Yuta Kawarasaki

Gene expression changes governing extreme dehydration tolerance in an Antarctic insect

Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world’s southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions.

Survival and energetic costs of repeated cold exposure in the Antarctic midge, Belgica antarctica : a comparison between frozen and supercooled larvae

SUMMARY In this study, we examined the effects of repeated cold exposure (RCE) on the survival, energy content and stress protein expression of larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae). Additionally, we compared results between larvae that were frozen at –5°C in the presence of water during RCE and those that were supercooled at –5°C in a dry environment. Although >95% of larvae survived a single 12 h bout of freezing at –5°C, after five cycles of RCE survival of frozen larvae dropped below 70%. Meanwhile, the survival of control and supercooled larvae was unchanged, remaining around 90% for the duration of the study. At the tissue level, frozen larvae had higher rates of cell mortality in the midgut than control and supercooled larvae. Furthermore, larvae that were frozen during RCE experienced a dramatic reduction in energy reserves; after five cycles, frozen larvae had 25% less lipid, 30% less glycogen and nearly 40% less trehalose than supercooled larvae. Finally, larvae that were frozen during RCE had higher expression of hsp70 than those that were supercooled, indicating a higher degree of protein damage in the frozen group. Results were similar between larvae that had accumulated 60 h of freezing at –5°C over five cycles of RCE and those that were frozen continuously for 60 h, suggesting that the total time spent frozen determines the physiological response. Our results suggest that it is preferable, both from a survival and energetic standpoint, for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain unfrozen during RCE.

Functional characterization of an aquaporin in the Antarctic midge Belgica antarctica

Aquaporins (AQPs) are water channel proteins facilitating movement of water across the cell membrane. Recent insect studies clearly demonstrate that AQPs are indispensable for cellular water management under normal conditions as well as under stress conditions including dehydration and cold. In the present study we cloned an AQP cDNA from the Antarctic midge Belgica antarctica (Diptera, Chironomidae) and investigated water transport activity of the AQP protein and transcriptional regulation of the gene in response to dehydration and rehydration. The nucleotide sequence and deduced amino acid sequence of the cDNA showed high similarity to AQPs in other insects and also showed characteristic features of orthodox AQPs. Phylogenetic analysis revealed that Belgica AQP is a homolog of dehydration-inducible AQP of another chironomid, Polypedilum vanderplanki. A swelling assay using a Xenopus oocyte expression system verified that Belgica AQP is capable of transporting water, but not glycerol or urea. The AQP mRNA was detected in various organs under non-stressed conditions, suggesting that this AQP plays a fundamental role in cell physiology. In contrast to our expectation, AQP transcriptional expression was not affected by either dehydration or rehydration.

Diatom assemblages promote ice formation in large lakes.

We present evidence for the directed formation of ice by planktonic communities dominated by filamentous diatoms sampled from the ice-covered Laurentian Great Lakes. We hypothesize that ice formation promotes attachment of these non-motile phytoplankton to overlying ice, thereby maintaining a favorable position for the diatoms in the photic zone. However, it is unclear whether the diatoms themselves are responsible for ice nucleation. Scanning electron microscopy revealed associations of bacterial epiphytes with the dominant diatoms of the phytoplankton assemblage, and bacteria isolated from the phytoplankton showed elevated temperatures of crystallization (Tc) as high as −3 °C. Ice nucleation-active bacteria were identified as belonging to the genus Pseudomonas, but we could not demonstrate that they were sufficiently abundant to incite the observed freezing. Regardless of the source of ice nucleation activity, the resulting production of frazil ice may provide a means for the diatoms to be recruited to the overlying lake ice, thereby increasing their fitness. Bacterial epiphytes are likewise expected to benefit from their association with the diatoms as recipients of organic carbon excreted by their hosts. This novel mechanism illuminates a previously undescribed stage of the life cycle of the meroplanktonic diatoms that bloom in Lake Erie and other Great Lakes during winter and offers a model relevant to aquatic ecosystems having seasonal ice cover around the world.

Expression of genes involved in energy mobilization and osmoprotectant synthesis during thermal and dehydration stress in the Antarctic midge, Belgica antarctica

The Antarctic midge, Belgica antarctica, experiences sub-zero temperatures and desiccating conditions for much of the year, and in response to these environmental insults, larvae undergo rapid shifts in metabolism, mobilizing carbohydrate energy reserves to promote synthesis of low-molecular-mass osmoprotectants. In this study, we measured the expression of 11 metabolic genes in response to thermal and dehydration stress. During both heat and cold stress, we observed upregulation of phosphoenolpyruvate carboxykinase (pepck) and glycogen phosphorylase (gp) to support rapid glucose mobilization. In contrast, there was a general downregulation of pathways related to polyol, trehalose, and proline synthesis during both high- and low-temperature stress. Pepck was likewise upregulated in response to different types of dehydration stress; however, for many of the other genes, expression patterns depended on the nature of dehydration stress. Following fast dehydration, expression patterns were similar to those observed during thermal stress, i.e., upregulation of gp accompanied by downregulation of trehalose and proline synthetic genes. In contrast, gradual, prolonged dehydration (both at a constant temperature and in conjunction with chilling) promoted marked upregulation of genes responsible for trehalose and proline synthesis. On the whole, our data agree with known metabolic adaptations to stress in B. antarctica, although a few discrepancies between gene expression patterns and downstream metabolite contents point to fluxes that are not controlled at the level of transcription.

