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Tardigrades are known for their ability to survive extreme conditions. Reports indicate that tardigrade thermal tolerance is enhanced in the desiccated state; however, these reports have almost always used a single tardigrade species and drying/heating methods vary between studies. Using six different species of tardigrades we confirm that desiccation enhances thermal tolerance in tardigrades. Furthermore, we show that differences in thermal tolerance exist between tardigrade species both when hydrated and desiccated. While Viridiscus viridianus survives the highest temperatures in the hydrated state of any species tested here, under hydrated conditions, the thermal tolerance of V. viridianus is restricted to an acute transient stress. Furthermore, unlike other stresses, such as desiccation, where mild initial exposure preconditions some species to survive subsequent harsher treatment, for V. viridianus exposure to mild thermal stress in the hydrated state does not confer protection to harsher heating. Our results suggest that while tardigrades have the capacity to tolerate mild thermal stress while hydrated, survival of high temperatures in a desiccated state is a by-product of tardigrades' ability to survive desiccation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris . Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 ( TDR1 ). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR., Competing Interests: MA, ED, QH, AB, ED, MA, XM, KL, CS, LJ, AA, SH, GT, SH, GG, IG, LP, MG, CB, YC, RG, LR, CG, AD, JC No competing interests declared, (© 2024, Anoud, Delagoutte, Helleu et al.)

A new protocol for preparation of tardigrades for scanning electron microscope (SEM) analysis is proposed. The more conventional protocols require various steps and a long time to obtain good drying of water bears, together with specific and uncommon instruments (i.e., critical point dryer) or highly volatile toxic compounds (i.e., hexametildisilazane). The new protocol can be performed using few and simple instruments and materials, all easily accessible, and produces a high yield in terms of dried animals in excellent condition for the observation of external morphological structures with SEM. The acquired data exhibit considerable promise, and the proposed methodology shows potential for application to other meiofaunal groups, including small arthropods, nematodes, and rotifers. RESEARCH HIGHLIGHTS: Cheap, safe, and fast new method for Tardigrada preparation for SEM. With the new protocol, the number of animals required for SEM studies is minimized. New protocol is potentially applicable to the study of other meiofaunal soft-bodied taxa., (© 2023 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Long-term environment acidifications due to decrease pH of the rainwaters affect both soils and water bodies. The organisms most likely to be affected by acid rain are the ones that possess vital organs made of calcium carbonate; among them are tardigrades, presenting aragonite piercing stylets in feeding apparatuses. A positive relationship between acidic rainfall and loss of tardigrades diversity has been already shown, but there is lack of knowledge of its lethal and sublethal effects. This study quantifies the effects of the acute exposure of three eutardigrade, Acutuncus antarcticus, Hypsibius exemplaris, and Macrobiotus cf. hufelandi, to synthetic acid rains and to organic and inorganic acids (hydrochloric, acetic, sulfuric, and nitric acids) naturally occurring in the environment. The cumulative proportion of dead animals in respect of exposition time was fitted to cumulative Weibull Distribution using a Bayesian framework. At the end of the experiments, animals were observed to investigate damages to their piercing stylets. Besides, stylets were finely morphologically described with Scanning Electron Microscopy. This study shows that acid rains and the other tested acids negatively affect tardigrades accordingly with pH, time of exposure, and tardigrade species. Freshwater species show a better resistance to acidity than the moss dwelling species, which can better acclimate over the time to low pH. The stylets resulted unaltered in almost all of the alive specimens. The results suggest that the tested tardigrades taxa have the ability to buffer the environmental proton change and the negative effect on their populations could be counteracted., (© 2023 The Authors. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

Water unavailability is an abiotic stress causing unfavourable conditions for life. Nevertheless, some animals evolved anhydrobiosis, a strategy allowing for the reversible organism dehydration and suspension of metabolism as a direct response to habitat desiccation. Anhydrobiotic animals undergo biochemical changes synthesizing bioprotectants to help combat desiccation stresses. One stress is the generation of reactive oxygen species (ROS). In this study, the eutardigrade Paramacrobiotus spatialis was used to investigate the occurrence of ROS associated with the desiccation process. We observed that the production of ROS significantly increases as a function of time spent in anhydrobiosis and represents a direct demonstration of oxidative stress in tardigrades. The degree of involvement of bioprotectants, including those combating ROS, in the P. spatialis was evaluated by perturbing their gene functions using RNA interference and assessing the successful recovery of animals after desiccation/rehydration. Targeting the glutathione peroxidase gene compromised survival during drying and rehydration, providing evidence for the role of the gene in desiccation tolerance. Targeting genes encoding glutathione reductase and catalase indicated that these molecules play roles during rehydration. Our study also confirms the involvement of aquaporins 3 and 10 during rehydration. Therefore, desiccation tolerance depends on the synergistic action of many different molecules working together., (© 2022. The Author(s).)

