppland and Skåne, while much lower species numbers are reported from the middle part of Sweden. This pattern probably represents biased sampling activities of biologists rather than real differences in biodiversity of tardigrades. In view of the few studies that have been made on tardigrade biodiversity in Sweden, the relatively high number of tardigrade species recorded, representing almost a tenth of the species recorded worldwide, indicates that many more species remain to be found. In this respect, more studies of the marine ecosystems along the Swedish west coast and the long Baltic Sea coastline would be of particular interest.

Two new tardigrade species from Romania (Eutardigrada: Milnesiidae, Macrobiotidae), with some remarks on secondary sex characters in Milnesium dornensis sp. nov.

In two moss and lichen samples collected in Romania, two new eutardigrade species were found. Milnesium dornensis sp. nov. belongs to the granulatum group and differs from most of other species in this group mainly by having a different claw configuration ([3-3]-[3-3]) and by some morphometric characters. Minibiotus diversus sp. nov. is very similar to M. gumersindoi Guil & Guidetti, 2005 and M. weglarskae Michalczyk et al., 2005, but differs from these and other congeners by the cuticular pore arrangement and morphometric characters of both adults and eggs. Males and females of the newly described Milnesium species differ not only by the shape and structure of claws I, but also by other morphometric characters. Males of Milnesium dornensis sp. nov. are smaller, more slender, have shorter papillae and relatively longer claws on legs III-IV.

The first record of Pyxidium tardigradum Van der Land, 1964 (Ciliophora) in Romania.

In three lichen samples collected from eastern part of Romania, three populations of Ramazzottius cf. oberhaeuseri (Doyère, 1840) infested by Pyxidium tardigradum Van der Land 1964 were found. In this short correspondence we present a first record of P. tardigradum in Romania and infestation rates in studied populations according to the different life stages.

Quisarctus yasumurai gen. et sp. nov. (Arthrotardigrada: Halechiniscidae) from a submarine cave, off Iejima, Ryukyu Islands, Japan.

Quisarctus yasumurai gen. et sp. nov. (Arthrotardigrada: Halechiniscidae) is described from the submarine cave 'Daidokutsu', off Iejima, Okinawa Islands, Ryukyu Islands, Japan. It is characterised by a cylindrical body, long primary clava and shorter lateral cirrus inserted on a common cirrophore, and simple digits of unequal lengths (without folds, peduncles, proximal pads, pretarsi, or wrinkles) that terminate in a sheathed, small, crescent-shaped claw with a minute calcar. Quisarctinae subfam. nov. is erected for this unique new genus.

New records of marine tardigrades from Moorea, French Polynesia, with the description of Styraconyx turbinarium sp. nov. (Arthrotardigrada, Halechiniscidae).

Five marine arthrotardigrade species are recorded from Moorea, Society Islands, French Polynesia. Four were collected from coral sand; two, Dipodarctus anaholiensis Pollock, 1995 and Florarctus kwoni Chang & Rho, 1997, are new records for the region, and two, Halechiniscus perfectus Schulz, 1955 and Styraconyx kristenseni kristenseni Renaud-Mornant, 1981, have been previously reported. The fifth, a new species Styraconyx turbinarium sp. nov., is described and was collected from the drifting brown alga Turbinaria ornata. The new species is characterized by the presence of peduncles on all digits, an elongate primary clava, and the lateral cirrus A arising from a common pedestal and enveloped by a common membrane extending almost to the claval tip. The new species differs from the most similar species, Styraconyx tyrrhenus D'Addabbo Gallo, Morone De Lucia & de Zio Grimaldi, 1989, by having longer and differently shaped primary clavae which are elongated in the new species and club-shaped in S. tyrrhenus. By having a dorsal cuticle that is coarsely punctated but without folds or other ornamentations, the new species can be easily distinguished from S. craticulus (Pollock, 1983), a species with similar primary clavae, but with cuticular dorsal folds ornamented with a grid-like pattern.

The genus Milnesium (Eutardigrada: Milnesiidae) in the Sierra Nevada de Santa Marta (Colombia), with the description of Milnesium kogui sp. nov.

A new species, Milnesium kogui sp. nov. is described from the Sierra Nevada de Santa Marta, Colombia. The new species belongs to the tardigradum group and is most similar, by the claw configuration [2-2]-[2-2], to Milnesium dujiangensis and Milnesium katarzynae. Milnesium kogui sp. nov. differs from M. dujiangensis mainly by the presence of primary branches on all legs and from M. katarzynae by the absence of dorsal sculpture. Additionally, in this paper we present a list of all Milnesium species recorded in Colombia including Milnesium cf. barbadosense Meyer & Hinton, 2012 and M. brachyungue Binda & Pilato, 1990, new additions to the recorded fauna of Colombia.

