Dehydration-inducible changes in expression of two aquaporins in the sleeping chironomid, Polypedilum vanderplanki

Aquaporin, AQP, is a channel protein that allows water to permeate across cell membranes. Larvae of the sleeping chironomid, Polypedilum vanderplanki, can withstand complete dehydration by entering anhydrobiosis, a state of suspended animation; however, the mechanism by which water flows out of the larval body during dehydration is still unclear. We isolated two cDNAs (PvAqp1 and PvAqp2) encoding water-selective aquaporins from the chironomid. When expressed in Xenopus oocytes, PvAQP1 and PvAQP2 facilitated permeation of water but not glycerol. Northern blots and in situ hybridization showed that expression of PvAqp1 was dehydration-inducible and ubiquitous whereas that of PvAqp2 was dehydration-repressive and fat body-specific. These data suggest distinct roles for these aquaporins in P. vanderplanki, i.e., PvAqp2 controls water homeostasis of fat body during normal conditions and PvAqp1 is involved in the removal of water during induction of anhydrobiosis.     

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.     

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.     

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.     

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.     

Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence.     

Dehydration in dormant insects

Many of the mechanisms used by active insects to maintain water balance are not available to dormant individuals. Physiological and biochemical mechanisms of dehydration tolerance and resistance in dormant insects and some other invertebrates are reviewed, as well as linkages of dehydration with energy use and metabolism, with cold hardiness, and with diapause. Many dormant insects combine several striking adaptations to maintain water balance that-in addition to habitat choice-may include especially reduction of body water content, decreased cuticular permeability, absorption of water vapour, and tolerance of low body water levels. Many such features require energy and hence that metabolism, albeit much reduced, continues during dormancy. Four types of progressively dehydrated states are recognized: water is managed internally by solute or ion transport; relatively high concentrations of solutes modify the behaviour of water in solutions; still higher concentrations of certain carbohydrates lead to plasticized rubbers or glasses with very slow molecular kinetics; and anhydrobiosis eliminates metabolism.     

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.     

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.     

Recipes for successful anhydrobiosis in bdelloid rotifers

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

Mechanisms of plant desiccation tolerance.

Anhydrobiosis ("life without water") is the remarkable ability of certain organisms to survive almost total dehydration. It requires a coordinated series of events during dehydration that are associated with preventing oxidative damage and maintaining the native structure of macromolecules and membranes. The preferential hydration of macromolecules is essential when there is still bulk water present, but replacement by sugars becomes important upon further drying. Recent advances in our understanding of the mechanism of anhydrobiosis include the downregulation of metabolism, dehydration-induced partitioning of amphiphilic compounds into membranes and immobilization of the cytoplasm in a stable multicomponent glassy matrix.     

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.     

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.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Water and ion balance in the tissues of the dehydrated cockroach, Periplaneta americana

Cockroaches dehydrated for 8 days lost nearly 50% of their haemolymph volume and approx 25% of their tissue water. Haemolymph osmolality and sodium, potassium, and chloride concentrations in the haemolymph and tissue water were all regulated within narrow limits. It is confirmed that sodium and potassium ions are sequestered within the fat body during periods of dehydration. The increase in sodium and potassium ions in the fat body is shown to arise from ionic regulation of haemolymph and other tissues. During periods of rehydration, sodium and potassium concentrations decrease in the fat body and haemolymph volume and ionic concentrations return to near original levels. A small proportion of the surplus haemolymph chloride ions is shown to be associated with the cuticle during times of water deprivation.