Like meiofauna in general, tardigrades are often neglected in ecological and environmental surveys. Tardigrades occur in all parts of the world, from deep marine sediments to alpine environments, and are present in most ecosystems. They are therefore potentially good candidates for biomonitoring programs. However, sampling of these minute animals is both tedious and time-consuming, impeding their inclusion in large-scale ecological surveys. In this study we argue that using a multi-marker metabarcoding approach on environmental DNA (eDNA) partly can overcome this barrier. Samples of moss, lichens, and leaf litter were investigated both by morphology-based methods and DNA metabarcoding, and the results were compared in terms of tardigrade diversity and community composition of the sampled microhabitats. DNA metabarcoding using three markers detected more species of tardigrades than identification by morphology in most samples. Also, metabarcoding detected the same community differences and microhabitat distribution patterns as morphology-based methods. In general, metabarcoding of litter samples was unreliable, with only one out of three markers consistently amplifying and detecting tardigrades. The low availability of tardigrade reference sequences in public databases restricts the taxonomic resolution in eDNA surveys, but this impediment is partly circumvented by utilizing multiple markers.

Symbiotic associations of metazoans with bacteria strongly influence animal biology since bacteria are ubiquitous and virtually no animal is completely free from them. Tardigrades are micrometazoans famous for their ability to undergo ametabolic states (cryptobiosis) but very little information is available on potential microbial associations. We characterized the microbiomes of six limnoterrestrial tardigrade species belonging to several phylogenetic lines in tandem with the microbiomes of their respective substrates. The experimental design enabled us to determine the effects of both the environment and the host genetic background on the tardigrade microbiome; we were able to define the microbial community of the same species sampled from different environments, and the communities of different species from the same environment. Our 16S rRNA gene amplicon approach indicated that the tardigrade microbiome is species-specific and well differentiated from the environment. Tardigrade species showed a much lower microbial diversity compared to their substrates, with only one significant exception. Forty-nine common OTUs (operational taxonomic units) were classified into six bacterial phyla, while four common OTUs were unclassified and probably represent novel bacterial taxa. Specifically, the tardigrade microbiome appears dominated by Proteobacteria and Bacteroidetes. Some OTUs were shared between different species from geographically distant samples, suggesting the associated bacteria may be widespread. Putative endosymbionts of tardigrades from the order Rickettsiales were identified. Our results indicated that like all other animals, tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.

Because conditions in continental Antarctica are highly selective and extremely hostile to life, its biota is depauperate, but well adapted to live in this region. Global climate change has the potential to impact continental Antarctic organisms because of increasing temperatures and ultraviolet radiation. This research evaluates how ongoing climate changes will affect Antarctic species, and whether Antarctic organisms will be able to adapt to the new environmental conditions. Tardigrades represent one of the main terrestrial components of Antarctic meiofauna; therefore, the pan-Antarctic tardigrade Acutuncus antarcticus was used as model to predict the fate of Antarctic meiofauna threatened by climate change. Acutuncus antarcticus individuals tolerate events of desiccation, increased temperature and UV radiation. Both hydrated and desiccated animals tolerate increases in UV radiation, even though the desiccated animals are more resistant. Nevertheless, the survivorship of hydrated and desiccated animals is negatively affected by the combination of temperature and UV radiation, with the hydrated animals being more tolerant than desiccated animals. Finally, UV radiation has a negative impact on the life history traits of successive generations of A. antarcticus , causing an increase in egg reabsorption and teratological events. In the long run, A. antarcticus could be at risk of population reductions or even extinction. Nevertheless, because the changes in global climate will proceed gradually and an overlapping of temperature and UV increase could be limited in time, A. antarcticus , as well as many other Antarctic organisms, could have the potential to overcome global warming stresses, and/or the time and capability to adapt to the new environmental conditions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

A new species, Doryphoribius chetumalensis, is described from specimens collected in the city of Chetumal (Quintana Roo state, Mexico). The species was found in a new and unusual habitat for urban tardigrades, i.e. the soil sediment accumulated on the border of streets. This discovery shows that tardigrades can live in this habitat, demonstrating once again the wide capacity of this taxon to tolerate adverse habitats, and to survive in environments with high anthropogenic impact. Doryphoribius chetumalensis sp. nov. differs from all the other species of the genus in having enlarged and wide bulbous base of the claws. Within Doryphoribius, it belongs to the zappalai group, and differs from the species in this group, not only in the claw shape, but also by the orange body colour, the smooth cuticle, the absence of a tooth in the wall of the buccal ring, and the absence of lunules under the claws. This is the first record of tardigrades, identified to species level, in Quintana Roo state. A taxonomic key of the Doryphoribius genus is also presented.

