Both heat-shock and cold-shock influence trehalose metabolism in an entomopathogenic nematode

Heat-shock response is highly conserved in animals and microorganisms, and it results in the synthesis of heat-shock proteins. In yeast, heat-shock response has also been reported to induce trehalose accumulation. We explored the relationship between heat- (35 C) or cold-shock (1 and 10 C) and trehalose metabolism in the entomopathogenic nematode, Heterorhabditis bacteriophora. Because both heat- and cold-shocks may precede desiccation stress in natural soil environments, we hypothesized that nematodes may accumulate a general desiccation protectant, trehalose, under both situations. Indeed, both heat- and cold-shocks influenced trehalose accumulation and activities of enzymes of trehalose metabolism in H. bacteriophora. Trehalose increased by 5- and 6-fold in heat- and cold-shocked infective juveniles, respectively, within 3 hr of exposure, compared with the nematodes maintained at 25 C (culture temperature). The activity of trehalose-6-phosphate synthase (T6PS), an enzyme involved in the synthesis of trehalose, also significantly increased in both heat- and cold-shocked nematodes during the first 3 hr of exposure. Generally, the trehalose levels and activities of T6PS declined to their original levels within 3 hr when nematodes were transferred back to 25 C. In both heat- and cold-shocked nematodes, trehalase activity decreased significantly within the first 3 hr and generally returned to the original levels within 3 hr when these nematodes were transferred back to 25 C. The results demonstrate that the trehalose concentrations in H. bacteriophora are influenced by both heat- and cold-shocks and are regulated by the action of 2 trehalose-metabolizing enzymes, T6PS and trehalase. The accumulated trehalose may enhance survival of nematodes under both cold and warm conditions, but it may also provide simultaneous protection against desiccation that may result from subsequent evaporation or freezing. This is the first report of the relationship between trehalose metabolism and heat-shock for the Nematoda.     

Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori

Recently, it was confirmed that embryos derived from diapausing eggs of the silkworm, Bombyx mori, begin their development and reach larval maturity on mulberry leaves, when the naked eggs are cultured in vitro. In this study, we found that the method of embryo culture is useful for determining the physiological regulation of diapause. We show that the development of embryos derived from diapausing eggs was strongly inhibited by the addition of either sorbitol or trehalose to the culture medium. Furthermore, this inhibitory effect disappeared when the embryos were cultured in a control medium which did not contain either sorbitol or trehalose, indicating that the inhibitory reactions caused by both substances are reversible. The minimal effective dose of either sorbitol or trehalose was approximately 0.2 M, a value similar to the in vivo concentration of sorbitol in diapausing eggs (0.2 M). Glycerol, mannitol or glucose were moderately effective for inhibition. Sorbitol present in diapausing silkworm eggs does not appear to serve as an antifreeze, but as an strong arresting factor of embryonic development. Furthermore, these results show that a decrease in sorbitol releases the embryos from diapause at the termination of diapause.     

Desiccated doubled-haploid embryos obtained from microspore culture of barley cv. Igri

 Barley microspore-derived doubled-haploid embryos have been produced in vitro. The development of embryo desiccation technology will allow long-term storage, germplasm preservation and low delivery cost. Treatment of the microspore-derived embryos was essential to induce desiccation tolerance and to arrest further development and plant regeneration. At the concentrations used, a treatment with trehalose was more efficient than with sucrose, and mannitol was harmful to the embryos. Up to 80% of the desiccated embryos produced complete green plants when transferred to regeneration medium, by the application of a 0.6 m trehalose or a 10^–5 m abscisic acid treatment to the embryos in the culture induction medium. The morphology of these plants was similar to plants produced directly from non-desiccated embryos. Received: 28 September 1998 / Revision received: 27 November 1998 / Accepted: 5 January 1999     

