Richtersius Research Articles

An integrative description of a new Richtersius species from Greece (Tardigrada: Eutardigrada: Richtersiusidae)

In this paper, we describe a new tardigrade species, Richtersius tertius sp. n., from Greece. The description is based on morphological and morphometric analysis using light and scanning electron microscopy as well as genetic analysis based on four molecular markers (DNA sequences of three nuclear, i.e., 18S rRNA, 28S rRNA, ITS-2 and one mitochondrial COI fragment). Morphological and morphometric differences, together with genetic comparisons, provide independent verifications of Richtersius tertius sp. n. as a species new to science. Phenotypically, the new taxon differs from Richtersius coronifer (Richters, 1903) and Richtersius ziemowiti Kayastha, Berdi, Miaduchowska, Gawlak, Łukasiewicz, Gołdyn, Jędrzejewski et Kaczmarek, 2020 mainly by the morphology and size of cuticular pores, present only in hatchlings (first instars), as well as some morphometric characters. The results presented herein contribute further to the recognition of the morphological variability and biodiversity within Richtersius, with Richtersius tertius sp. n. being the third species formally described within the genus.

Diversity in Expression Biases of Lineage-Specific Genes During Development and Anhydrobiosis Among Tardigrade Species.

Lineage-specific genes can contribute to the emergence and evolution of novel traits and adaptations. Tardigrades are animals that have adapted to tolerate extreme conditions by undergoing a form of cryptobiosis called anhydrobiosis, a physical transformation to an inactive desiccated state. While studies to understand the genetics underlying the interspecies diversity in anhydrobiotic transitions have identified tardigrade-specific genes and family expansions involved in this process, the contributions of species-specific genes to the variation in tardigrade development and cryptobiosis are less clear. We used previously published transcriptomes throughout development and anhydrobiosis (5 embryonic stages, 7 juvenile stages, active adults, and tun adults) to assess the transcriptional biases of different classes of genes between 2 tardigrade species, Hypsibius exemplaris and Ramazzottius varieornatus. We also used the transcriptomes of 2 other tardigrades, Echiniscoides sigismundi and Richtersius coronifer, and data from 3 non-tardigrade species (Adenita vaga, Drosophila melanogaster, and Caenorhabditis elegans) to help identify lineage-specific genes. We found that lineage-specific genes have generally low and narrow expression but are enriched among biased genes in different stages of development depending on the species. Biased genes tend to be specific to early and late development, but there is little overlap in functional enrichment of biased genes between species. Gene expansions in the 2 tardigrades also involve families with different functions despite homologous genes being expressed during anhydrobiosis in both species. Our results demonstrate the interspecific variation in transcriptional contributions and biases of lineage-specific genes during development and anhydrobiosis in 2 tardigrades.

Macrobiotus coronifer Richters, 1903 (type species for Richtersius Pilato amp; Binda, 1989): designating a new neotype from the original type locality described within the integrative taxonomy framework.

The designation of a neotype for Macrobiotus coronifer Richters, 1903 (now the type species of the genus Richtersius Pilato Binda, 1989) by Maucci Ramazzotti (1981) with type locality Bodø in Norway is shown to be invalid as it does not comply with the International Code of Zoological Nomenclature (Article 75.3.4). Furthermore, the specimen selected by Maucci Ramazzotti (1981) is not from the original type locality, and the superficial and outdated documentation prevent a reliable identification of the species. A Code-compliant neotype is therefore designated. The new neotype was collected from the original locus typicus in Svalbard and described with standard light microscopy, detailed scanning electron microscopy imaging, DNA barcodes and a transcriptome, which makes it ideally suited for stabilising the taxonomy and nomenclature of Richtersius coronifer (Richters, 1903).