The cuticular portion of the tardigrade feeding apparatus is a complex structure that can be schematically divided into four parts: a buccal ring, a buccal tube, a stylet system (formed by two piercing stylets, each within a stylet coat, and two stylet supports), and the lining of a myoepithelial sucking pharynx. To better understand the function and evolution of the feeding apparatus, the morpho-functional traits and chemical composition of the structures forming the feeding apparatuses of eight different species of tardigrades were analyzed. These eight species are representative of almost all main phylogenetic lineages of the phylum. The calcium and chitin in the feeding apparatus were examined by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, energy dispersive X-ray spectroscopy, and Raman microspectroscopy (Raman). In all species, the feeding apparatus had been subjected to biomineralization due to CaCO3 encrustations organized in the crystalline form of aragonite. Aragonite and chitin are present in different concentrations in the feeding apparatus according to the structures and species considered. Generally, where the structures are rigid there is more aragonite than chitin, and vice versa. The buccal tube and piercing stylets are rich in calcium, with the piercing stylets apparently composed exclusively of aragonite. In eutardigrades, chitin is in higher concentration in the structures subject to higher mechanical stresses, such as the crests of the buccal crown and the condyles of the stylet furca., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

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.

Anhydrobiosis is an adaptive strategy that allows withstanding almost complete body water loss. It has been developed independently by many organisms belonging to different evolutionary lines, including tardigrades. The loss of water during anhydrobiotic processes leads to oxidative stress. To date, the metabolism of free radicals in tardigrades remained unclear. We present a method for in vivo monitoring of free radical production in tardigrades, based on electron paramagnetic resonance and spin-trap DEPMPO, which provides simultaneous identification of various spin adducts (i.e., different types of free radicals). The spin trap can be easily absorbed in animals, and tardigrades stay alive during the measurements and during 24-h monitoring after the treatment. The results show that hydrated specimens of the tardigrade Paramacrobiotus richtersi produce the pure superoxide anion radical ((•)O2(-)). This is an unexpected result, as all previously examined animals and plants produce both superoxide anion radical and hydroxyl radical ((•)OH) or exclusively hydroxyl radical.

The TARDIKISS (Tardigrades in Space) experiment was part of the Biokon in Space (BIOKIS) payload, a set of multidisciplinary experiments performed during the DAMA (Dark Matter) mission organized by Italian Space Agency and Italian Air Force in 2011. This mission supported the execution of experiments in short duration (16 days) taking the advantage of the microgravity environment on board of the Space Shuttle Endeavour (its last mission STS-134) docked to the International Space Station. TARDIKISS was composed of three sample sets: one flight sample and two ground control samples. These samples provided the biological material used to test as space stressors, including microgravity, affected animal survivability, life cycle, DNA integrity, and pathways of molecules working as antioxidants. In this paper we compared the molecular pathways of some antioxidant molecules, thiobarbituric acid reactive substances, and fatty acid composition between flight and control samples in two tardigrade species, namely, Paramacrobiotus richtersi and Ramazzottius oberhaeuseri. In both species, the activities of ROS scavenging enzymes, the total content of glutathione, and the fatty acids composition between flight and control samples showed few significant differences. TARDIKISS experiment, together with a previous space experiment (TARSE), further confirms that both desiccated and hydrated tardigrades represent useful animal tool for space research.

An extensive study of the phylogeny of Eutardigrada, the largest class of Tardigrada, has been performed analyzing one hundred and forty sequences (eighty of which newly obtained) representative of one hundred and twenty-nine specimens belonging to all families (except Necopinatidae) of this class. The molecular (18S and 28S rRNA) results were compared with new and previous morphological data, allowing us to find new phylogenetic relationships, to identify new phylogenetic lineages, to erect new taxa for some lineages, and to find several morphological synapomorphies supporting the identified clusters. The class Eutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamilies Macrobiotoidea, Hypsibioidea, Isohypsibioidea, and Eohypsibioidea, and all the families and subfamilies considered, although with emended diagnoses in several cases. In addition, new taxa have been erected: the new subfamily Pilatobiinae (Hypsibiidae) with the new genus Pilatobius, as well as an upgrading of Diphascon and Adropion to genus level, previously considered subgenera of Diphascon. Our results demonstrate that while molecular analysis is an important tool for understanding phylogeny, an integrative and comparative approach using both molecular and morphological data is necessary to better elucidate evolutionary relationships., (Copyright © 2014 Elsevier Inc. All rights reserved.)