The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause

An annual life cycle is characterized by growth, maturity, and reproduction condensed into a single, short season favourable to development, with production of embryos (seeds, cysts, or eggs) capable of surviving harsh conditions which juveniles or adults cannot tolerate. More typically associated with plants in desert environments, or temperate‐zone insects exposed to freezing winters, the evolution of an annual life cycle in vertebrates is fairly novel. Killifish, small sexually dimorphic fishes in the Order Cyprinodontiformes, have adapted to seasonally ephemeral water bodies across much of Africa and South America through the independent evolution of an annual life history. These annual killifish produce hardy desiccation‐resistant eggs that undergo diapause (developmental arrest) and remain buried in the soil for long periods when fish have perished due to the drying of their habitat. Killifish are found in aquatic habitats that span a continuum from permanent and stable to seasonal and variable, thus providing a useful system in which to piece together the evolutionary history of this life cycle using natural comparative variation. I first review adaptations for life in ephemeral aquatic environments in killifish, with particular emphasis on the evolution of embryonic diapause. I then bring together available evidence from a variety of approaches and provide a scenario for how this annual life cycle evolved. There are a number of features within Aplocheiloidei killifish including their inhabitation of marginal or edge aquatic habitat, their small size and rapid attainment of maturity, and egg properties that make them particularly well suited to the colonization of ephemeral waters.     

Diapausing pupae of the flesh fly Sarcophaga crassipalpis (Diptera: Sarcophagidae) are more resistant to inoculative freezing than non-diapausing pupae

The resistance of diapausing (overwintering) and non‐diapausing (summer) Sarcophaga crassipalpis (Diptera: Sarcophagidae) pupae to inoculative freezing was examined. Although both types of pupae resisted inoculative freezing after 24‐h submergence in water, diapausing pupae were overall significantly more resistant than non‐diapausing pupae. Exposing the thin pupal cuticle by removing the ends of the puparial case eliminated the capacity of both pupal types to resist inoculative freezing, indicating that resistance to inoculative freezing resides with the puparium. Pupae submerged in surfactant solution were significantly less resistant to inoculative freezing than those submerged in water. Furthermore, the puparial water content of pupae submerged in surfactant solution was significantly greater than that of puparia from pupae submerged in water. Surfactant may have promoted inoculative freezing by facilitating the spread of water over the surface of and into the puparium, thereby creating bridges between external ice and pupal body fluids. Extracting puparial surface lipids with chloroform/methanol (2 : 1, v:v) decreased the resistance of non‐diapausing pupae to inoculative freezing but did not significantly affect that of diapausing pupae. This finding indicates that the puparium of diapausing pupae contains protection against inoculative freezing separate from its surface lipids. This barrier may be important in protecting the freezing‐intolerant overwintering pupae against inoculative freezing within their soil hibernaculum.     

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H₂O₂) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.     

Protecting activity of desiccated enzymes

Protein‐based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation‐, freezing‐, and lyophilization‐induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.     

Variation in ephippial buoyancy in Daphnia pulicaria

1. Freshwater zooplankton often produce diapausing eggs to survive environmental stress. The diapausing eggs of Daphnia (Crustacea, Cladocera) are encased in an ephippium that either floats at the surface or sinks to the sediment. These two types of ephippia may represent different strategies between spatial (floaters) and temporal (sinkers) dispersal of offspring. 2. We observed floating and sinking characteristics of ephippia obtained from eight lakes. We then conducted an experiment with 26 Daphnia pulicaria clones obtained from six of these lakes and observed the production of buoyant and non‐buoyant ephippia under high and low food conditions. 3. Ephippia were more often non‐buoyant than buoyant both from females caught in nature and those reared in the laboratory. The experiment revealed that each clone was able to produce both types of ephippia, but that there was considerable among‐clone variation in the percentage of non‐buoyant ephippia produced. 4. We conclude that production of non‐buoyant versus buoyant ephippia may be driven by both genetic and environmental factors.     