Some Tardigrades from Nepal (Asia) with Integrative Description of Macrobiotus wandae sp. nov. (Macrobiotidae: Hufelandi Group)

Global knowledge of tardigrade diversity is limited, and there still are regions with poorly studied fauna, such as Nepal. We applied an integrative taxonomy approach to describe a new tardigrade species from this area. The presented analysis was based on morphological and morphometrical data (using both light and scanning electron microscopy) and combined with multilocus molecular data (nuclear barcode sequences, i.e. 18S rRNA, 28S rRNA and ITS-2, and one mitochondrial COI sequences). The p-distances enabled an independent verification of Macrobiotus wandaesp. nov. as a species new to science. Molecular data allowed us to determine the phylogenetic position of Mac. wandaesp. nov. within the hufelandi group. Based on egg morphology, i.e. porous egg chorion and processes in shape of cones with terminal discs, new species is most similar to Mac. biserovi, Mac. denticulus, Mac. diversus, Mac. glebkai, Mac. macrocalix, Mac. maculatus and Mac. ramoli. But it differs from them mainly by some morphological and morphometrical characters of eggs (number of egg processes on circumference, details of egg shell sculpture, and morphology of terminal discs of eggs) and adults (oral cavity armature type, morphology of lunule and presence or absence of leg granulation). Moreover, we report from Nepal six other tardigrade taxa: Calohypsibius cf. maliki, Echiniscus blumi, Echiniscus nepalensis, Mesobiotus cf. furciger, Richtersius coronifer and Stellariscus cf. latifasciatus.

A new redescription of Richtersius coronifer, supported by transcriptome, provides resources for describing concealed species diversity within the monotypic genus Richtersius (Eutardigrada)

Richtersius coronifer, the nominal species for the family Richtersiidae and a popular laboratory model, exemplifies a common problem in modern tardigrade taxonomy. Despite undeniable progress in the field, many old and incomplete descriptions of taxa hinder both species delimitation and the estimation of species diversity and distribution. Although for over a century this species has been recorded throughout the world, recent research indicates that records to date are likely to represent a species complex rather than a single cosmopolitan species. However, in order to recognise and name species diversity within the complex, an integrative redescription of the nominal species is first needed. Here, we describe an R. coronifer population collected from Spitsbergen, i.e., one of the two localities mentioned in the original description, with detailed morphological and morphometric data associated with standard DNA sequences of four standard genetic markers (18S rRNA, 28S rRNA, ITS-2, and COI) and supported by transcriptome sequencing. We propose replacement of the neotype designated in 1981 by Maucci and Ramazzotti, as it is impossible to verify whether the existing neotype is conspecific with specimens studied by Richters in 1903 and 1904. Finally, using newly obtained cytochrome c oxidase subunit I (COI) sequences of populations from Spitsbergen, Italy, Poland, and Greece together with sequences deposited in GenBank (China, Greenland, Italy, Mongolia), we performed genetic species delimitation, which indicated seven distinct potential species within the genus Richtersius, in addition to the nominal taxon. This study marks a starting point for further research on the taxonomy of and species diversity within the genus. Moreover, this work has the potential to be the first tardigrade redescription to provide both genetic barcodes and a transcriptome of the species in question.

A method for studying the metabolic activity of individual tardigrades by measuring oxygen uptake using microrespirometry.

Studies of tardigrade biology have been severely limited by the sparsity of appropriate quantitative techniques, informative on a single-organism level. Therefore, many studies rely on motility-based survival scoring and quantifying reproductive success. Measurements of O2 respiration rates, as an integrating expression of the metabolic activity of single tardigrades, would provide a more comprehensive insight into how an individual tardigrade is responding to specific environmental factors or changes in life stages. Here, we present and validate a new method for determining the O2 respiration rate (nmolO2mg-1h-1) of single tardigrades under steady state, using O2 microsensors. As an example, we show that the O2 respiration rate determined in MilliQ water for individuals of Richtersius coronifer and of Macrobiotus macrocalix at 22°C was 10.8±1.84 and 13.1±2.19 nmol O2mg-1h-1, respectively.

A comparative ultrastructure study of storage cells in the eutardigrade Richtersius coronifer in the hydrated state and after desiccation and heating stress.

