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.     

Anhydrobiosis: Inside yeast cells

Under natural conditions yeast cells as well as other microorganisms are regularly subjected to the influence of severe drought, which leads to their serious dehydration. The dry seasons are then changed by rains and there is a restoration of normal water potential inside the cells. To survive such seasonal changes a lot of vegetative microbial cells, which belong to various genera and species, may be able to enter into a state of anhydrobiosis, in which their metabolism is temporarily and reversibly suspended or delayed. This evolutionarily developed adaptation to extreme conditions of the environment is widely used for practical goals – for conservation of microorganisms in collections, for maintenance and long storage of different important strain-producers and for other various biotechnological purposes. This current review presents the most important data obtained mainly in the studies of the structural and functional changes in yeast cells during dehydration. It describes the changes of the main organelles of eukaryotic cells and their role in cell survival in a dry state. The review provides information regarding the role of water in the structure and functions of biological macromolecules and membranes. Some important intracellular protective reactions of eukaryotic organisms, which were revealed in these studies and may have more general importance, are also discussed. The results of the studies of yeast anhydrobiosis summarized in the review show the possibilities of improving the conservation and long-term storage of various microorganisms and of increasing the quality of industrially produced dry microbial preparations.     

Is there a single biochemical adaptation to anhydrobiosis?

Even though water is required for the maintenance of biologicalintegrity, numerous organisms are capable of surviving lossof virtually all their cellular water and existing in a stateknown as anhydrobiosis. Over the past three decades we and othershave established that disaccharides such as trehalose and sucroseare almost certainly involved in stabilizing the dry cells.We discuss here some of the evidence behind the mechanism ofthis stabilization. Until the past few years this mechanismhas been sufficiently appealing that a consensus has been developingthat acquisition of these sugars in the cytoplasm may be bothnecessary and sufficient for anhydrobiosis. We show here thatthere are other routes to achieve the effects conferred by thesugars and that other adaptations are almost certainly required,at least in environmental conditions that are less than optimal.Under optimal storage conditions, the presence of the sugarsalone may be sufficient to stabilize even mammalian cells inthe dry state, findings that are already finding use in humanclinical medicine.     

Microbial anhydrobiosis

The loss of cellular water (desiccation) and the resulting low cytosolic water activity are major stress factors for life. Numerous prokaryotic and eukaryotic taxa have evolved molecular and physiological adaptions to periods of low water availability or water-limited environments that occur across the terrestrial Earth. The changes within cells during the processes of desiccation and rehydration, from the activation (and inactivation) of biosynthetic pathways to the accumulation of compatible solutes, have been studied in considerable detail. However, relatively little is known on the metabolic status of organisms in the desiccated state; that is, in the sometimes extended periods between the drying and rewetting phases. During these periods, which can extend beyond decades and which we term ‘anhydrobiosis’, organismal survival could be dependent on a continued supply of energy to maintain the basal metabolic processes necessary for critical functions such as macromolecular repair. Here, we review the state of knowledge relating to the function of microorganisms during the anhydrobiotic state, highlighting substantial gaps in our understanding of qualitative and quantitative aspects of molecular and biochemical processes in desiccated cells.     