As part of the All Taxa Biodiversity Inventory (http://www.dlia.org), an extensive survey of tardigrades has been conducted in the Great Smoky Mountains National Park (GSMNP) in Tennessee and North Carolina, U.S.A., by Bartels and Nelson. Freshwater tardigrades include three species in the aquatic genus Thulinius (Eutardigrada, Isohypsibiidae). A new species, Thulinius romanoi, described from stream sediment, is distinguished from all other congeners by having a sculptured cuticle. In addition, the presence of Thulinius augusti (Murray, 1907) was verified by combined morphological and molecular analysis, and nine specimens of a third species, Thulinius cf. saltursus, were also found. Thulinius augusti is a new record for the United States. Thulinius saltursus (Schuster, Toftner & Grigarick, 1978) was previously recorded in California and Ohio, but our specimens vary slightly in morphology. The list of tardigrades from streams in the GSMNP was updated to a total of 44 species, 22 of which were predominantly or exclusively aquatic.

The taxonomy of tardigrades is challenging as these animals demonstrate a limited number of useful morphological characters, therefore several species descriptions are supported by only minor differences. For example, Echiniscus oihonnae and Echiniscus multispinosus are separated exclusively by the absence or presence of dorsal spines at position Bd. Doubts were raised on the validity of these two species, which were often sampled together. Using an integrative approach, based on genetic and morphological investigations, we studied two new Portuguese populations, and compared these with archived collections. We have determined that the two species must be considered synonymous with Echiniscus oihonnae the senior synonym. Our study showed generally low genetic distances of cox1 gene (with a maximum of 4.1%), with specimens displaying both morphologies sharing the same haplotype, and revealed character Bd to be variable. Addition-ally, a more detailed morphological and phylogenetic study based on the 18S gene uncovered a new evolutionary line within the Echiniscidae, which justified the erection of Diploechiniscus gen. nov. The new genus is in a sister group relationship with Echiniscus and is, for the moment, composed of a single species.

Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes.

In this review we analyze the dormancy strategies of metazoans inhabiting "hostile to life" habitats, which have a strong impact on their ecology and in particular on the traits of their life history. Tardigrades are here considered a model animal, being aquatic organisms colonizing terrestrial habitats. Tardigrades evolved a large variety of dormant stages that can be ascribed to diapause (encystment, cyclomorphosis, resting eggs) and cryptobiosis (anhydrobiosis, cryobiosis, anoxibiosis). In tardigrades, diapause and cryptobiosis can occur separately or simultaneously, consequently the adoption of one adaptive strategy is not necessarily an alternative to the adoption of the other. Encystment and cyclomorphosis are characterized by seasonal cyclic changes in morphology and physiology of the animals. They share several common features and their evolution is strictly linked to the molting process. A bet-hedging strategy with different patterns of egg hatching time has been observed in a tardigrade species. Four categories of eggs have been identified: subitaneous, delayed-hatching, abortive and diapause resting eggs, which needs a stimulus to hatch (rehydration after a period of desiccation). Cryptobiotic tardigrades are able to withstand desiccation (anhydrobiosis) and freezing (cryobiosis) at any stage of their life-cycle. This ability involves a complex array of factors working at molecular (bioprotectans), physiological and structural levels. Animal survival and the accumulation of molecular damage are related to the time spent in the cryptobiotic state, to the abiotic parameters during the cryptobiotic state, and to the conditions during initial and final phases of the process. Cryptobiosis evolved independently at least two times in tardigrades, in eutardigrades and in echiniscoids. Within each evolutionary line, the absence of cryptobiotic abilities is more related to selective pressures to local habitat adaptation than to phylogenetic relationships. The selective advantages of cryptobiosis (e.g. persistency in "hostile to life" habitats, reduction of competitors, parasites and predators, escaping in time from stressful conditions) could explain the high tardigrade species diversity and number of specimens found in habitats that dry out compared to freshwater habitats., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The seasonality and unpredictability of environmental conditions at high altitudes and latitudes govern the life cycle patterns of organisms, giving rise to stresses that cause death or development of specific adaptations. Ice formation is a major variable affecting the survival of both freshwater fauna and fauna inhabiting lichens, mosses and leaf litter. Tardigrades occupy a wide range of niches in marine, freshwater and terrestrial environments. The highest number of species is found in terrestrial habitats thanks to their ability to enter anhydrobiosis and cryobiosis. The cryobiotic ability of tardigrade species from polar regions is well known. Consequently, we focused our research on the ability to survive freezing in the active hydrated state using seven tardigrade species differing in phylogenetic position and collected at various altitudes and from different habitats in a temperate area. Specimens were cooled at different cooling rates (from 0.31° C min(-1) to 3.26° C min(-1)). Even though the final survival and the time required by animals to recover to active life were both inversely related to the cooling rate, highly significant interspecific differences were found. Species survival ability ranged from excellent to none. Species living in xeric habitats withstood freezing better than those living in hygrophilous habitats, while true limnic species did not exhibit any cryobiotic ability. The ability to withstand freezing seems linked to the anhydrobiotic ability. The differences in cryptobiotic performance among tardigrade species seem more influenced by selective pressures linked to local adaptation to habitat characteristics than by phylogenetic relationships., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

