Anhydrobiosis in bdelloid species, populations and individuals

Bdelloid rotifers are aquatic microinvertebrates common in waterbodies and in unstable "terrestrial" habitats, such as mossesand lichens. The key to the adaptability to live in unstablehabitats is their capacity to tolerate habitat desiccation throughanhydrobiosis, that is assumed apomorphic to the taxon. Thelife history traits of some "moss" and "water" species of bdelloidare compared, showing that the water species have shorter lifespan, higher fecundity and earlier age at first reproductionthan the moss species. These traits are discussed in the lightof current life history theories. Contrary to the assumptionsof the models, anhydrobiosis of bdelloids does not appear toimply energy demand. Past research on bdelloid anhydrobiosisis briefly reviewed, focusing on the factors that affect anhydrobiosissuccess, like morphological and physiological adjustments, andon the effect of events of anhydrobiosis during life time. Desiccationproduces a time shift on the age of the bdelloid, which disregardsthe time spent as anhydrobiotic, following the so-called "SleepingBeauty" model. Average fecundity is never found to decreaseas a consequence of anhydrobiosis, but is either equal or evenhigher than that of a hydrated rotifer. Bdelloid populationsseem to benefit from anhydrobiosis; fitness of a bdelloid isfound to decline, if populations are maintained hydrated forseveral generations, but not if populations are cyclically desiccated.We hypothesize that anhydrobiosis can be an essential eventfor long-term survival of bdelloid populations.     

Anhydrobiotic Survival in Populations of the Tardigrades Richtersius coronifer and Ramazzottius oberhaeuseri from Italy and Sweden

We report a study on anhydrobiotic survival in the eutardigrades Richtersius coronifer and Ramazzottius oberhaeuseri. In each of these species, we investigated the anhydrobiotic survival of two populations, one from Sweden and one from Italy. We found that anhydrobiotic survival was similar in the Swedish and the Italian populations in both species, indicating no divergence with respect to anhydrobiotic capacity. Body size had a strong effect on the probability to recover from anhydrobiosis, but the effect was in opposite direction in the two tardigrade species. Ramazzottius oberhaeuseri had a considerable higher overall survival (66%) than R. coronifer (40%). This result is in line with earlier studies that found R. oberhaeuseri to have a very high water-retentive capacity.     

Anhydrobiosis quotient: a novel approach to evaluate stability in desiccated bacterial cells

Aim:  The major objective of this study was the development of a methodology to quantify the anhydrobiotic ability of bacteria and its application to evaluate the stability of desiccated bacterial cells using the biocontrol agent Tsukamurella paurometabola C-924 as a model of anhydrobiote.
Methods and Results:  Tsukamurella paurometabola C-924 was desiccated by spray-drying. Samples of desiccated cells were stored at several temperatures and viability and residual moisture were measured at different intervals of time. The term anhydrobiosis quotient (ε) was defined, and a scale of anhydrobiotic ability for classifying micro-organisms in terms of tolerance to desiccation was established (1 ≤  ε  ≤ 15). The anhydrobiosis quotient was used to evaluate the stability of the anhydrobiotic cells. As a main result, changes in the anhydrobiosis quotient at several temperatures were fitted using a reparameterized Weibull model, which was found to be robust for the prediction of the stability at 4°C.
Conclusions:  A novel methodology was developed to evaluate the desiccated state in bacteria. The anhydrobiosis quotient allows the quantitative estimation of the anhydrobiotic ability, and the mathematical model developed allows the prediction of the desiccated state of bacterial populations.
Significance and Impact of the Study:  The new methodology could be applied in studying the anhydrobiosis state of bacterial populations as a predictive tool for industrial and environmental microbiology.     

Stress during dormancy: effect on recovery rates and life-history traits of anhydrobiotic animals

To test the influence of storage conditions on recovery and life-history traits of dormant animals we subjected cohorts of a bdelloid rotifer (Macrotrachela quadricornifera) and a nematode (Panagrolaimus rigidus) to three different conditions during a 26-d anhydrobiosis period. These conditions were (1) standard conditions in our laboratory (AC), (2) a flight to the U.S.A. and storage there (KSC), and (3) a flight to the U.S.A. followed by a flight on a Shuttle-to-Mir mission (Shuttle). Another cohort (HC) was maintained under hydrated conditions and served as reference for life-history traits of the desiccated samples. The recovery after dormancy and the residual life-history traits of the different cohorts were compared. Results indicate that (a) during dormancy the animals were indifferent to the test conditions, as evidenced from both recovery capacity and life history performances, (b) the anhydrobiotic period affected the experimental species differently. At recovery both animals resumed their life cycle, but while the rotifer appeared indifferent to the time spent in desiccation, the nematode modified its life history according to the duration of anhydrobiosis. Third, (c) induction of anhydrobiosis can act as a switching mechanism for animals capable of desiccation. Anhydrobiosis probably represents a suitable method to preserve organisms during stressful conditions allowing them to return to a normal life when convenient.     

