The structure of the desiccated <Emphasis Type="Italic">Richtersius coronifer</Emphasis> (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.     

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.     

Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer

The physiology and biochemistry behind the extreme tolerance to desiccation shown by the so-called anhydrobiotic animals represents an exciting challenge to biology. The current knowledge suggests that both carbohydrates and proteins are often involved in protecting the dry cell from damage, or in the repair of induced damage. Tardigrades belong to the most desiccation-tolerant multicellular organisms, but very little research has been reported on the biochemistry behind desiccation tolerance in this group. We quantified the induction of the heat-shock protein Hsp70, a very wide-spread stress protein, in response to desiccation, ionising radiation, and heating, in the anhydrobiotic tardigrade Richtersius coronifer using an immuno-westernblot method. Elevated levels of Hsp70 were recorded after treatment of both heat and ionising radiation, and also in rehydrated tardigrades after a period of desiccation. In contrast, tardigrades in the desiccated (dry) state had reduced Hsp70 levels compared to the non-treated control group. Our results suggest that Hsp70 may be involved in the physiological and biochemical system underlying desiccation (and radiation) tolerance in tardigrades, and that its role may be connected to repair processes after desiccation rather than to biochemical stabilization in the dry state.     

Experimentally induced anhydrobiosis in the tardigrade Richtersius coronifer: phenotypic factors affecting survival

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

Anhydrobiosis increases survival of trichostrongyle nematodes

This study demonstrates that infective-stage larvae of 2 trichostrongyle ruminant gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus colubriformis, can enter into anhydrobiotic states when completely desiccated. Larvae of control trichostrongyle species, Heligmosomoides polygyrus and Nippostrongylus brasiliensis, that infect mice were unable to survive desiccation or to enter into anhydrobiosis. Ruminant larvae were able to survive up to 7 desiccation/rehydration cycles, and, during anhydrobiosis, metabolic activity was decreased and survival of the larvae was prolonged both in the laboratory and in the field. Relative humidity had no effect on ruminant larval survival after anhydrobiosis compared with controls. Temperature had a significant effect, 85.8 +/- 2.3% of larvae in anhydrobiosis could survive low temperatures (0 C) that killed all control larvae. Metabolic activity, measured by changes in lipid content and CO2 respiration, was significantly lower in larvae that entered anhydrobiosis compared with controls (P < 0.05). In field experiments using open-meshed chambers under ambient environmental conditions, larvae in anhydrobiosis had significantly higher survival rates in the field compared with controls (P < 0.05) during summer and winter trials. These data suggest that anhydrobiosis in ruminant larvae promotes survival at freezing temperatures, decreases metabolic activity, and prolongs survival under natural field conditions.     

Anhydrobiosis in tardigrades--the last decade

The current state of knowledge about anhydrobiosis in tardigrades is presented. In response to adverse environmental conditions tardigrades arrest their metabolic activity and after complete dehydration enter the so-called “tun” state. In this ametabolic state they are able to tolerate exposure to various chemical and physical extremes. These micrometazoans have evolved various kinds of morphological, physiological and molecular adaptations to reduce the effects of desiccation. In this review we address behavioral adaptation, morphological features and molecules which determine the anhydrobiotic survival. The influence of the time spent in anhydrobiotic state on the lifespan and DNA and the role of the antioxidant defense system are also considered. Finally we summarize recent input from the “omics” sciences.     

Morphological response of a bdelloid rotifer to desiccation

We desiccated bdelloid rotifers (Macrotrachela quadricornifera), submitting the animals to four desiccation procedures (protocols A, B, C, D) that differed in the rate of water evaporation, in the time of desiccation, and in the substrates provided. We observed external morphological changes of the rotifer bodies during drying with scanning electron microscopy and, in parallel, assessed rates of recovery after a 7-day period of dormancy. Two protocols produced disorganized morphologies of the anhydrobiotic animals, with no (A) or very poor (B) recovery. Protocols C and D gave rather high rates of recovery and dry rotifers appeared unaltered and well organized. The different protocols affected rotifer morphology during the 7-day anhydrobiosis and rates of recovery after the 7-day anhydrobiosis; high recovery rates corresponded to well-organized morphologies of anhydrobiotic bdelloids, suggesting that a proper contraction of the body into a tun shape and probably a rigorous packing of internal structures are necessary for survival after anhydrobiosis. These features are affected by the time between water shortage and full desiccation, but also by the surrounding relative humidity and by the nature of the substrate. Possible adaptations of anhydrobiotic rotifers are discussed.     

DNA damage in storage cells of anhydrobiotic tardigrades

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 ± 1.98% DNA in tail, compared to 0.44 ± 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 ± 6.41% of DNA was found in the comet tail. After ten months, 23.66 ± 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.     

Dynamics of Long-term Anhydrobiotic Survival of Lichen-dwelling Tardigrades

It is not rare to find in references that anhydrobiotic tardigrades can survive for more than a century. However, a closer look at the empirical evidence provides very little support that tardigrades are capable of surviving dried for such a long time. In order to resolve this discrepancy, we carried out a study to evaluate the long-term survival of naturally dried tardigrades. A large fragment of dry lichen (Xanthoria parietina) was collected in the field two days after a rainy day in 1999. The dry lichen was stored inside a paper bag in the laboratory at room temperature and humidity and under atmospheric oxygen exposure. Replicates of the dry lichen were re-hydrated after various time periods of storage, with all tardigrades extracted and the survivors enumerated. Five species of tardigrades were found, but two of them only occasionally. Ramazzottius oberhaeuseri, Echiniscus testudo and Echiniscus trisetosus were sufficiently represented for statistical analysis. At the beginning of the experiment 91.1% of R. oberhaeuseri and 71.7% of Echiniscus spp. were alive. R. oberhaeuseri survived up to 1604 days, while Echiniscus spp. lived up to 1085 days. Recovery after four years of anhydrobiosis has to be considered a very good long-term survival, which is important from an ecological and evolutionary point of view.     