Energetic consequences of repeated and prolonged dehydration in the Antarctic midge, Belgica antarctica.

Larvae of the Antarctic midge, Belgica antarctica, routinely face periods of limited water availability in their natural environments on the Antarctic Peninsula. As a result, B. antarctica is one of the most dehydration-tolerant insects studied, surviving up to 70% loss of its body water. While previous studies have characterized the physiological effects of a single bout of dehydration, in nature larvae are likely to experience multiple bouts of dehydration throughout their lifetime. Thus, we examined the physiological consequences of repeated dehydration and compared results to larvae exposed to a single, prolonged period of dehydration. For the repeated dehydration experiment, larvae were exposed to 1-5 cycles of 24 h dehydration at 75% RH followed by 24 h rehydration. Each bout of dehydration resulted in 30-40% loss of body water, with a concomitant 2- to 3-fold increase in body fluid osmolality. While nearly 100% of larvae survived a single bout of dehydration, <65% of larvae survived five such cycles. Larvae subjected to multiple bouts of dehydration also experienced severe depletion of carbohydrate energy reserves; glycogen and trehalose content decreased with each successive cycle, with larvae losing 89% and 48% of their glycogen and trehalose, respectively, after five cycles of dehydration/rehydration. Larvae exposed to prolonged dehydration (99% RH for 10d) had 26% less water, 43% less glycogen, and 27% less lipid content than controls, but did not experience any mortality. Thus, both repeated and prolonged dehydration results in substantial energetic costs that are likely to negatively impact fitness.

Alternative overwintering strategies in an Antarctic midge: freezing vs. cryoprotective dehydration

Summary 1. Cryoprotective dehydration is a relatively new addition to our understanding of freeze avoidance strategies employed by polar invertebrates. Although the underlying cellular processes associated with this strategy are similar to those of freeze tolerance, little is known about potential trade-offs of overwintering in these physiological states. 2. This study compares the potential of larvae of the terrestrial midge Belgica antarctica (Diptera, Chironomidae) to overwinter in these two states. As the only insect with the capacity to tolerate freezing and to cryoprotectively dehydrate, it is an ideal model to compare the benefits and costs of these strategies. 3. Compared to summer-acclimated larvae, supercooling points of winter-acclimatized larvae were significantly depressed and were lower than observed minima for their microhabitat temperatures. Thus, if larvae avoid inoculative freezing from environmental ice, they could remain unfrozen via cryoprotective dehydration. 4. Both frozen and cryoprotectively dehydrated larvae readily survived a 32-day exposure to simulated overwintering temperatures. 5. Freezing had little effect on larval body water content and haemolymph osmolality. In contrast, cryoprotective dehydration at 5 °C resulted in a progressive loss of body water, ultimately reducing larval water content by 62%. This level of dehydration corresponded to an increase in haemolymph osmolality to c. 2750 mOsm kg 1 , depressing the haemolymph melting point to 49 °C. 6. Freezing and cryoprotective dehydration resulted in distinctly different patterns of glycogen breakdown. Whereas the glycogen content decreased only during the first 14 days in cryoprotectively dehydrated larvae, frozen larvae continued to break down glycogen throughout the 32-day subzero exposure. However, after recovery at 0 °C for 5 days, glycogen levels were similar in these two groups, as were the levels of total lipids. 7. Our results indicate that freezing and cryoprotective dehydration are both effective in promoting winter survival of larvae, with surprisingly few differences in energetic costs. Whether larvae freeze or become cryoprotectively dehydrated ultimately depends on the hydric condition of their microhabitat. The physiological flexibility of B. antarctica to overwinter in these alternative states likely contributed to its range distribution that extends further south than any other free-living insect.

The protective effect of rapid cold-hardening develops more quickly in frozen versus supercooled larvae of the Antarctic midge, Belgica antarctica

SUMMARY During the austral summer, larvae of the terrestrial midge Belgica antarctica (Diptera: Chironomidae) experience highly variable and often unpredictable thermal conditions. In addition to remaining freeze tolerant year-round, larvae are capable of swiftly increasing their cold tolerance through the rapid cold-hardening (RCH) response. The present study compared the induction of RCH in frozen versus supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater level of cryoprotection in frozen versus supercooled larvae. Furthermore, RCH in frozen larvae could be induced at temperatures as low as −12°C, which is the lowest temperature reported to induce RCH. Remarkably, as little as 15 min at −5°C significantly enhanced larval cold tolerance. Not only is protection from RCH acquired swiftly, but it is also quickly lost after thawing for 2 h at 2°C. Because the primary difference between frozen and supercooled larvae is cellular dehydration caused by freeze concentration of body fluids, we also compared the effects of acclimation in dehydrated versus frozen larvae. Because slow dehydration without chilling significantly increased larval survival to a subsequent cold exposure, we hypothesize that cellular dehydration caused by freeze concentration promotes the rapid acquisition of cold tolerance in frozen larvae.