Tardigrades represent an invertebrate phylum with no circulatory or respiratory system. Their body cavity is filled with free storage cells of the coelomocyte-type, which are responsible for important physiological functions. We report a study comparing the ultrastructure of storage cells in anhydrobiotic and hydrated specimens of the eutardigrade Richtersius coronifer. We also analysed the effect of temperature stress on storage cell structure. Firstly, we verified two types of ultrastructurally different storage cells, which differ in cellular organelle complexity, amount and content of reserve material and connection to oogenetic stage. Type I cells were found to differ ultrastructurally depending on the oogenetic stage of the animal. The main function of these cells is energy storage. Storage cells of Type I were also observed in the single male that was found among the analysed specimens. The second cell type, Type II, found only in females, represents young undifferentiated cells, possibly stem cells. The two types of cells also differ with respect to the presence of nucleolar vacuoles, which are related to oogenetic stages and to changes in nucleolic activity during oogenesis. Secondly, this study revealed that storage cells are not ultrastructurally affected by six months of desiccation or by heating following this desiccation period. However, heating of the desiccated animals (tuns) tended to reduce animal survival, indicating that long-term desiccation makes these animals more vulnerable to heat stress. We confirmed the degradative pathways during the rehydration process after desiccation and heat stress. Our study is the first to document two ultrastructurally different types of storage cells in tardigrades and reveals new perspectives for further studies of tardigrade storage cells.

3D printed microchannels for sub-nL NMR spectroscopy.

Nuclear magnetic resonance (NMR) experiments on subnanoliter (sub-nL) volumes are hindered by the limited sensitivity of the detector and the difficulties in positioning and holding such small samples in proximity of the detector. In this work, we report on NMR experiments on liquid and biological entities immersed in liquids having volumes down to 100 pL. These measurements are enabled by the fabrication of high spatial resolution 3D printed microfluidic structures, specifically conceived to guide and confine sub-nL samples in the sub-nL most sensitive volume of a single-chip integrated NMR probe. The microfluidic structures are fabricated using a two-photon polymerization 3D printing technique having a resolution better than 1 μm3. The high spatial resolution 3D printing approach adopted here allows to rapidly fabricate complex microfluidic structures tailored to position, hold, and feed biological samples, with a design that maximizes the NMR signals amplitude and minimizes the static magnetic field inhomogeneities. The layer separating the sample from the microcoil, crucial to exploit the volume of maximum sensitivity of the detector, has a thickness of 10 μm. To demonstrate the potential of this approach, we report NMR experiments on sub-nL intact biological entities in liquid media, specifically ova of the tardigrade Richtersius coronifer and sections of Caenorhabditis elegans nematodes. We show a sensitivity of 2.5x1013 spins/Hz1/2 on 1H nuclei at 7 T, sufficient to detect 6 pmol of 1H nuclei of endogenous compounds in active volumes down to 100 pL and in a measurement time of 3 hours. Spectral resolutions of 0.01 ppm in liquid samples and of 0.1 ppm in the investigated biological entities are also demonstrated. The obtained results may indicate a route for NMR studies at the single unit level of important biological entities having sub-nL volumes, such as living microscopic organisms and eggs of several mammalians, humans included.

Tolerance to X-rays and Heavy Ions (Fe, He) in the Tardigrade Richtersius coronifer and the Bdelloid Rotifer Mniobia russeola.