An important, but generally overlooked source in intraspecific variability of characters used in tardigrades taxonomy is sexual dimorphism. Dioecious tardigrades species with unisexual or bisexual populations are common, but external sexual dimorphism is rarely observed. The discovery and integrative analysis of a Diploechiniscus species population with high sexual dimorphism from Calvert Island (Canada) has allowed its description as a new species. Diploechiniscus dimorphus n. sp. is characterized by a different chaetotaxy in males and females, and by enlarged cephalic sensory appendages in the males. The discovery of this new species shows that it is important to consider sexual dimorphism as a source of intraspecific variability in tardigrade taxonomy due to its implications on the use of traits commonly used to delineate species such as chaetotaxy. Lastly, the finding of individuals of Diploechiniscus horningi (Schuster and Grigarick, 1971), a species previously synonymized with Diploechiniscus oihonnae (Richters, 1903), allows us to prove that it is genetically a separate species and to reinstate it. [ABSTRACT FROM AUTHOR]

Tardigrades are common in many terrestrial environments and habitats. Although little is known on their ecological preferences, previous studies found diversity and community composition significantly affected by various variables. This study associated tardigrade species' records with climatic variables, forest type, and substrate categories exploring tardigrade diversity and species communities to find associations with ecological characteristics of Norwegian forests. A total of 17 473 specimens were identified, encompassing 131 species (including putatively new species) from 305 samples of different substrates (leaf litter, bryophytes, and lichens). Bryophytes and lichens of samples were classified according to the main species, and growth form, and associated with tardigrade species and sample metadata. Tardigrade species' richness was related to climatic variables and forest type, increased with precipitation, decreased with summer temperature, and not varied with precipitation seasonality. Although there was an unbalanced representation of substrate categories in the different forest types, some tendencies were detectable. Mixed oak and birch forests reached the highest species' richness. Tardigrade community composition varied between substrate categories and, to a lesser degree, between forest types, but not with climatic variables. Our study highlights the importance of large-scale variables on tardigrade diversity, and substrate categories for tardigrade community composition. [ABSTRACT FROM AUTHOR]

The genera Xerobiotus and Pseudohexapodibius are xerophilic and characterized by reduced appendages and claws as adaptations to move between small interstices. To increase the knowledge of their biodiversity, biogeography and phylogeny, several specimens from European countries and Australia were analysed using an integrative approach, i.e. morphological, karyological and molecular studies (18S, 28S, cox1 , cytb and ITS2). The phylogenetic position of Xerobiotus with respect to the three evolutionary lineages of Macrobiotus remains unchanged with respect to what was previously identified in the phylogeny of Macrobiotidae. The following new Xerobiotus species are described: Xerobiotus litus sp. nov. Xerobiotus arenosum sp. nov. and Xerobiotus reductus sp. nov. It is proposed that Macrobiotus naginae should be transferred to Xerobiotus (Xerobiotus naginae comb. nov.). Pseudohexapodibius degenerans clusters within Xerobiotus and shares morphological and genetic characters with this genus. Some discrepancies between genetic and phenotypic data are found among the analysed species. Even between the results obtained with analysed genes disagreements are found, with cox1 and cytb generally agreeing with phenotypic results more than ITS2. Genotypic and phenotypic data are useful tools for species identification, but they have to be evaluated critically to obtain reliable results. [ABSTRACT FROM AUTHOR]

Chitin is a structural sugar broadly distributed among all kingdoms, and it is present in Tardigrada as α-chitin. Its presence in the buccopharyngeal apparatus of tardigrades was confirmed in previous studies and associated with autofluorescence emission, and within this emission three components were identified. This study aimed to localize the chitin in the buccopharyngeal apparatus and to understand the nature of the other autofluorescent components. Calcofluor white staining of three tardigrade species (Echiniscus sp. Milnesium sp. Macrobiotus sp.) allowed the chitin localization in the feeding structures and the separation of its signal from the whole autofluorescence emission via confocal laser scanning microscopy. Comparing different fluorochromes found in tardigrades and in the nematode Panagrellus pycnus , lambda and square lambda analyses showed that part of the autofluorescent signal is imputable to a collagen-like compound. The images acquired were analysed for morphological, functional, and evolutionary purposes. Chitin and the other autofluorescent compound were localized in the structures of the buccopharyngeal apparatus. Their distribution highlighted the presence of different levels of 'flexibility/strength' of the structures providing better understanding of their mechanical properties and relating them to the characteristics of the three major limnoterrestrial clades of the phylum Tardigrada. [ABSTRACT FROM AUTHOR]

