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.     

Factors Inducing Successful Anhydrobiosis in the African Chironomid Polypedilum vanderplanki: Significance of the Larval Tubular Nest

The African chironomid Polypedilum vanderplanki exhibits anhydrobiosis,i.e., the larvae can survive complete desiccation. Recoveryrate and trehalose content were investigated in larvae desiccatedslowly or at a rate more than 3 times faster. Upon slow desiccation(evaporation rate 0.22 ml day^–1) larvae synthesized 38µg trehalose/individual before complete desiccation, andall of them recovered after rehydration, whereas larvae thatwere dehydrated quickly (evaporation rate 0.75 ml day^–1)accumulated only 6.8 µg trehalose/individual and noneof them revived after rehydration. In the pools that are theirnatural habitat P. vanderplanki larvae make tubes by incorporatingdetritus or soil with their sticky saliva. This tubular structureis a physical barrier not only to protect the larva from naturalenemies but also induces successful anhydrobiosis by reducingthe dehydration rate. When larvae were dehydrated with 100 µldistilled water (DW) in soil tubes, they accumulated 37 µgtrehalose/individual and more than half of them could reviveafter rehydration, whereas larvae without tubes accumulatedlower level of trehalose and none recovered after rehydration.     

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.     

Induction of anhydrobiosis in fat body tissue from an insect

The larva of the African chironomid Polypedilum vanderplanki can withstand complete desiccation. Our previous reports revealed that even when the larva is dehydrated without a brain, it accumulated a great amount of trehalose and successfully went into anhydrobiosis. In this paper we determined the viability after rehydration in tissues from the larvae followed by complete dehydration. Only fat-body tissues that were the main producer of trehalose could be preserved in a dry state at room temperature for an extended period of more than 18 months in a viable form. Thus we have confirmed that the central nervous system is not involved in the induction of anhydrobiosis, even in this complex multicellular organism.     

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.     

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.     

Trehalose and anhydrobiosis in tardigrades--evidence for divergence in responses to dehydration

To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non-reducing disaccharide trehalose to protect against dehydration damage. The trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.     

The effect of different periods of dehydrationhehydration upon the ability of second stage larvae of Anguina tritici to survive desiccation at 0% relative humidity

The effect of various desiccation regimes on the ability of second stage larvae of Anguina tritici (Steinbuch, 1799) to survive drying at 0% relative humidity has been studied. It was found that both repeated dehydrationhehydration cycles and the length of the desiccation period itself decreased viability of the larvae. The results are discussed in relation to the mechanisms which may facilitate anhydrobiosis.     

Anhydrobiosis without trehalose in bdelloid rotifers

Eukaryotes able to withstand desiccation enter a state of suspended animation known as anhydrobiosis, which is thought to require accumulation of the non-reducing disaccharides trehalose (animals, fungi) and sucrose (plants), acting as water replacement molecules and vitrifying agents. We now show that clonal populations of bdelloid rotifers Philodina roseola and Adineta vaga exhibit excellent desiccation tolerance, but that trehalose and other disaccharides are absent from carbohydrate extracts of dried animals. Furthermore, trehalose synthase genes (tps) were not found in rotifer genomes. This first observation of animal anhydrobiosis without trehalose challenges our current understanding of the phenomenon and calls for a re-evaluation of existing models.     

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.     

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.     

高温对家蚕三品系血淋巴中糖水平的影响（英文）

家蚕Bombyx mori的两个二化性品系热耐受型NB4D2和热敏感型CSR2均适合于温带气候,而多化性的PM（Pure Mysore) 品系适合于热带气候,将这3种品系5龄幼虫分别置于32℃和36℃的高温下,观察高温对其5龄幼虫至蛹期血淋巴中糖含量及海藻糖酶活性的影响。结果表明： PM幼虫和蛹的死亡率均小于NB4D2和CSR2。在蜕皮期间血淋巴海藻糖水平较高,而葡萄糖水平及海藻糖酶活性较低。32℃和36℃的高温下,幼虫蜕皮期间血淋巴中糖含量及海藻糖酶活性仅在其各自的水平上表现为小幅度的增加。蜕皮后幼虫血淋巴中海藻糖含量显著下降,而葡萄糖含量和海藻糖酶活性显著上升。在较高温度下,蜕皮后幼虫血淋巴中海藻糖含量下降幅度更大,而葡萄糖含量及海藻糖酶活性上升水平也更加显著。25±1℃下取食幼虫血淋巴中葡萄糖含量显著下降,海藻糖含量显著上升;3℃和36℃下PM 和NB4D2取食幼虫血淋巴葡萄糖和海藻糖含量以及海藻糖酶活性增加,而CSR2均减少或降低。吐丝幼虫血淋巴中葡萄糖含量及海藻糖酶活性显著下降,海藻糖小幅度下降。而在较高温度下,耐热型PM 和NB4D2吐丝家蚕血淋巴糖含量含量和海藻糖酶活性明显增加,而热敏感型CSR2的则明显下降。这3种品系蛹发育期的血淋巴糖含量及海藻糖酶活性均下降。在两较高温度下,PM蛹期血淋巴糖和海藻糖酶活性增加,而NB4D2 36℃时增加幅度小于32℃时。对于CSR2,32℃时观察到其血淋巴葡萄糖含量增加,但当环境温度增加到36℃时其血淋巴葡萄糖含量降至正常水平下。然而,当CSR2的蛹置于32℃和36℃时血淋巴海藻糖含量及其酶活性下降,且36℃时下降幅度更大。因此,桑蚕对高温的适应取决于家蚕的品系及发育阶段,并可通过其血淋巴糖及海藻糖酶活性水平进行验证。     

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.     