The aim of this study was to analyze tolerance to heavy ions in desiccated animals of the eutardigrade Richtersius coronifer and the bdelloid rotifer Mniobia russeola within the STARLIFE project. Both species were exposed to iron (Fe) and helium (He) ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) in Chiba, Japan, and to X-rays at the German Aerospace Center (DLR) in Cologne, Germany. Results show no effect of Fe and He on viability up to 7 days post-rehydration in both R. coronifer and M. russeola, while X-rays tended to reduce viability in R. coronifer at the highest doses. Mean egg production rate tended to decline with higher doses in R. coronifer for all radiation types, but the pattern was not statistically confirmed. In M. russeola, there was no such tendency for a dose response in egg production rate. These results confirm the previously reported high tolerance to high linear energy transfer (LET) radiation in tardigrades and show for the first time that bdelloid rotifers are also very tolerant to high-LET radiation. These animal phyla represent the most desiccation- and radiation-tolerant animals on Earth and provide excellent eukaryotic models for astrobiological research. Key Words: Tardigrada-Rotifera-Radiation tolerance-Heavy ions-X-rays. Astrobiology 17, 163-167.

Morphological and molecular analyses onRichtersius(Eutardigrada) diversity reveal its new systematic position and lead to the establishment of a new genus and a new family within Macrobiotoidea

Important contributions have been made to the systematics of Eutardigrada in recent years, but these have also revealed that several taxa are polyphyletic and that cryptic species are present. To shed light on the taxonomy and systematic position of the genus Richtersius (Eutardigrada, Macrobiotoidea), six populations attributed to Richtersius coronifer were collected and analysed from morphological (light and scanning electron microscopy) and molecular (mitochondrial cytochrome oxidase subunit 1, 18S, 28S) points of view. In particular, a new morphometric index (claw common tract: length of the common tract of the claw/total claw length × 100) and a new morphological character (stalk system) were introduced. Our integrative study was able to unveil the ‘cryptic’ species diversity within Richtersius, showing that the genus contains more than one evolutionary lineage. A morphological peculiarity in the animals of all lineages is the dimorphism in the morphology of the cuticle. Cuticular pores are present in the newborns and are lost with the first moult; this morphological change represents a novelty in the life cycle of eutardigrades. The phylogenetic analyses carried out on Richtersius populations and other Macrobiotoidea show that Richtersius is closely related to Macrobiotus islandicus, whereas Adorybiotus granulatus is more related to Richtersius and M. islandicus than to other members of the genus Macrobiotus (type genus of Macrobiotidae); therefore, the genus Macrobiotus and the family Macrobiotidae are not monophyletic. Based on these results, the new genus Diaforobiotus (for M. islandicus) and the new family Richtersiidae (composed of Richtersius, Diaforobiotus gen. nov., and Adorybiotus) are established.

Experimentally Induced Repeated Anhydrobiosis in the Eutardigrade Richtersius coronifer.

Tardigrades represent one of the main animal groups with anhydrobiotic capacity at any stage of their life cycle. The ability of tardigrades to survive repeated cycles of anhydrobiosis has rarely been studied but is of interest to understand the factors constraining anhydrobiotic survival. The main objective of this study was to investigate the patterns of survival of the eutardigrade Richtersius coronifer under repeated cycles of desiccation, and the potential effect of repeated desiccation on size, shape and number of storage cells. We also analyzed potential change in body size, gut content and frequency of mitotic storage cells. Specimens were kept under non-cultured conditions and desiccated under controlled relative humidity. After each desiccation cycle 10 specimens were selected for analysis of morphometric characteristics and mitosis. The study demonstrates that tardigrades may survive up to 6 repeated desiccations, with declining survival rates with increased number of desiccations. We found a significantly higher proportion of animals that were unable to contract properly into a tun stage during the desiccation process at the 5th and 6th desiccations. Also total number of storage cells declined at the 5th and 6th desiccations, while no effect on storage cell size was observed. The frequency of mitotic storage cells tended to decline with higher number of desiccation cycles. Our study shows that the number of consecutive cycles of anhydrobiosis that R. coronifer may undergo is limited, with increased inability for tun formation and energetic constraints as possible causal factors.

The structure of the desiccated Richtersius coronifer (Richters, 1903).