Biochemical and genomic regulation of the trehalose cycle in yeast: review of observations and canonical model analysis

The physiological hallmark of heat-shock response in yeast is a rapid, enormous increase in the concentration of trehalose. Normally found in growing yeast cells and other organisms only as traces, trehalose becomes a crucial protector of proteins and membranes against a variety of stresses, including heat, cold, starvation, desiccation, osmotic or oxidative stress, and exposure to toxicants. Trehalose is produced from glucose 6-phosphate and uridine diphosphate glucose in a two-step process, and recycled to glucose by trehalases. Even though the trehalose cycle consists of only a few metabolites and enzymatic steps, its regulatory structure and operation are surprisingly complex. The article begins with a review of experimental observations on the regulation of the trehalose cycle in yeast and proposes a canonical model for its analysis. The first part of this analysis demonstrates the benefits of the various regulatory features by means of controlled comparisons with models of otherwise equivalent pathways lacking these features. The second part elucidates the significance of the expression pattern of the trehalose cycle genes in response to heat shock. Interestingly, the genes contributing to trehalose formation are up-regulated to very different degrees, and even the trehalose degrading trehalases show drastically increased activity during heat-shock response. Again using the method of controlled comparisons, the model provides rationale for the observed pattern of gene expression and reveals benefits of the counterintuitive trehalase up-regulation.     

Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.     

3D Multi-isotope Imaging Mass Spectrometry Reveals Penetration of (18)O-Trehalose in Mouse Sperm Nucleus

The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.     

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage,diapause III

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage, diapause III. The embryo, surrounded by a transparent vitelline envelope, is in the pre‐hatching stage. A prominent eye and part of the pigmented body and tail are apparent. Why annual fishes? Annual fishes (Order Cyprinodontiformes) are a special kind of teleost, found in Africa and South America, with developmental strategies closely related to their life cycle. These fishes inhabit temporary pools that undergo drying during summer, when all adults die. The embryos remain buried in the bottom mud and are resistant to desiccation. In the subsequent rainy season they hatch a few hours after the pool is flooded and a new reproductive cycle begins. This developmental pattern is characterized by the presence of a unique stage between cleavage and embryogenesis, dispersion‐aggregation of blastomeres and because the embryos show reversible developmental arrests (diapauses) at different stages. Annual fish embryos are transparent, large, hardy and easy to maintain in the laboratory. Adults show continuous production of eggs and juveniles reach sexual maturity a few weeks after hatching (an unusual condition in fishes). Their particular developmental features confer unique opportunities for research on cell behavior during early development, the effect of environmental factors on development, the regulation of diapauses and the mechanisms involved in sex determination, among others topics. Image provided by Nibia Berois, Universidad de la República, Montevideo, Uruguay.     

梨小食心虫滞育与非滞育幼虫过冷却能力与体内主要生化物质含量

梨小食心虫Grapholitha molesta(Busck)是我国北方果园中重要的果树害虫,现已成为泰安肥城桃园中为害最为严重的一种食心虫。本文主要测定了梨小食心虫滞育与非滞育幼虫的过冷却能力与体内主要生化物质的含量。研究结果表明;在26℃的温度条件下,滞育幼虫的过冷却点与结冰点均低于非滞育幼虫,但差异均不显著。滞育幼虫体内的含水量、糖原和蛋白质含量均极显著低于非滞育幼虫,但是滞育幼虫体内的总脂肪、甘油和海藻糖含量均极显著高于非滞育幼虫。研究结果说明,滞育幼虫在体内生理生化代谢做了调整,以应对不利环境条件的来临。     

Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

Comparisons of stress proteins and soluble carbohydrate in encysted embryos of Artemia franciscana and two species of Parartemia