Divergence of larval resource acquisition for water conservation and starvation resistance in Drosophila melanogaster

Laboratory selection experiments have evidenced storage of energy metabolites in adult flies of desiccation and starvation resistant strains of D. melanogaster but resource acquisition during larval stages has received lesser attention. For wild populations of D. melanogaster, it is not clear whether larvae acquire similar or different energy metabolites for desiccation and starvation resistance. We tested the hypothesis whether larval acquisition of energy metabolites is consistent with divergence of desiccation and starvation resistance in darker and lighter isofemale lines of D. melanogaster. Our results are interesting in several respects. First, we found contrasting patterns of larval resource acquisition, i.e., accumulation of higher carbohydrates during 3rd instar larval stage of darker flies versus higher levels of triglycerides in 1st and 2nd larval instars of lighter flies. Second, 3rd instar larvae of darker flies showed ~40?h longer duration of development at 21°C; and greater accumulation of carbohydrates (trehalose and glycogen) in fed larvae as compared with larvae non-fed after 150?h of egg laying. Third, darker isofemale lines have shown significant increase in total water content (18%); hemolymph (86%) and dehydration tolerance (11%) as compared to lighter isofemale lines. Loss of hemolymph water under desiccation stress until death was significantly higher in darker as compared to lighter isofemale lines but tissue water loss was similar. Fourth, for larvae of darker flies, about 65% energy content is contributed by carbohydrates for conferring greater desiccation resistance while the larvae of lighter flies acquire 2/3 energy from lipids for sustaining starvation resistance; and such energy differences persist in the newly eclosed flies. Thus, larval stages of wild-caught darker and lighter flies have evolved independent physiological processes for the accumulation of energy metabolites to cope with desiccation or starvation stress.     

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.     

Divergence of water balance mechanisms in two melanic Drosophila species from the western Himalayas

Drosophila busckii is more abundant under colder and drier montane habitats in the western Himalayas as compared to Drosophila melanogaster but the mechanistic basis of such climatic adaptations is largely unknown. We tested the hypothesis whether genetic variation or phenotypic plasticity of cuticular traits confer adaptive protection against desiccation stress in two melanic Drosophila species living under drier montane localities. For D. melanogaster, changes in melanisation are known to be associated with reduced water loss but there are no data on D. busckii. We investigated changes in body melanisation, cuticular lipids, desiccation resistance, water loss, extractable hemolymph volume (%), and dehydration tolerance in six sympatric populations of D. busckii and D. melanogaster over an altitudinal range of 640-2236 m. D. busckii is a melanic species but changes in cuticular water loss are negatively correlated with cuticular lipid mass and not with body melanisation. In D. melanogaster, there are no plastic effects (14-28 °C) for cuticular lipid mass but variation in body melanisation is associated with desiccation-related traits. Effects of organic solvents (hexane or chloroform: methanol), developmental plasticity and seasonal variation in cuticular lipids affect body water loss in D. busckii but no such changes occur in D. melanogaster. Thus, sympatric populations of D. busckii and D. melanogaster have evolved different water balance mechanisms under shared environmental conditions in the western Himalayas. Multiple measures of desiccation resistance in these species show clinal variation with altitude, consistent with adaptation to increased desiccation stress.     

Cellular localization and proposed function of midgut trehalase in the silkworm larva, Bombyx mori

A sorbitol density gradient analysis with the aid of several marker enzymes demonstrated that midgut trehalase of the silkworm larvae. Bombyx mori, was localized in the microsomal membranes, but not in mitochondria, lysosomes and microvilli at the apical surface. Electron microscopic examination showed that trehalase-enriched membrane fraction consisted of heterogeneous mixtures of membrane vesicles derived from the endoplasmic reticulum and plasma membrane parts other than the microvillus membrane. The enzyme-histochemical stains of trehalase activity on the midgut section could be detected only at the basal surface of the epithelium against haemocoel. Such a specific localization was further confirmed by immunohistochemistry with the peroxidase-conjugated antibody technique. Thus, it is concluded that midgut trehalase of silkworm larvae is situated on the plasma membrane at the basal surface of the epithelium. An intact preparation of midgut incubated in vitro in the medium containing [(14)C]trehalose could hydrolyse trehalose into glucose and take it up into the cell, although some glucose was liberated into the medium when incubated for extended periods. These results suggest that midgut trehalase plays a physiological role in utilization of haemolymph trehalose not in nutrient absorption.     

Studies of sibling Drosophila species from Laguna Verde, Veracruz, Mexico: I. Species frequencies, viability, desiccation resistance, and vagility

The sibling species Drosophila melanogaster and D. simulans were collected at Laguna Verde, Veracruz, Mexico. D. melanogaster was found in significantly greater frequency than was D. simulans. Ten isofemale lines of each species were tested for egg to adult viability, desiccation resistance, and vagility. D. melanogaster surpassed D. simulans in all three characteristics. The findings are discussed with reference to the climatic conditions at Laguna Verde and the expected effect of such an environment on the relative frequencies of these species. The dichotomous results in regard to desiccation resistance and vagility that were observed between recently collected D. melanogaster and the Oregon-R laboratory stock of that species are also discussed.     

Anhydrobiotic engineering of gram-negative bacteria

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.