Tun formation is an essential morphological adaptation for entering the anhydrobiotic state in tardigrades, but its internal structure has rarely been investigated. We present the structure and ultrastructure of organs and cells in desiccated Richtersius coronifer by transmission and scanning electron microscopy, confocal microscopy, and histochemical methods. A 3D reconstruction of the body organization of the tun stage is also presented. The tun formation during anhydrobiosis of tardigrades is a process of anterior-posterior body contraction, which relocates some organs such as the pharyngeal bulb. The cuticle is composed of epicuticle, intracuticle and procuticle; flocculent coat; and trilaminate layer. Moulting does not seem to restrict the tun formation, as evidenced from tardigrade tuns that were in the process of moulting. The storage cells of desiccated specimens filled up the free inner space and surrounded internal organs, such as the ovary and digestive system, which were contracted. All cells (epidermal cells, storage cells, ovary cells, cells of the digestive system) underwent shrinkage, and their cytoplasm was electron dense. Lipids and polysaccharides dominated among reserve material of storage cells, while the amount of protein was small. The basic morphology of specific cell types and organelles did not differ between active and anhydrobiotic R. coronifer.

Remains of non-pollen-palynomorphs – tardigrades from Spitsbergen found during pollen analyses

ABSTRACTPollen- and non-pollen-palynomorphs (NPP) analytical studies of the northwestern part of Spitsbergen were conducted between 1988 and 1991. As well as pollen from local native flora and more dispersed species, some well preserved remains of tardigrada exuvia, buccal tubes and eggs were found. This study reviewed the remains of at least six tardigrade taxa reported:Dactylobiotus ambiguous, Paramacrobiotus richtersigroup,Richtersius coronifer, Macrobiotus hufelandigroup,Macrobiotus peterseniandMinibiotuscf.intermedius, which are reassessed and determined more accurately. These findings provide some new insights into the past environmental conditions and changes for Spitsbergen. Based on the present research it can be concluded that tardigrade remains are frequent NPP elements of pollen analyses from lake, peat bogs and detritus sub-fossil sediment cores, at least in polar regions. It can also be stated that tardigrades can be considered indicators in further palaeontological studies helping to reconstruct past environmental conditions (for example humidity) for some regions. However, the knowledge of tardigrades in these types of analyses is still rather poor.

Desiccation Tolerance in the Tardigrade Richtersius coronifer Relies on Muscle Mediated Structural Reorganization

Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted to handle large fluctuations in physical and chemical parameters. Tardigrades are microscopic ecdysozoans well known for their ability to endure hostile conditions, such as complete desiccation – a phenomenon called anhydrobiosis. During dehydration, anhydrobiotic animals undergo a series of anatomical changes. Whether this reorganization is an essential regulated event mediated by active controlled processes, or merely a passive result of the dehydration process, has not been clearly determined. Here, we investigate parameters pivotal to the formation of the so-called "tun", a state that in tardigrades and rotifers marks the entrance into anhydrobiosis. Estimation of body volume in the eutardigrade Richtersius coronifer reveals an 87 % reduction in volume from the hydrated active state to the dehydrated tun state, underlining the structural stress associated with entering anhydrobiosis. Survival experiments with pharmacological inhibitors of mitochondrial energy production and muscle contractions show that i) mitochondrial energy production is a prerequisite for surviving desiccation, ii) uncoupling the mitochondria abolishes tun formation, and iii) inhibiting the musculature impairs the ability to form viable tuns. We moreover provide a comparative analysis of the structural changes involved in tun formation, using a combination of cytochemistry, confocal laser scanning microscopy and 3D reconstructions as well as scanning electron microscopy. Our data reveal that the musculature mediates a structural reorganization vital for anhydrobiotic survival, and furthermore that maintaining structural integrity is essential for resumption of life following rehydration.