Long‐term environment acidifications due to decrease pH of the rainwaters affect both soils and water bodies. The organisms most likely to be affected by acid rain are the ones that possess vital organs made of calcium carbonate; among them are tardigrades, presenting aragonite piercing stylets in feeding apparatuses. A positive relationship between acidic rainfall and loss of tardigrades diversity has been already shown, but there is lack of knowledge of its lethal and sublethal effects. This study quantifies the effects of the acute exposure of three eutardigrade, Acutuncus antarcticus, Hypsibius exemplaris, and Macrobiotus cf. hufelandi, to synthetic acid rains and to organic and inorganic acids (hydrochloric, acetic, sulfuric, and nitric acids) naturally occurring in the environment. The cumulative proportion of dead animals in respect of exposition time was fitted to cumulative Weibull Distribution using a Bayesian framework. At the end of the experiments, animals were observed to investigate damages to their piercing stylets. Besides, stylets were finely morphologically described with Scanning Electron Microscopy. This study shows that acid rains and the other tested acids negatively affect tardigrades accordingly with pH, time of exposure, and tardigrade species. Freshwater species show a better resistance to acidity than the moss dwelling species, which can better acclimate over the time to low pH. The stylets resulted unaltered in almost all of the alive specimens. The results suggest that the tested tardigrades taxa have the ability to buffer the environmental proton change and the negative effect on their populations could be counteracted. Research Highlights: Tardigrades exhibit differential sensitivity toacids.Aragonite piercing stylets of feeding apparatus exposed to acids.Tardigrades can to moderate/high acidity [ABSTRACT FROM AUTHOR]

Simple Summary: Tardigrades are small micrometazoans able to resist several environmental stresses in any stage of their life cycle. The integrated molecular and morphological analysis of tardigrade specimens collected in Tsukuba (Japan) revealed that this population represents a new species, Macrobiotus kyoukenus sp. nov., belonging to the genus Macrobiotus, one of the most speciose and widespread water bear taxon. The stress resistance capabilities of M. kyoukenus sp. nov. have been tested by submitting animals to extreme desiccation, rapid freezing, and high levels of ultraviolet radiations (UVB and UVC). Animals were able to survive desiccation and freezing, and both hydrated and desiccated animals showed a high tolerance to increasing UV radiations. Overall, our findings contribute to the discovery of a larger tardigrade biodiversity in Japan, and the tolerance capabilities of M. kyoukenus sp. nov. show that this new species could become an emerging model for stress resistance studies. Tardigrades are small micrometazoans able to resist several environmental stresses in any stage of their life cycle. An integrated analysis of tardigrade specimens collected in Tsukuba (Japan) revealed a peculiar morphology and a new sensory field in the cloaca. Molecular taxonomy and phylogenetic analysis on different genes (COI, ITS2, 18S and 28S) confirmed that this population is a new species, Macrobiotus kyoukenus sp. nov., belonging to the widespread Macrobiotus hufelandi group. The stress resistance capabilities of M. kyoukenus sp. nov. have been tested by submitting animals to extreme desiccation, rapid freezing, and high levels of ultraviolet radiations (UVB and UVC). Animals were able to survive desiccation (survivorship 95.71 ± 7.07%) and freezing up to −80 °C (82.33 ± 17.11%). Both hydrated and desiccated animals showed a high tolerance to increasing UV radiations: hydrated animals survived to doses up to 152.22 kJ m−2 (UVB) and up to 15.00 kJ m−2 (UVC), while desiccated specimens persisted to radiations up to 165.12 kJ m−2 (UVB) and up to 35.00 kJ m−2 (UVC). Present data contribute to the discovery of a larger tardigrade biodiversity in Japan, and the tolerance capabilities of M. kyoukenus sp. nov. show that it could become a new emerging model for stress resistance studies. [ABSTRACT FROM AUTHOR]

The temperature experienced by micro-invertebrates in extreme environments (such as those of Antarctica) is a pivotal parameter regarding these animals' ecology and physiology. However, at present, detailed knowledge of microhabitat physical conditions in Antarctica is limited, as well as being biased towards sub-Antarctic and Maritime Antarctic regions. To better understand the temperature conditions experienced in the microhabitats of Continental Antarctica by the native microfauna, we recorded temperatures year round in ponds and soils in an area of the Victoria Land coast and compared these measurements with air temperature data from the closest automatic weather station. We identified an important difference in temperature dynamics between the air, soil and pond datasets. Ponds were the warmest sites overall, differing by up to 7.5°C in comparison with the air temperature due to their greater thermal capacity, which also drove their patterns of freeze-thaw cycles and mean daily thermal excursion. [ABSTRACT FROM AUTHOR]