We compared stress proteins (p26, artemin, hsp70) and alcohol-soluble carbohydrates (ASC) in cysts of Artemia franciscana and two as yet un-named species populations of Parartemia, the brine shrimp endemic to Australia. The small stress proteins and molecular chaperones, p26 and artemin, previously thought to be restricted to Artemia, and present in very large amounts in its encysted embryos (cysts), were also detected by western blotting in Parartemia cysts, even though roughly 85-100 million years have passed since these genera diverged. We interpret this finding as further evidence for the adaptive importance of these proteins in coping with the severe stresses these encysted embryos endure. As expected, hsp70 was present in all three groups of cysts, but apparently at somewhat lower concentrations in those of Parartemia. Based on measurements of ASC we propose that the disaccharide trehalose, critical for desiccation tolerance in many animal cells, has probably also been maintained in the metabolic repertoire of Parartemia whose cysts have well developed tolerance to severe desiccation.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

Combining endocytic and freezing‐induced trehalose uptake for cryopreservation of mammalian cells

Fibroblasts take up trehalose during freezing and thawing, which facilitates cryosurvival of the cells. The aim of this study was to investigate if trehalose uptake via fluid‐phase endocytosis prefreeze increases cryosurvival. To determine endocytic trehalose uptake in attached as well as suspended fibroblasts, intracellular trehalose concentrations were determined during incubation at 37°C using an enzymatically based trehalose assay. In addition, freezing‐induced trehalose uptake of extracellularly added trehalose was determined. Cryosurvival rates were determined via trypan blue staining. Intracellular trehalose contents of attached as well as suspended cells were found to increase linearly with time, consistent with fluid‐phase endocytosis. Furthermore, the intracellular trehalose concentration increased with increasing extracellular trehalose concentration (0–100 mM) in a linear fashion. Prefreeze loading of cells with trehalose via fluid‐phase endocytosis only showed increased cryosurvival rates at extracellular trehalose concentrations lower than 50 mM in the cryopreservation medium. To obtain satisfactory cryosurvival rates after endocytic preloading, extracellular trehalose is needed to prevent efflux of trehalose during freezing and thawing and for freezing‐induced trehalose uptake. At trehalose concentrations greater than 100 mM, cryosurvival rates were similar or slightly higher if cells were not loaded with trehalose prefreeze. Cells that were grown in the presence of trehalose showed a tendency to aggregate after harvesting. It is concluded that it is particularly freezing‐induced trehalose uptake that facilitates cryosurvival when trehalose is used as the sole cryoprotectant for cryopreservation of fibroblasts. Preloading with trehalose does not increase cryosurvival rates if trehalose is also added as extracellular protectant. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:229–230, 2017     

The influence of pool volume and summer desiccation on the production of the resting and dispersal stage in a Daphnia metapopulation

Dispersal is a key process in metapopulations, as migrants genetically connect populations and enable the colonization of empty habitat patches. Sub-populations may differ in their numerical contribution of migrants within a metapopulation. This has strong implications on evolutionary and ecological dynamics and has led to two different hypotheses about the Daphnia metapopulation studied here: the assessment by some authors is that sub-populations contribute equally to the production of migrants, while others have postulated long-lived core populations in large "mainland" habitat patches as the dominant source of migrants. We have studied the resting and dispersal stage (ephippium) in a natural Daphnia metapopulation and in mesocosm experiments, and tested for effects of habitat size and summer desiccation. We found that a 1000-fold increase in rock pool volume resulted on average in only in a 2.8-fold increase in ephippium production. Mesocosm experiments confirmed these results: a 1000-fold increase of the mesocosms' volume resulted in a 7.2-fold increase in ephippium production. Additionally, we showed that ephippium production did not depend on the initial population size. Thus, populations in small pools may contribute only marginal fewer potential migrants in the whole metapopulation than populations in large pools. In a second mesocosm experiment we found that summer desiccation, which is a typical occurrence in small pools, is not detrimental for the populations. Daphnia hatched out of ephippia that were produced earlier within the same season and built up viable populations again. The substantial production of ephippia by populations in small pools suggests that these populations might be important for both the dynamics and global stability of metapopulations.