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis

In 1702, Van Leeuwenhoek was the first to describe the phenomenon of anhydrobiosis in a species of bdelloid rotifer, Philodina roseola. It is the purpose of this review to examine what has been learned since then about the extreme desiccation tolerance in rotifers and how this compares with our understanding of anhydrobiosis in other organisms. Remarkably, much of what is known today about the requirements for successful anhydrobiosis, and the degree of biostability conferred by the dry state, was already determined in principle by the time of Spallanzani in the late 18th century. Most modern research on anhydrobiosis has emphasized the importance of the non-reducing disaccharides trehalose and sucrose, one or other sugar being present at high concentrations during desiccation of anhydrobiotic nematodes, brine shrimp cysts, bakers' yeast, resurrection plants and plant seeds. These sugars are proposed to act as water replacement molecules, and as thermodynamic and kinetic stabilizers of biomolecules and membranes. In apparent contradiction of the prevailing models, recent experiments from our laboratory show that bdelloid rotifers undergo anhydrobiosis without producing trehalose or any analogous molecule. This has prompted us to critically re-examine the association of disaccharides with anhydrobiosis in the literature. Surprisingly, current hypotheses are based almost entirely on in vitro data: there is very limited information which is more than simply correlative in the literature on living systems. In many species, disaccharide accumulation occurs at approximately the same time as desiccation tolerance is acquired. However, several studies indicate that these sugars are not sufficient for anhydrobiosis; furthermore, there is no conclusive evidence, through mutagenesis or functional knockout experiments, for example, that sugars are necessary for anhydrobiosis. Indeed, some plant seeds and micro-organisms, like the rotifer, exhibit excellent desiccation tolerance in the absence of high intracellular sugar concentrations. Accordingly, it seems appropriate to call for a re-evaluation of our understanding of anhydrobiosis and to embark on new experimental programmes to determine the key molecular mechanisms involved.     

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.     

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.     

Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation

Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis [1]. It is assumed that sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions [2]. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a "dry, yet alive" larva from a "dry and dead" one.     

Cryptobiosis--a peculiar state of biological organization

David Keilin (Proc. Roy. Soc. Lond. B, 150, 1959, 149–191) coined the term ‘cryptobiosis’ (hidden life) and defined it as ‘the state of an organism when it shows no visible signs of life and when its metabolic activity becomes hardly measurable, or comes reversibly to a standstill.’ I consider selected aspects of the 300 year history of research on this unusual state of biological organization. Cryptobiosis is peculiar in the sense that organisms capable of achieving it exhibit characteristics that differ dramatically from those of living ones, yet they are not dead either, so one may propose that cryptobiosis is a unique state of biological organization. I focus chiefly on animal anhydrobiosis, achieved by the reversible loss of almost all the organism's water. The adaptive biochemical and biophysical mechanisms allowing this to take place involve the participation of large concentrations of polyhydroxy compounds, chiefly the disaccharides trehalose or sucrose. Stress (heat shock) proteins might also be involved, although the details are poorly understood and seem to be organism-specific. Whether the removal of molecular oxygen (anoxybiosis) results in the reversible cessation of metabolism in adapted organisms is considered, with the result being ‘yes and no’, depending on how one defines metabolism. Basic research on cryptobiosis has resulted in unpredicted applications that are of substantial benefit to the human condition and a few of these are described briefly.     

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.     

Trehalose and vitreous states: desiccation tolerance of dormant stages of the crustaceans Triops and Daphnia

Several aquatic organisms are able to withstand extreme desiccation in at least one of their life stages. This is commonly known as "anhydrobiosis." It was often thought that to tolerate such a desiccated state required high amounts of compatible solutes such as the nonreducing disaccharide trehalose, which protects cellular structures by water replacement and glass formation. Trehalose levels of dormant eggs and cysts of five freshwater crustaceans (Daphnia magna, Daphnia pulex, Triops longicaudatus, Triops cancriformis, and Triops australiensis) were observed in different states of hydration and dehydration. Although trehalose was detected in all species, the concentration was under 0.5% of the dry weight (0.05 μg/μg protein), and no change between the different states was observed. Differential scanning calorimetry (DSC) measurements indicated that dried cysts of all Triops species were in a glassy state, supporting the vitrification hypothesis. No indication for a vitreous state was found in dried resting eggs of Daphnia.     

Molecular Strategies of the Caenorhabditis elegans Dauer Larva to Survive Extreme Desiccation

Massive water loss is a serious challenge for terrestrial animals, which usually has fatal consequences. However, some organisms have developed means to survive this stress by entering an ametabolic state called anhydrobiosis. The molecular and cellular mechanisms underlying this phenomenon are poorly understood. We recently showed that Caenorhabditis elegans dauer larva, an arrested stage specialized for survival in adverse conditions, is resistant to severe desiccation. However, this requires a preconditioning step at a mild desiccative environment to prepare the organism for harsher desiccation conditions. A systems approach was used to identify factors that are activated during this preconditioning. Using microarray analysis, proteomics, and bioinformatics, genes, proteins, and biochemical pathways that are upregulated during this process were identified. These pathways were validated via reverse genetics by testing the desiccation tolerances of mutants. These data show that the desiccation response is activated by hygrosensation (sensing the desiccative environment) via head neurons. This leads to elimination of reactive oxygen species and xenobiotics, expression of heat shock and intrinsically disordered proteins, polyamine utilization, and induction of fatty acid desaturation pathway. Remarkably, this response is specific and involves a small number of functional pathways, which represent the generic toolkit for anhydrobiosis in plants and animals.     

