Antarctica's terrestrial environment is a challenge to which very few animals have adapted. The largest, free-living animal to inhabit the continent year-round is a flightless midge, Belgica antarctica. Larval midges survive the lengthy austral winter encased in ice, and when the ice melts in summer, the larvae complete their 2-yr life cycle, and the wingless adults form mating aggregations while subjected to surprisingly high substrate temperatures. Here we report a dichotomy in survival strategies exploited by this insect at different stages of its life cycle. Larvae constitutively up-regulate their heat shock proteins (small hsp, hsp70, and hsp90) and maintain a high inherent tolerance to temperature stress. High or low temperature exposure does not further upregulate these genes nor does it further enhance thermotolerance. Such ''preemptive'' synthesis of hsps is sufficient to prevent irreversible protein aggregation in response to a variety of common environmental stresses. Conversely, adults exhibit no constitutive up-regulation of their hsps and have a lower intrinsic tolerance to high temperatures, but their hsps can be thermally activated, resulting in enhanced thermotolerance. Thus, the midge larvae, but not the adults, have adopted the unusual strategy of expressing hsps continuously, possibly to facilitate proper protein folding in a cold habitat that is more thermally stable than that of the adults but a habitat subjected frequently to freeze-thaw episodes and bouts of pH, anoxic, and osmotic stress.Antarctica ͉ cold tolerance ͉ protein aggregation ͉ temperature stress ͉ thermotolerance

Rapid cold-hardening (RCH) is well known to increase the tolerance of chilling or cold shock in a diverse array of invertebrate systems at both organismal and cellular levels. Here, we report a novel role for RCH by showing that RCH also increases freezing tolerance in an Antarctic midge, Belgica antarctica (Diptera, Chironomidae). The RCH response of B. antarctica was investigated under two distinct physiological states: summer acclimatized and cold acclimated. Summer-acclimatized larvae were less cold tolerant, as indicated by low survival following exposure to -10°C for 24·h; by contrast, nearly all coldacclimated larvae survived -10°C, and a significant number could survive -15°C. Cold-acclimated larvae had higher supercooling points than summer larvae. To evaluate the RCH response in summer-acclimatized midges, larvae and adults, maintained at 4°C, were transferred to -5°C for 1·h prior to exposures to -10, -15 or -20°C. RCH significantly increased survival of summer-acclimatized larvae frozen at -10°C for 1·h compared with larvae receiving no cold-hardening treatment, but adults, which live for only a week or so in the austral summer, lacked the capacity for RCH. In coldacclimated larvae, RCH significantly increased freeze tolerance to both -15 and -20°C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at -20°C for 24·h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance.

No abstract

SUMMARYDuring winter, larvae of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), must endure 7-8 months of continuous subzero temperatures, encasement in a matrix of soil and ice, and severely desiccating conditions. This environment, along with the fact that larvae possess a high rate of water loss and are extremely tolerant of desiccation, may promote the use of cryoprotective dehydration as a strategy for winter survival. This study investigates the capacity of larvae to resist inoculative freezing and undergo cryoprotective dehydration at subzero temperatures. Slow cooling to -3°C in an environment at equilibrium with the vapor pressure of ice reduced larval water content by ~40% and depressed the body fluid melting point more than threefold to -2.6°C. This melting point depression was the result of the concentration of existing solutes (i.e. loss of body water) and the de novo synthesis of osmolytes. By day 14 of the subzero exposure, larval survival was still >95%, suggesting larvae have the capacity to undergo cryoprotective dehydration. However, under natural conditions the use of cryoprotective dehydration may be constrained by inoculative freezing as result of the insectʼs intimate contact with environmental ice. During slow cooling within a substrate of frozen soil, the ability of larvae to resist inoculative freezing and undergo cryoprotective dehydration was dependent upon the moisture content of the soil. As detected by a reduction of larval water content, the percentage of larvae that resisted inoculative freezing increased with decreasing soil moisture. These results suggest that larvae of the Antarctic midge have the capacity to resist inoculative freezing at relatively low soil moisture contents and likely undergo cryoprotective dehydration when exposed to subzero temperatures during the polar winter.

No abstract

We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene's responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins (smHsp, Hsp70, Hsp90), antioxidants (superoxide dismutase, catalase), detoxification (metallothionein, cytochrome p450), genes involved in altering cell membranes (fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton (actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment.

No abstract

SUMMARYSurvival of freezing not only requires organisms to tolerate ice formation within their body, but also depends on the rapid redistribution of water and cryoprotective compounds between intra-and extracellular compartments. Aquaporins are transmembrane proteins that serve as the major pathway through which water and small uncharged solutes (e.g. glycerol) enter and leave the cell. Consequently, we examined freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, to determine whether aquaporins are present and if their presence promotes freeze tolerance of specific tissues. Immunoblotting with mammalian anti-AQP2, -AQP3 and -AQP4 revealed corresponding aquaporin homologues in E. solidaginis, whose patterns of expression varied depending on acclimation temperature and desiccation treatment. To examine the role of aquaporins in freeze tolerance, we froze fat body, midgut and salivary gland tissues in the presence and absence of mercuric chloride, an aquaporin inhibitor. Survival of fat body and midgut cells was significantly reduced when mercuric chloride was present. In contrast, survival of the salivary gland did not decrease when it was frozen with mercuric chloride. Overall, this study supports our hypothesis that naturally occurring aquaporins in E. solidaginis are regulated during desiccation and promote cell survival during freezing.