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.     

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.     

Cells from an anhydrobiotic chironomid survive almost complete desiccation

Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science. As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210. Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply. We also attempted dry-preservation of organs isolated from P. vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation.     

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.     

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.     

A new anhydrobiotic midge from Malawi,Polypedilum pembai sp.n. (Diptera: Chironomidae), closely related to the desiccation tolerant midge,Polypedilum vanderplanki Hinton

The sleeping chironomid (Polypedilum vanderplanki Hinton) lives on temporary rock pools in the semi‐arid tropical regions of Africa. Its larvae are able to survive the dry season in a completely desiccated ametabolic state known as anhydrobiosis. So far, P. vanderplanki was the only species among all insects showing demonstrated anhydrobiotic ability. Here, we show that a new related species originating from Malawi, Polypedilum pembai sp.n. , is also anhydrobiotic and that its desiccation tolerance mechanism is probably similar to what is observed in P. vanderplanki. The new species, P. pembai sp.n. , is described with special attention to the common and different morphological features, compared with P. vanderplanki. Phylogenetic analysis showed that both species are closely related, suggesting that anhydrobiosis evolved only once in their common ancestor about 49 Ma somewhere in Africa, before the divergence of two species, one in the sub‐Saharan area and another in southeastern Africa.     

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

Background

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

Results

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

Conclusions

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

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.     

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line,Pv11

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5–34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

     

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.     

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.     

Molecular anhydrobiology: identifying molecules implicated in invertebrate anhydrobiosis

Studies in anhydrobiotic plants have defined many genes whichare upregulated during desiccation, but comparable studies ininvertebrates are at an early stage. To develop a better understandingof invertebrate anhydrobiosis, we have begun to characterisedehydration-inducible genes and their proteins in anhydrobioticnematodes and bdelloid rotifers; this review emphasises recentfindings with a hydrophilic nematode protein. Initial work withthe fungivorous nematode Aphelenchus avenae led to the identificationof two genes, both of which were markedly induced on slow drying(90–98% relative humidity, 24 hr) and also by osmoticstress, but not by heat or cold or oxidative stresses. The firstof these genes encodes a novel protein we have named anhydrin;it is a small, basic polypeptide, with no counterparts in sequencedatabases, which is predicted to be natively unstructured andhighly hydrophilic. The second is a member of the Group 3 LEAprotein family; this and other families of LEA proteins arewidely described in plants, where they are most commonly associatedwith the acquisition of desiccation tolerance in maturing seeds.Like anhydrin, the nematode LEA protein, Aav-LEA-1, is highlyhydrophilic and a recombinant form has been shown to be unstructuredin solution. In vitro functional studies suggest that Aav-LEA-1is able to stabilise other proteins against desiccation-inducedaggregation, which is in keeping with a role of LEA proteinsin anhydrobiosis. In vivo, however, Aav-LEA-1 is apparentlyprocessed into smaller forms during desiccation. A processingactivity was found in protein extracts of dehydrated, but nothydrated, nematodes; these shorter polypeptides are also activeanti-aggregants and we hypothesise that processing LEA proteinserves to increase the number of active molecules availableto the dehydrating animal. Other LEA-like proteins are beingidentified in nematodes and it seems likely therefore that theywill play a major role in the molecular anhydrobiology of invertebrates,as they are thought to do in plants.     

Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed “anhydrobiosis”. Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.     

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.     

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.     

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.