Stabilization of dry Mammalian cells: lessons from nature

The Center for Biostabilization at UC Davis is attempting tostabilize mammalian cells in the dry state. We review here someof the lessons from nature that we have been applying to thisenterprise, including the use of trehalose, a disaccharide foundat high concentrations in many anhydrobiotic organisms, to stabilizebiological structures, both in vitro and in vivo. Trehalosehas useful properties for this purpose and in at least in onecase—human blood platelets—introducing this sugarmay be sufficient to achieve useful stabilization. Nucleatedcells, however, are stabilized by trehalose only during theinitial stages of dehydration. Introduction of a stress proteinobtained from an anhydrobiotic organism, Artemia, improves thestability markedly, both during the dehydration event and followingrehydration. Thus, it appears that the stabilization will requiremultiple adaptations, many of which we propose to apply fromstudies on anhydrobiosis.     

Trehalose as an indicator of desiccation stress in Drosophila melanogaster larvae: a potential marker of anhydrobiosis

In the current scenario of global climate change, desiccation is considered as one of the major environmental stressors for the biota exposed to altered levels of ambient temperature and humidity. Drosophila melanogaster, a cosmopolitan terrestrial insect has been chosen as a humidity-sensitive bioindicator model for the present study since its habitat undergoes frequent stochastic and/or seasonally aggravated dehydration regimes. We report here for the first time the occurrence of anhydrobiosis in D. melanogaster larvae by subjecting them to desiccation stress under laboratory conditions. Larvae desiccated for ten hours at <5% relative humidity could enter anhydrobiosis and could revive upon rehydration followed by resumption of active metabolism. As revealed by FTIR and HPLC analyzes, our findings strongly indicated the synthesis and accumulation of trehalose in the desiccating larvae. Biochemical measurements pointed out the desiccation-responsive trehalose metabolic pathway that was found to be coordinated in concert with the enzymes trehalose 6-phosphate synthase and trehalase. Further, an inhibitor-based experimental approach using deoxynojirimycin, a specific trehalase inhibitor, demonstrated the pivotal role of trehalose in larval anhydrobiosis of D. melanogaster. We therefore propose trehalose as a potential marker for the assessment of anhydrobiosis in Drosophila. The present findings thus add to the growing list of novel biochemical markers in specific bioindicator organisms for fulfilling the urgent need of environmental biomonitoring of climate change.     

Application of anhydrobiosis and dehydration of yeasts for non-conventional biotechnological goals

Dehydration of yeast cells causes them to enter a state of anhydrobiosis in which their metabolism is temporarily and reversibly suspended. This unique state among organisms is currently used in the production of active dry yeasts, mainly used in baking and winemaking. In recent decades non-conventional applications of yeast dehydration have been proposed for various modern biotechnologies. This mini-review briefly summarises current information on the application of dry yeasts in traditional and innovative fields. It has been shown that dry yeast preparations can be used for the efficient protection, purification and bioremediation of the environment from heavy metals. The high sorption activity of dehydrated yeasts can be used as an interesting tool in winemaking due to their effects on quality and taste. Dry yeasts are also used in agricultural animal feed. Another interesting application of yeast dehydration is as an additional stage in new methods for the stable immobilisation of microorganisms, especially in cases when biotechnologically important strains have no affinity with the carrier. Such immobilisation methods also provide a new approach for the successful conservation of yeast strains that are very sensitive to dehydration. In addition, the application of dehydration procedures opens up new possibilities for the use of yeast as a model system. Separate sections of this review also discuss possible uses of dry yeasts in biocontrol, bioprotection and biotransformations, in analytical methods as well as in some other areas.     