Identification of Anhydrobiosis-related Genes from an Expressed Sequence Tag Database in the Cryptobiotic Midge Polypedilum vanderplanki (Diptera; Chironomidae)

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.     

Aggregation effects on anhydrobiotic survival in the tardigrade Richtersius coronifer

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

A Free-living Panagrolaimus sp. from Armenia Can Survive in Anhydrobiosis for 8.7 Years

Although the ability of plant-parasitic nematodes to survive in a dehydrated or anhydrobiotic state for long periods of time has been well documented, the ability of free-living nematodes has not. Here we report on the survival of a free-living nematode, Panagrolaimus sp., from Armenia in the anhydrobiotic state for 8.7 years. This Panagrolaimus sp. can be cultured and maintained readily and may provide a good system for studying anhydrobiosis in nematodes.     

Studies on Anhydrobiosis of Pratylenchus thornei

Large populations of Pratylenchus thornei, a winter pest of cereals, legumes, and potatoes in the northern Negev region of Israel, survive 7-8 months of summer drought and return to full activity at the beginning of the rainy season. To demonstrate that it survives the summer in an anhydrobiotic state, all developmental stages of P. thornei were exposed to gradually reduced relative humidity (RH) using glycerin water solutions. At 97.7% RH the nematodes were coiled and able to survive exposure to 0% RH. About 40% of artificially desiccated nematodes could be reactivated by gradually increasing the humidity to the final water environment. Desiccated nematodes could withstand temperatures up to 40 C. Reactivated individuals showed intestines apparently devoid of reserve materials. Only 3% survived three cycles of desiccation and reactivation. P. thornei reactivated after anhydrobiosis multiplied twice as much within Vicia sativa roots as did fresh nematodes.     

Anhydrobiosis in Pratylenchus penetrans

Anhydrobiotic survival of Pratylenchus penetrans was compared in several soil moisture regimes. Bodies of anhydrobiotic nematodes were coiled. In slow-dried soils, Vineland silt loam (VSL) and Fox loamy sand (FLS), 70 and 58% of the total P. penetrans populations were anhydrobiotic when soil moistures reached ca. 3% and water potential 15 kPa or greater. Coiling began at a much lower water potential in FLS than in VSL. In fast-dried soils, only 31 and 22% of the P. penetrans populations in the same two soil types had entered the anhydrobiotic state at comparable moistures. In the above soils, 76-96% of the P. penetrans were alive immediately after entering the anhydrobiotic state. In slow-dried VSL, some nematodes (1%) survived 770 days. In the other soils, all anhydrobiotic nematodes were dead after 438 days. Anhydrobiosis increased the ability of nematodes to survive subzero temperatures, but it did not increase their ability to survive temperatures above 40 C. Infectivity and reproductivity of rehydrated P. penetrans were not affected by anhydrobiosis.     

A molecular analysis of desiccation tolerance mechanisms in the anhydrobiotic nematode Panagrolaimus superbus using expressed sequenced tags

Background

Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. Panagrolaimus superbus is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that P. superbus uses to combat the potentially lethal effects of cellular dehydration may include the constitutive and inducible expression of protective molecules, along with behavioural and/or morphological adaptations that slow the rate of cellular water loss. In addition, inducible repair and revival programmes may also be required for successful rehydration and recovery from anhydrobiosis.

Results

To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of P. superbus. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at http://www.nematodes.org/nembase4/species_info.php?species=PSC. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from P. superbus. Notable among those is a putative lineage expansion of the lea (late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific sxp/ral-2 family that is highly expressed in parasitic nematodes and secreted onto the surface of the nematodes' cuticles. There were 2,059 novel unigenes (51.7% of the total), 149 of which are predicted to encode intrinsically disordered proteins lacking a fixed tertiary structure. One unigene may encode an exo-??-1,3-glucanase (GHF5 family), most similar to a sequence from Phytophthora infestans. GHF5 enzymes have been reported from several species of plant parasitic nematodes, with horizontal gene transfer (HGT) from bacteria proposed to explain their evolutionary origin. This P. superbus sequence represents another possible HGT event within the Nematoda. The expression of five of the 19 putative stress response genes tested was upregulated in response to desiccation. These were the antioxidants glutathione peroxidase, dj-1 and 1-Cys peroxiredoxin, an shsp sequence and an lea gene.

Conclusions

P. superbus appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of lea genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of P. superbus.     

Tardigrades survive exposure to space in low Earth orbit

Vacuum (imposing extreme dehydration) and solar/galactic cosmic radiation prevent survival of most organisms in space [1]. Only anhydrobiotic organisms, which have evolved adaptations to survive more or less complete desiccation, have a potential to survive space vacuum, and few organisms can stand the unfiltered solar radiation in space. Tardigrades, commonly known as water-bears, are among the most desiccation and radiation-tolerant animals and have been shown to survive extreme levels of ionizing radiation [2-4]. Here, we show that tardigrades are also able to survive space vacuum without loss in survival, and that some specimens even recovered after combined exposure to space vacuum and solar radiation. These results add the first animal to the exclusive and short list of organisms that have survived such exposure.     

Demographic processes underlying fitness restoration in bdelloid rotifers emerging from dehydration

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