The integration of morphological data and data from molecular genetic markers is important for examining the taxonomy of meiofaunal animals, especially for eutardigrades, which have a reduced number of morphological characters. This integrative approach has been used more frequently, but several tardigrade taxa lack molecular confirmation. Here, we describe Crenubiotus ruhesteini sp. nov. from the Black Forest (Germany) integratively, with light and electron microscopy and with sequences of four molecular markers (18S, 28S, ITS2, cox1 genes). Molecular genetic markers were also used to confirm the recently described Crenubiotus genus and to establish its phylogenetic position within the Macrobiotoidea (Eutardigrada). The erection of Crenubiotus and its place in the family Richtersiidae are confirmed. Richtersiidae is redescribed as Richtersiusidae fam. nov. because its former name was a junior homonym of a nematode family. [ABSTRACT FROM AUTHOR]

Kristianstads Vattenrike Biosphere Reserve [KVBR] is a UNESCO designated area of Sweden possessing high biological value. Although several studies on tardigrades inhabiting Sweden have been performed, the KVBR area has been neglected. The current study investigates the tardigrade fauna of five areas of the biosphere reserve and includes 34 samples of different substrates analysed quantitatively and qualitatively. In total, 33 species of tardigrades were found in the samples, including 22 new records for the Skåne region, 15 new records for Sweden, and four species new to science. Mesobiotus emiliae sp. nov., Xerobiotus gretae sp. nov., Itaquascon magnussoni sp. nov., and Thulinius gustavi sp. nov. were described with an integrative approach (when possible) using morphological characters (light, electron scanning, and confocal laser scanning microscopies) and molecular markers (ITS2, 18S, 28S, cox1). A new protocol to increase morphological data was developed recovering mounted specimens within old slides for SEM analysis. Emended diagnoses for the genus Itaquascon and the transfer of Platicrista itaquasconoide to the genus Meplitumen are proposed. This study enriches the knowledge of the tardigrade biodiversity both within the KVBR and in Sweden and contributes to the rapidly increasing number of tardigrade species reported worldwide. The 33 species identified in the KVBR area represents 28% of all water bear species found in Sweden so far. The restricted study areas and limited number of samples collected suggests that the KVBR is very rich of tardigrades. [ABSTRACT FROM AUTHOR]

Background. The phylum Tardigrada consists of over 1,300 species that inhabit terrestrial, freshwater and marine environments throughout the world. In terrestrial habitats they live primarily in mosses, lichens, leaf litter and soil, whereas tardigrades in freshwater and marine environments are mainly found in sediments and on aquatic plants. More than 65 species have been previously reported in the state of Tennessee, USA. Methods. Tardigrades present in moss cushions (Grimmia sp.) collected from a xerothermic habitat on the East Tennessee State University campus, Johnson City, TN, USA, were extracted, mounted on slides, identified, and counted. Additional samples of fresh dried moss were used for integrative analyses, including morphological analysis with phase contrast (PCM) and scanning electron microscopy (SEM), as well as molecular analyses of COI, 18S rRNA, 28S rRNA, and ITS-2 of the Macrobiotus and Milnesium species. Results. Five species were found, including two species new to science: Viridiscus miraviridis sp. nov. and Macrobiotus basiatus sp. nov. Viridiscus miraviridis sp. nov. differs from other members of the genus mainly by having a different type of dorsal cuticle and some other, more subtle, morphometric characters. In addition to the two new species, Viridiscus perviridis and Viridiscus viridissimus were present, and males of Vir. viridissimus were found for the first time, the first record of males in the genus Viridiscus. Macrobiotus basiatus sp. nov. is most similar to Macrobiotus nelsonae, but it differs from Mac. nelsonae mainly by the stylet supports being situated in a more anterior position, shorter and narrower egg processes, and a smaller number of areoles around the egg processes. Moreover, the identification of Milnesium inceptum was confirmed as the first record for the USA by analysis of COI. [ABSTRACT FROM AUTHOR]

