Is rapid cold-hardening an aerobic process? Characterization of changes in metabolic activity during its induction and effects of anoxia in flesh fly.

Rapid cold-hardening (RCH) is a type of phenotypic plasticity that promotes a swift improvement of cold tolerance in insects. A brief exposure to mild cold dramatically increases insect survival to a subsequent cold exposure that would be lethal otherwise. In adult male flesh fly, Sarcophaga bullata, as little as 15 min at 5 °C significantly improved organismal survival at -7°C from 0 to 66.7 ± 11.1%. In this study, we investigated whether this RCH response is an aerobic process in S. bullata by characterizing changes in metabolic activity during its induction. At the level of whole organism, CO2 production continued at a level above our detection limit, and a relatively greater rate was observed during the early phase before it stabilized after ~1 h of the RCH induction. Similarly, in isolated flight muscle tissues, those maintained at 5 °C for 10 min exhibited significantly greater rates of oxygen consumption, compared to those maintained at 5 °C for 1 h (2.82 ± 0.29 vs. 1.36 ± 0.22 µl O2 mg-1 DM h-1). When these tissues were exposed to LaCl3, a treatment that should inhibit RCH ex vivo, oxygen consumption rates of the muscles were reduced significantly to a level similar to those that had been maintained at 5 °C for 1 h. Interestingly, however, the RCH response was still evident among individuals exposed to chilling under anoxia. Compared to those exposed to anoxia for 30 min only at 25 °C, flies exposed to 5 °C for 2 h under anoxia following the initial exposure exhibited a significantly greater level of cold tolerance at -7.5 °C (41.7 ± 7.1 vs. 91.8 ± 3.9%). