Volume and morphology changes of a bdelloid rotifer species (Macrotrachela quadricornifera) during anhydrobiosis

Following a study on the changes occurring in a bdelloid species (Macrotrachela quadricornifera, Rotifera, Bdelloidea) when entering anhydrobiosis, we investigated the changes in morphology, including weight and volume during the transition from the active hydrated to the dormant anhydrobiotic state by scanning electron microscopy, confocal microscopy and light microscopy. We compared sizes and morphologies of hydrated extended, hydrated contracted and anhydrobiotic specimens. Bdelloid musculature is defined: longitudinal muscles are contracted in the hydrated contracted animal (head and foot are retracted inside the trunk), but appear loose in the anhydrobiotic animal. When anhydrobiotic, M. quadricornifera appears much smaller in size, with a volume reduction of about 60% of the hydrated volume, and its internal organization undergoes remarkable modifications. Internal body cavities, clearly distinguishable in the hydrated extended and contracted specimens, are no longer visible in the anhydrobiotic specimen. Concomitantly, M. quadricornifera loses more than 95% of its weight when anhydrobiotic; this is more than expected from the volume reduction data and could indicate the presence of space-filling molecular species in the dehydrated animal. We estimate that the majority of body mass loss and volume reduction can be ascribed to the water loss from the body cavity during desiccation.     

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.     

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.     

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 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.     

The induction of anhydrobiosis in the sleeping chironomid: current status of our knowledge

An African chironomid, Polypedilum vanderplanki, is the only insect known to be capable of extreme desiccation tolerance, or anhydrobiosis. In the 1950s and 1960s, Hinton strenuously studied anhydrobiosis in this insect from a physiological standpoint; however, nobody has afterward investigated the phenomenon. In 2000, research on mechanisms underlying anhydrobiosis was resumed due to successful establishment of a rearing system for P. vanderplanki. This review is focused on the latest findings on the physiological and molecular mechanisms underlying the induction of anhydrobiosis in P. vanderplanki. Early experiments demonstrated that the induction of anhydrobiosis was possible in isolated tissues and independent from the control of central nervous system. However, to achieve successful anhydrobiosis, larvae need a slow regime of desiccation, allowing them to synthesize molecules, which will protect cells and tissues against the deleterious effects of dehydration. Trehalose, a nonreducing disaccharide, which accumulates in P. vanderplanki larvae up to 20% of the dry body mass, is thought to replace the water in its tissues. Similarly, highly hydrophilic proteins called the late embryogenesis abundant (LEA) proteins are expressed in huge quantities and act as a molecular shield to protect biological molecules against aggregation and denaturation. This function is shared by heat shock proteins, which are also upregulated during the desiccation process. At the same time, desiccating larvae express various antioxidant molecules and enzymes, to cope with the massive oxidative stress, which is responsible for general damage to membranes, proteins, and DNA in dehydrating cells. Finally, specific water channels, called aquaporins, accelerate dehydration, and trehalose together with LEA proteins forms a glassy matrix, which protects the biological molecules and the structural integrity of larvae in the anhydrobiotic state.     

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.     

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.     

A Putative LEA Protein,but no Trehalose,is Present in Anhydrobiotic Bdelloid Rotifers

Some eukaryotes, including bdelloid rotifer species, are able to withstand desiccation by entering a state of suspended animation. In this ametabolic condition, known as anhydrobiosis, they can remain viable for extended periods, perhaps decades, but resume normal activities on rehydration. Anhydrobiosis is thought to require accumulation of the non-reducing disaccharides trehalose (in animals and fungi) or sucrose (in plant seeds and resurrection plants), which may protect proteins and membranes by acting as water replacement molecules and vitrifying agents. However, in clone cultures of bdelloid rotifers Philodina roseola and Adineta vaga, we were unable to detect trehalose or other disaccharides in either control or dehydrating animals, as determined by gas chromatography. Indeed, trehalose synthase genes (tps) were not detected in these rotifer genomes, suggesting that bdelloids might not have the capacity to produce trehalose under any circumstances. This is in sharp contrast to other anhydrobiotic animals such as nematodes and brine shrimp cysts, where trehalose is present during desiccation. Instead, we suggest that adaptations involving proteins might be more important than those involving small biochemicals in rotifer anhydrobiosis: on dehydration, P. roseola upregulates a hydrophilic protein related to the late embryogenesis abundant (LEA) proteins associated with desiccation tolerance in plants. Since LEA-like proteins have also been implicated in the desiccation tolerance of nematodes and micro-organisms, it seems that hydrophilic protein biosynthesis represents a common element of anhydrobiosis across several biological kingdoms.     

Anhydrobiosis-associated nuclear DNA damage and repair in the sleeping chironomid: linkage with radioresistance

Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions ((4)He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3-4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation.     

Anhydrobiosis in bacteria: from physiology to applications

Anhydrobiosis is a phenomenon related to the partial or total desiccation of living organisms, keeping their vital functions after rehydration. The desiccated state in prokaryotes has been widely studied, mainly due to the broad spectrum of the anhydrobiosis applications. In this review, we present the basic theoretical concepts related to anhydrobiosis, focusing on bacterial species. An update about desiccation tolerance in bacteria is given; and the general mechanisms of desiccation tolerance and desiccation damage are described. In addition, we show how the study of anhydrobiosis in prokaryotes has established the theoretical and practical basis for the development of the drying technologies. With regard to the desiccation tolerance in bacteria, although many mechanisms remain undiscovered at the molecular level, important research about the physiology of the anhydrobiotic state and its applications has been performed, and here we provide the most recent information about this subject. On the other hand, the most widely used drying technologies and their particular applications in several fields are described (e.g. medicine, agriculture and food industry). Finally, topics on the stability of desiccated bacterial cells are treated, concluding with the necessity of focusing the research on the mathematical modelling of the desiccated state in bacteria.     

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.     

Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition

Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva, which can then further survive harsh desiccation in an anhydrobiotic state. We have previously identified the genetic and biochemical pathways essential for survival—but without detailed knowledge of their material properties, the mechanistic understanding of this intriguing phenomenon remains incomplete. Here we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. ODT revealed that the properties of the dauer larvae undergo a dramatic transition upon harsh desiccation. Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that could not be observed by conventional microscopy. Our advance opens a door to quantitatively assessing the transitions in material properties and structure necessary to fully understand an organism on the verge of life and death.     

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely alpha-helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, AavLEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in alpha-helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.     

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.