Two new freshwater eutardigrade species from Sicily.

Two new species of freshwater Eutardigrada are described from Sicily: Isohypsibius rusticus sp. nov. and Isohypsibius zappalai sp. nov. The former species has eye spots, ornamented body surface with many, variously sized tubercles; bucco-pharyngeal apparatus of the Isohypsibius type; pharyngeal bulb with apophyses and two rod-shaped macroplacoids; microplacoid absent; claws, of the Isohypsibius type, well developed, with long and thin common basal portion; main branches of all claws without free accessory points; very thin lunules present; cuticular bars on the legs absent. Isohypsibius zappalai sp. nov. has eye spots; entire body surface with small tubercles rounded in shape, fairly uniformly sized and tending to form transverse lines; bucco-pharyngeal apparatus of the Isohypsibius type, pharyngeal bulb with apophyses and two macroplacoids; microplacoid absent; claws of the Isohypsibius type, well developed, with long common basal portion and both main and secondary branches with a wide proximal portion. Main branches of all claws with accessory points; small, flexible lunules present; cuticular bars on the legs absent.

A new tardigrade, Mutaparadoxipus duodigifinis gen. nov., sp. nov. (Heterotardigrada: Arthrotardigrada), from the Southeastern United States.

A new genus and species of Arthrotardigrada is described from Florida, USA based on its unique adhesive pad/claw combinations. Mutaparadoxipus duodigifinis gen. nov., sp. nov., is characterized by well-developed, ventral secondary clavae that are adjacent to the mouth, pointed lateral and caudal alae, seminal receptacles with coiled ducts opening lateral to the gonopore, and all legs with digits bearing proximal adhesive pads. Distal claws are present on digits I-III of legs I-III, but are missing from digit IV. On leg IV, distal claws are present only on digits II & III. A single accessory point is present on claws II & III only. This is the fourth species discovered to date with proximal adhesive pads, increasing support for a clade of adhesive-padded arthrotardigrades, and is likely the sister taxon of Paradoxipus orzeliscoides. The incomplete set of claws may represent an evolutionary step in a progressive loss of claws hypothesized to have occurred within the Halechiniscidae. The subfamily Orzeliscinae is amended as a result.

Tardigrada from a sub-Andean forest in the Sierra Nevada de Santa Marta (Colombia) with the description of Itaquascon pilatoi sp. nov.

Currently only 32 species of limno-terrestrial tardigrades have been reported in the literature for Colombia. Our study focused on both heterotardigrades and eutardigrades, which were extracted from eight samples of bryophytes and lichens collected in a sub-Andean forest transect in the Sierra Nevada de Santa Marta, Colombia. Fourteen species were found, six of which are new records for Colombia: Echiniscus madonnae Michalczyk & Kaczmarek, 2006, Echiniscus virginicus Riggin, 1962, Milnesium krzysztofi Kaczmarek & Michalczyk, 2007, Doryphoribius amazzonicus Lisi, 2011, Isohypsibius sattleri (Richters, 1902) and Diphascon higginsi Binda, 1971; and one new to science. Itaquascon pilatoi sp. nov., is characterized by having smooth cuticle, no eyes, buccal tube almost as long as the pharyngeal tube, well developed, obvious stylet furcae with long branches, slender claws, no lunules and no cuticular bars on the legs. The new species differs from I. umbellinae Barros, 1939, the most similar species, in having the stylet supports inserted precisely at the border between buccal and pharyngeal tube, more slender claws and more pronounced length differential between the external and internal claws of each leg. The total number of Colombian limno-terrestrial tardigrade species is raised to 37. 

A new Stygarctus (Arthrotardigrada: Stygarctidae) from Japan, with entangled seminal receptacle ducts.

Stygarctus ayatori sp. nov. (Arthrotardigrada: Stygarctidae), is described from a sandy beach located at Okinoshima, Tateyama Bay, Boso Peninsula, Honshu, Japan. The most prominent characters were observed in the female genital structure, with the seminal receptacle ducts forming a three-dimensional entanglement near the exterior opening, and internal thickening situated peripheral to the gonopore and between the gonopore and anus. The new species is distinguished from the congeners by these characters; excluding S. abornatus McKirdy et al., 1976 for which there is no information on the genital structure. However, S. ayatori sp. nov. and S. abornatus can be differentiated by the presence of dorsal spines on the former species, which are absent from the latter.

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

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