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.     

Resistance of Anhydrobiotic Aphelenchus avenae to Methyl Bromide Fumigation

The effect of methyl bromide (MB) was tested on active and anhydrobiotic Aphelenchus avenae. A. avenae was induced into anhydrobiosis by three different techniques. Both active and anhydrobiotic nematodes were subjected to 3,000 μ1 MB/liter air for 14 periods from 0 to 82 h. Anhydrobiotic nematodes were more resistant to fumigation than active nematodes, regardless of the technique used to induce anhydrobiosis. The percent survival decreased with increasing MB exposures (μ1 MB × h). For an LD₉₅ of 45,000-54,000 μ1/1 × h were required for active nematodes and >279,000 μ1/1 × h for anhydrobiotic nematodes.     

Recipes for successful anhydrobiosis in bdelloid rotifers

We tested the effect of several environmental variables on the ability of three bdelloid rotifers (Macrotrachela quadricornifera, Philodina roseola and Adineta oculata) to recover from the anhydrobiotic state. The variables we examined were (1) rate of water evaporation, (2) relative humidity during anhydrobiosis, (3) temperature during anhydrobiosis, (4) duration of anhydrobiosis, and (5) rehydration rate. Our results indicate that bdelloids can regulate to some degree net water balance during onset and termination of anhydrobiosis.     

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.     

Physiological changes leading to anhydrobiosis improve radiation tolerance in Polypedilum vanderplanki larvae

High tolerance against various extreme environments exhibited by some anhydrobionts might be due to being almost completely desiccated, a state where little or no chemical reactions occur. We have shown that anhydrobiotic larvae of Polypedilum vanderplanki have higher tolerance against both high- and low-linear energy transfer (LET) radiation than hydrated larvae. It is of great interest to know how the desiccating larvae gain radiation tolerance. We therefore examined effects of high-LET radiation on four kinds of larvae: (1) normal hydrated (intact) larva, (2) intermediates between the anhydrobiotic and normal hydrated state, (3) almost completely dehydrated (anhydrobiotic) larvae, and (4) immediately rehydrated larvae that are assumed to have a similar molecular profile to anhydrobiotic larvae. The intermediates and immediately rehydrated larvae survived longer after high-LET radiation than intact larvae, indicating that radiation tolerance could be enhanced even in hydrated larvae. Physiological changes toward anhydrobiosis, e.g. accumulation of protectants or increasing damage repair capacity, correlate with improved radiation tolerance in hydrated larvae. In addition, almost complete desiccation further enhanced radiation tolerance, possibly in a different way from the hydrated larvae.     

Anhydrobiosis and Freezing-Tolerance: Adaptations That Facilitate the Establishment of Panagrolaimus Nematodes in Polar Habitats

Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. Panagrolaimus davidi, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent “ecological fitting” of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.     

Anhydrobiotic engineering of bacterial and mammalian cells: is intracellular trehalose sufficient?

Anhydrobiotic engineering aims to confer a high degree of desiccation tolerance on otherwise sensitive living organisms and cells by adopting the strategies of anhydrobiosis. Nonreducing disaccharides such as trehalose and sucrose are thought to play a pivotal role in resistance to desiccation stress in many microorganisms, invertebrates, and plants, and in vitro trehalose is known to confer stability on dried biomolecules and biomembranes. We have therefore tested the hypothesis that intracellular trehalose (or a similar molecule) may be not only necessary for anhydrobiosis but also sufficient. High concentrations of trehalose were produced in bacteria by osmotic preconditioning, and in mammalian cells by genetic engineering, but in neither system was desiccation tolerance similar to that seen in anhydrobiotic organisms, suggesting that trehalose alone is not sufficient for anhydrobiosis. In Escherichia coli such desiccation tolerance was achievable, but only when bacteria were dried in the presence of both extracellular trehalose and intracellular trehalose. In mouse L cells, improved osmotolerance was observed with up to 100 mM intracellular trehalose, but desiccation was invariably lethal even with extracellular trehalose present. We conclude that anhydrobiotic engineering of at least some microorganisms is achievable with present technology, but that further advances are needed for similar desiccation tolerance of mammalian cells.     