Comparative analyses of life-history theory studies are based on the characteristics of the life cycles of different species. For tardigrades, life-history traits are available only from laboratory cultures, most of which have involved parthenogenetic species. The discovery of a new gonochoristic bisexual Mesobiotus species in a moss collected on the island of Elba (Italy) provides us with the opportunity to describe Mesobiotus joenssoni sp. nov. and to collect data on the life-history traits of cultured specimens to increase our knowledge of the life-history strategies present in tardigrades. This new species is differentiated from all other species of the genus by the presence of granules (~1 µm in diameter) on the dorsal cuticle of the last two body segments, two large bulges (gibbosities) on the hindlegs and long, conical egg processes. The species exhibits sexual dimorphism in body length, with females being longer than males of the same age. The mean lifespan of specimens was 86 days, with a maximum of 150 days. The mean age at first oviposition was 19.8 days and the mean egg hatching time 15.4 days. The life-cycle traits correspond to those collected for the only other two macrobiotid species with gonochoric amphimictic reproduction examined so far. [ABSTRACT FROM AUTHOR]

Data from a previous study showed that microbiomes of six tardigrade species are species-specific and distinct from associated environmental microbes. We here performed a more in-depth analyses of those data, to identify and characterize new potential symbionts. The most abundant bacterial operational taxonomic units (OTUs) found in tardigrades are classified, and their prevalence in other environments is assessed using public databases. A subset of OTUs was selected for molecular phylogenetic analyses based on their affiliation with host-associated bacterial families in tardigrades. Almost 22.6% of the most abundant OTUs found do not match any sequence at 99% identity in the IMNGS database. These novel OTUs include four putative tardigrade endosymbionts from Alphaproteobacteria (Anaplasmataceae and Candidatus Tenuibacteraceae), which are characterized by 16S rRNA gene analysis and investigated for their infection rates in: Echiniscus trisetosus , Richtersisus coronifer and Macrobiotus macrocalix. These putative endosymbionts have an infection prevalence between 9.1% and 40.0%, and are, therefore, likely secondary symbionts, not essential for tardigrade survival and reproduction. Using fluorescence in situ hybridization (FISH), we detected bacteria on the cuticle and within the ovary of E. trisetosus , suggesting possible vertical transmission. This study highlights the great contribution in biodiversity discovery that neglected phyla can provide in microbiome and symbiosis studies. [ABSTRACT FROM AUTHOR]

Pseudechiniscus is the second most species-rich genus in Heterotardigrada and in the family Echiniscidae. However, previous studies have pointed out polyphyly and heterogeneity in this taxon. The recent erection of the genus Acanthechiniscus was another step in making Pseudechiniscus monophyletic, but species identification is still problematic. The present investigation aims at clarifying biodiversity and taxonomy of Pseudechiniscus taxa, with a special focus on species pertaining to the so-called ' suillus – facettalis group', by using an integrated approach of morphological and molecular investigations. The analysis of sequences from specimens sampled in Europe and Asia confirms the monophyly of the genus Pseudechiniscus. Inside the genus, two main evolutionary lineages are recognizable: the P. novaezeelandiae lineage and the P. suillus–facettalis group lineage. Inside the P. suillus–facettalis group, COI molecular data points out a very high variability between sampled localities, but in some cases also among specimens sampled in the same locality (up to 33.3% p-distance). The integrated approach to the study of Pseudechiniscus allows confirmation of its monophyly and highlights the relationships in the taxon, pointing to its global distribution. [ABSTRACT FROM AUTHOR]

The underestimation of biodiversity and scarce knowledge of the biogeographic distributions of terrestrial meiofaunal component limit our understanding of the origin of Antarctic fauna. One of its main components is tardigrades; nonetheless studies on tardigrade diversity are still few in the continental Antarctic area. In order to increase our knowledge of the underreported terrestrial meiofaunal communities within continental Antarctica, as well as to provide new information for biogeographic and evolutionary analysis of these communities, the tardigrade diversity of 11 samples collected along the coast of Victoria Land was considered. The application of an integrative approach in which morphological (Light – LM, scanning electron – SEM, and confocal laser scanning microscopy – CSLM) and molecular analysis (18S, 28S, cox1 genes) were combined allowed us to characterize the tardigrade fauna inhabiting mosses, lichens, and cyanobacterial mats of Victoria Land. These analyses allowed the detection of two tardigrade species new to science (Cryoconicus antiarktos sp. nov., Ramazzottius sabatiniae sp. nov.), and the emendation of known species ((Hebesuncus ryani Dastych and Harris, 1994, Ramazzottius nivalis Dastych, 2006, Cryoconicus ljudmilae comb. nov. (Biserov, 1997/98), and Cryoconicus cataphractus (Maucci, 1974)), together with the genus Cryoconicus. The Ramazzottius type of claws was redefined identifying three subtypes. Also, we investigated the phylogenetic position of some problematic/unresolved lineages all belonging to the family Ramazzottiidae. These data increased the knowledge of the biodiversity in Victoria Land, the number of endemic tardigrades in Antarctica, and provided evidence on the origin of Antarctic endemism. Lastly, new methods for integrative taxonomic studies on tardigrades were presented and discussed. [ABSTRACT FROM AUTHOR]