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.     

Identification of novel smORFs and microprotein acting in response to rehydration of Nostoc flagelliforme

Nostoc flagelliforme, a terrestrial cyanobacterium spread throughout arid and semi-arid areas, has been long known for its outstanding adaptability to extremely dry conditions. This microorganism is able to recover biological activities within hours after months of anhydrobiosis state, attracting investigation through proteomic analysis. Except for canonical proteome, microproteins encoded by small ORFs (smORFs) have recently been regarded as indispensable participants in metabolic processes. However, the involvement of smORFs in N. flagelliforme remains unknown. Here we first constructed a smORF database in N. flagelliforme using bioinformatic prediction, resulting in 6072 novel smORFs. Then LS-MS/MS analysis was applied to identify expression patterns of microproteins and seek smORFs and their encoded microprotein playing a role during rehydration. In total, 18 novel microproteins were mined based on a smORF searching strategy combined with three proteomic assays, of which five were annotated as ribosomal proteins, one as RNA polymerase subunit, and one as acetohydroxy acid isomeroreductase. We also suggested the possible functions of smORFs according to their expression pattern and discovered two neighboring and homologous smORFs. All these results will expand our knowledge of smORFs-encoded microproteins and their relation to the stress response of extremophilic microorganisms.     

Looking beyond sugars: the role of amphiphilic solutes in preventing adventitious reactions in anhydrobiotes at low water contents

Plants and animals that can survive dehydration accumulate high concentrations of disaccharides in their cells and tissues during desiccation. These sugars are necessary both for the depression of the membrane phase transition temperature of the dry lipid and for the formation of a carbohydrate glass. In the past decade, however, it has become clear that certain types of adventitious enzymatic reactions are possible at low water contents, which along with free-radical mediated damage, can cause hydrolysis of lipids and loss of membrane barrier function. Disaccharides do not necessarily prevent these types of reactions, which suggests that other compounds might also be necessary for protecting organisms from this type of degradation during anhydrobiosis. Arbutin, one possible example, accumulates in large quantities in certain resurrection plants and has been shown to inhibit phospholipase A(2) activity at low water contents. The direct effect of arbutin on membranes under stress conditions depends on the membrane lipid composition. It can serve a protective function during desiccation- or freeze/thaw-induced stress in the presence of nonbilayer-forming lipids or a disruptive function in their absence. Other possible amphiphiles, including certain naturally occurring flavonols, may serve as anti-oxidants and some might have similar lipid composition-dependent effects. Such compounds, therefore, are likely to be localized near specific membranes, where they might provide the greatest benefit at the least liability to the organism.     

Activity of the α-glucoside transporter Agt1 in Saccharomyces cerevisiae cells during dehydration-rehydration events

Microbial cells can enter a state of anhydrobiosis under desiccating conditions. One of the main determinants of viability during dehydration-rehydration cycles is structural integrity of the plasma membrane. Whereas much is known about phase transitions of the lipid bilayer, there is a paucity of information on changes in activity of plasma membrane proteins during dehydration-rehydration events. We selected the α-glucoside transporter Agt1 to gain insights into stress mechanisms/responses and ecophysiology during anhydrobiosis. As intracellular water content of S. cerevisiae strain 14 (a strain with moderate tolerance to dehydration-rehydration) was reduced to 1.5 g water/g dry weight, the activity of the Agt1 transporter decreased by 10–15 %. This indicates that functionality of this trans-membrane and relatively hydrophobic protein depends on water. Notably, however, levels of cell viability were retained. Prior incubation in the stress protectant xylitol increased stability of the plasma membrane but not Agt1. Studies were carried out using a comparator yeast which was highly resistant to dehydration-rehydration (S. cerevisiae strain 77). By contrast to S. cerevisiae strain 14, there was no significant reduction of Agt1 activity in S. cerevisiae strain 77 cells. These findings have implications for the ecophysiology of S. cerevisiae strains in natural and industrial systems.