Cover Picture: J. Biophotonics 10/2013

AbstractMicroscopic image of Richtersius coronifer, a species of tardigrades. Tardigrades are microscopic metazoans which are able to survive extreme physical and chemical conditions by entering a stress tolerant state called cryptobiosis. H. Kneipp et al. show that surface enhanced Raman scattering supported by plasmonic gold nanoparticles can measure molecular constituents and their local distribution in live tardigrades. Surface enhanced Raman signatures allow to differentiate between two species and indicate molecular structural differences between tardigrades in water and in a dry state.(Picture: H. Kneipp et al., pp. 759–764 in this issue)

Element analysis of the eutardigrades Richtersius coronifer and Milnesium cf. asiaticum using Particle Induced X-ray Emission (PIXE)

Semi-terrestrial tardigrades are well-known for their tolerance to a variety of environmental extremes, including desiccation, freezing and radiation. Despite several attempts to reveal the genetic and molecular mechanisms behind the resilience of tardigrades, it is still unknown how these animals are able to maintain the integrity of their cellular components under severe stress. Quantitative or qualitative changes in molecular compounds (e.g., carbohydrates, proteins) are expected, and have been the main line of research towards understanding the tolerance of tardigrades. In radiation tolerant bacteria, a tolerance mechanism based on manganese has been proposed. We evaluate this hypothesis in tardigrades and provide the first data on element composition in desiccated and non-desiccated specimens of two eutardigrade species, Richtersius coronifer and Milnesium cf. asiaticum. A focused 2 MeV proton microbeam was utilised to determine the elemental content, distributions and concentrations, using the ion beam analytical technique particle induced X-ray emission (PIXE). The presence of six elements - phosphorus, sulphur, chlorine, potassium, calcium and iron - were confirmed in all tardigrade specimens, at levels up to a few mg g(-1). However, manganese was found in less than 10% of the analysed specimens, and in low amounts, thus our study provides no evidence for the manganese hypothesis. We also show that the distributions and/or concentrations of some elements differ between the two species as well as between the dehydrated and hydrated state. In particular, very low levels of iron were found in dehydrated M. cf. asiaticum. Our analysis shows that the PIXE technique is a useful tool for investigating questions on the distribution of elements both in dehydrated and hydrated tardigrades.

Tolerance to gamma-irradiation in eggs of the tardigrade Richtersius coronifer depends on stage of development

Tardigrades are known as one of the most radiation tolerant animals on Earth, and several studies on tolerance in adult tardigrades have been published. In contrast, very few studies on radiation tolerance of embryonic stages have been reported. Here we report a study on tolerance to gamma irradiation in eggs of the eutardigrade Richtersius coronifer. Irradiation of eggs collected directly from a natural substrate (moss) showed a clear dose-response, with a steep decline in hatchability at doses up to 0.4 kGy followed by a relatively constant hatchability around 25% up to 2 kGy, and a decline to ca. 5% at 4 kGy above which no eggs hatched. Analysis of the time required for eggs to hatch after irradiation (residual development time) showed that hatching of eggs after exposure to high doses of gamma radiation was associated with short residual development time. Since short residual development time means that the egg was irradiated at a late developmental stage, this suggests that eggs were more tolerant to radiation late in development. This was also confirmed in another experiment in which stage of development at irradiation was controlled. No eggs irradiated at the early developmental stage hatched, and only one egg at middle stage hatched, while eggs irradiated in the late stage hatched at a rate indistinguishable from controls. This suggests that the eggs are more sensitive to radiation in the early stages of development, or that tolerance to radiation is acquired only late in development, shortly before the eggs hatch, hypotheses that are not mutually exclusive. Our study emphasizes the importance of considering specific cell cycle phases and developmental stages in studies of tolerance to radiation in tardigrades, and the potential importance of embryonic studies in revealing the mechanisms behind the radiation tolerance of tardigrades and other cryptobiotic animals.