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.     

Soil nematodes and desiccation survival in the extreme arid environment of the antarctic dry valleys

Soil nematodes are capable of employing an anhydrobiotic survivalstrategy in response to adverse environmental conditions. TheMcMurdo Dry Valleys of Antarctica represent a unique environmentfor the study of anhydrobiosis because extremes of cold, salinity,and aridity combine to limit biological water availability.We studied nematode anhydrobiosis in Taylor Valley, Antarctica,using natural variation in soil properties. The coiled morphologyof nematodes extracted from dry valley soils suggests that theyemploy anhydrobiosis, and these coiled nematodes showed enhancedrevival when re-hydrated in water as compared to vermiform nematodes.Nematode coiling was correlated with soil moisture content,salinity, and water potential. In the driest soils studied (gravimetricwater content <2%), 20–80% of nematodes were coiled.Soil water potential measurements also showed a high degreeof variability. These measurements reflect microsite variationin soil properties that occurs at the scale of the nematode.We studied nematode anhydrobiosis during the austral summer,and found that the proportion of nematodes coiled can vary diurnally,with more nematodes vermiform and presumably active at the warmesttime of day. However, dry valley nematodes uncoiled rapidlyin response to soil wetting from snowmelt, and most nematodeactivity in the Dry Valleys may be confined to periods followingrare snowfall and melting events. Anhydrobiosis represents animportant temporal component of a dry valley nematode's lifespan. The ability to utilize anhydrobiosis plays a significantrole in the widespread distribution and success of these organismsin the Antarctic Dry Valleys and beyond.     

Ecology of bdelloids: how to be successful

Bdelloids inhabit many different environments. The entire taxon is an order belonging to the phylum Rotifera. In spite of its wide distribution, it has a very uniform morphology, suggesting that natural selection has had almost no effect on its morphological characteristics.This paper reviews the geographical and ecological distributions of bdelloids and their ability to tolerate different conditions is discussed. Two characteristics account for the wide distribution of bdelloids, parthenogenetic reproduction and their ability to withstand unfavorable conditions through anhydrobiosis. The former is an apomictic thelytoky which may ensure genetic homogeneity within clones. However, evidence which suggests variability among and within parthenogenetic clones is discussed. Some recent experimental evidence indicates that anhydrobiosis does not affect the life history of the surviving individuals. The effects of anhydrobiosis on bdelloid populations are considered.     

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.     

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 5^th and 6^th desiccations. Also total number of storage cells declined at the 5^th and 6^th 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.     

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.     

Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae

Some organisms can survive exposure to extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living nematode Aphelenchus avenae can be induced to enter the anhydrobiotic state by exposure to a moderate reduction in relative humidity. During this preconditioning period, the nematode accumulates large amounts of the disaccharide trehalose, which is thought to be necessary, but not sufficient, for successful anhydrobiosis. To identify other adaptations that are required for anhydrobiosis, we developed a novel SL1-based mRNA differential display technique to clone genes that are upregulated by dehydration in A. avenae. Three such genes, Aav-lea-1, Aav-ahn-1, and Aav-glx-1, encode, respectively, a late embryogenesis abundant (LEA) group 3 protein, a novel protein that we named anhydrin, and the antioxidant enzyme glutaredoxin. Strikingly, the predicted LEA and anhydrin proteins are highly hydrophilic and lack significant secondary structure in the hydrated state. The dehydration-induced upregulation of Aav-lea-1 and Aav-ahn-1 was confirmed by Northern hybridization and quantitative PCR experiments. Both genes were also upregulated by an osmotic upshift, but not by cold, heat, or oxidative stress. Experiments to investigate the relationship between mRNA levels and protein expression for these genes are in progress. LEA proteins occur commonly in plants, accumulating during seed maturation and desiccation stress; the presence of a gene encoding an LEA protein in an anhydrobiotic nematode suggests that some mechanisms of coping with water loss are conserved between plants and animals.