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.     

The Genetic Improvement of Entomopathogenic Nematodes and Their Symbiont Bacteria: Phenotypic Targets, Genetic Limitations and an Assessment of Possible Hazards

The methodologies of classical genetics and genetic engineering can be used for the genetic improvement of entomopathogenic nematodes (EPNs) and their symbiont bacteria. Many of the complex behavioural and physiological traits which are targets for genetic improvement are likely to be controlled polygenically, thus selective breeding for improvements to these traits would be appropriate. Much basic research needs to be carried out before researchers will be able to effect improvements to EPNs and their symbionts by genetic engineering. There is a lack of basic information on the genetics and biochemistry of the characteristics that might be altered by transgenic methods in EPNs, and their bacteria, and existing transformation protocols need to be made more effective.     

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.     

Effect of anaerobiosis and anhydrobiosis on the extent of glycolytic enzyme binding in Artermia embryos

The effect of anaerobiosis and anhydrobiosis on the extent of binding of glycolytic enzymes to the particulate fraction of the cell was studied in Artemia salina embryos. During control aerobic development, trehalase, phosphofructokinase and pyruvate kinase showed an increase in the percentage associated with the particulate fraction which is consistent with the carbohydrate-based metabolism of Artemia embryos. However, anaerobiosis resulted in decreased enzyme binding for six glycolytic enzymes; hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase were the exceptions. Decreased enzyme binding was also observed after exposure to dehydrating conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms in Artemia embryos. This reversible interaction of glycolytic enzymes with structural proteins may account for part of the metabolic arrest observed during anaerobic dormancy and anhydrobiosis.Abbreviation pH_i intracellular concentration of H⁺ ions     

A LEA model peptide protects the function of a red fluorescent protein in the dry state

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide.     

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.     

On dormancy strategies in tardigrades

In this review we analyze the dormancy strategies of metazoans inhabiting “hostile to life” habitats, which have a strong impact on their ecology and in particular on the traits of their life history. Tardigrades are here considered a model animal, being aquatic organisms colonizing terrestrial habitats. Tardigrades evolved a large variety of dormant stages that can be ascribed to diapause (encystment, cyclomorphosis, resting eggs) and cryptobiosis (anhydrobiosis, cryobiosis, anoxibiosis). In tardigrades, diapause and cryptobiosis can occur separately or simultaneously, consequently the adoption of one adaptive strategy is not necessarily an alternative to the adoption of the other. Encystment and cyclomorphosis are characterized by seasonal cyclic changes in morphology and physiology of the animals. They share several common features and their evolution is strictly linked to the molting process. A bet-hedging strategy with different patterns of egg hatching time has been observed in a tardigrade species. Four categories of eggs have been identified: subitaneous, delayed-hatching, abortive and diapause resting eggs, which needs a stimulus to hatch (rehydration after a period of desiccation). Cryptobiotic tardigrades are able to withstand desiccation (anhydrobiosis) and freezing (cryobiosis) at any stage of their life-cycle. This ability involves a complex array of factors working at molecular (bioprotectans), physiological and structural levels. Animal survival and the accumulation of molecular damage are related to the time spent in the cryptobiotic state, to the abiotic parameters during the cryptobiotic state, and to the conditions during initial and final phases of the process. Cryptobiosis evolved independently at least two times in tardigrades, in eutardigrades and in echiniscoids. Within each evolutionary line, the absence of cryptobiotic abilities is more related to selective pressures to local habitat adaptation than to phylogenetic relationships. The selective advantages of cryptobiosis (e.g. persistency in “hostile to life” habitats, reduction of competitors, parasites and predators, escaping in time from stressful conditions) could explain the high tardigrade species diversity and number of specimens found in habitats that dry out compared to freshwater habitats.     

Subcellular integrities in Chroococcidiopsis sp. CCMEE 029 survivors after prolonged desiccation revealed by molecular probes and genome stability assays

Desiccation-tolerant cells must either protect their cellular components from desiccation-induced damage and/or repair it upon rewetting. Subcellular damage to the anhydrobiotic cyanobacterium Chroococcidiopsis sp. CCMEE 029 stored in the desiccated state for 4 years was evaluated at the single-cell level using fluorescent DNA strand breakage labelling, membrane integrity and potential related molecular probes, oxidant-sensing fluorochrome and redox dye. Covalent modifications of dried genomes were assessed by testing their suitability as PCR template. Results suggest that desiccation survivors avoid/and or limit genome fragmentation and genome covalent modifications, preserve intact plasma membranes and phycobiliprotein autofluorescence, exhibit spatially-reduced ROS accumulation and dehydrogenase activity upon rewetting. Damaged cells undergo genome fragmentation, loss of plasma membrane potential and integrity, phycobiliprotein bleaching, whole-cell ROS accumulation and lack respiratory activity upon rewetting. The co-occurrence of live and dead cells within dried aggregates of Chroococcidiopsis confirms that desiccation resistance is not a simple process and that subtle modifications to the cellular milieu are required to dry without dying. It rises also intriguing questions about the triggers of dead cells in response to drying. The capability of desiccation survivors to avoid and/or reduce subcellular damage, shows that protection mechanisms are relevant in the desiccation tolerance of this cyanobacterium. This paper is dedicated to the memory of E. Imre Friedmann and his wife Roseli, who pioneered researches on Chroococcidiopsis and life in desert environments.     

Physiological changes leading to anhydrobiosis improve radiation tolerance in Polypedilum vanderplanki larvae

High tolerance against various extreme environments exhibited by some anhydrobionts might be due to being almost completely desiccated, a state where little or no chemical reactions occur. We have shown that anhydrobiotic larvae of Polypedilum vanderplanki have higher tolerance against both high- and low-linear energy transfer (LET) radiation than hydrated larvae. It is of great interest to know how the desiccating larvae gain radiation tolerance. We therefore examined effects of high-LET radiation on four kinds of larvae: (1) normal hydrated (intact) larva, (2) intermediates between the anhydrobiotic and normal hydrated state, (3) almost completely dehydrated (anhydrobiotic) larvae, and (4) immediately rehydrated larvae that are assumed to have a similar molecular profile to anhydrobiotic larvae. The intermediates and immediately rehydrated larvae survived longer after high-LET radiation than intact larvae, indicating that radiation tolerance could be enhanced even in hydrated larvae. Physiological changes toward anhydrobiosis, e.g. accumulation of protectants or increasing damage repair capacity, correlate with improved radiation tolerance in hydrated larvae. In addition, almost complete desiccation further enhanced radiation tolerance, possibly in a different way from the hydrated larvae.