Inorganic ion composition in Tardigrada: cryptobionts contain large fraction of unidentified organic solutes

Many species of tardigrades are known to tolerate extreme environmental stress, yet detailed knowledge of the mechanisms underlying the remarkable adaptations of tardigrades is still lacking, as are answers to many questions regarding their basic biology. Here, we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) using high-performance liquid chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na(+) and Cl(-) are the principal inorganic ions in tardigrade fluids, albeit other ions, i.e. K(+), NH4(+), Ca(2+), Mg(2+), F(-), SO4(2-) and PO4(3-) were also detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared with that of the external medium (Na(+), ×70-800; K(+), ×20-90; Ca(2+) and Mg(2+), ×30-200; F(-), ×160-1040, Cl(-), ×20-50; PO4(3-), ×700-2800; SO4(2-), ×30-150). In contrast, in the marine species H. crispae, Na(+), Cl(-) and SO4(2-) are almost in ionic equilibrium with (brackish) salt water, while K(+), Ca(2+), Mg(2+) and F(-) are only slightly concentrated (×2-10). An anion deficit of ~120 mEq l(-1) in M. tardigradum and H. crispae indicates the presence of unidentified ionic components in these species. Body fluid osmolality ranges from 361±49 mOsm kg(-1) in R. coronifer to 961±43 mOsm kg(-1) in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not lose large quantities of inorganic ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicate that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.

Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit

Abstract Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state termed anhydrobiosis in response to desiccation. In theanhydrobiotic state, tardigrades display an incredible capacity to tolerate extreme environmental stress, not necessarily encountered in theirnatural habitat. In this study, we determine the effect of different extreme stresses on initial survival, long-term survival and fecundity of selectedspecies of limno-terrestrial tardigrades. The primary focus was to assess the effect of cosmic radiation. This was achieved through the RoTaRad(Rotifers, Tardigrades and Radiation) project on the BIOPAN 6 mission, funded by Agenzia Spaziale Italiana under the European Space Agency.To test their tolerance of space environment, tardigrades were sent into low earth orbit, and exposed to cosmic radiation and a microgravityenvironment. Experiments on Whatman-3 filters show an effect of cosmic radiation on the survival of the eutardigrade Richtersius coronifer justafter returning to Earth; however, after 2 years of desiccation on Whatman-3 filters, none of the tardigrades previously exposed to cosmicradiation could be revived. In a microcosmos experiment, the tardigrades R. coronifer, Ramazzottius oberhauseri and Echiniscus testudo weredesiccated on a moss substrate together with rotifers and nematodes. Very low survival rates were observed in this experiment, likely due to theapplied desiccation protocol. Embryos of the tardigrade Milnesium tardigradum were also exposed to cosmic radiation; they all hatched in thelaboratory after the flight. In addition, experiments testing extreme cold and vacuum tolerance in R. coronifer show that tardigrades inanhydrobiosis are unaffected by these conditions.Key words: Tardigrada – anhydrobiosis – cosmic radiation – extreme freezing – vacuum

Tolerance to proton irradiation in the eutardigrade Richtersius coronifer – a nuclear microprobe study

Purpose: The tardigrade Richtersius coronifer has previously been shown to tolerate very high doses of low linear energy transfer (low-LET) radiation (gamma rays). The purpose of this study was to extend our knowledge on radiation tolerance in this species by investigating the dose-response to high-LET radiation in terms of protons.Materials and methods: Dehydrated tardigrades of the species R. coronifer were irradiated with 2.55 MeV (megaelectronvolts) protons at doses ranging from 500 gray (Gy) to 15,000 Gy, to investigate the dose-viability relationship. In addition, a focused proton microbeam was utilised to determine the areal mass distribution, using the ion beam analytical technique STIM (Scanning Transmission Ion Microscopy).Results: The experiment suggests that R. coronifer is unaffected by doses of proton irradiation up to 10,000 Gy, but shows very little viability at higher doses. The STIM analysis revealed that the thickness of the dehydrated tardigrades exceeds 150 μm, and that a fraction of the protons may not be fully absorbed.Conclusion: Our results are in line with previous studies of exposure to high-LET radiation in tardigrades, indicating that these animals are equally or even more tolerant to high-LET compared to low-LET gamma radiation. The physiological background to this remarkable result is currently unknown, but deserves investigation.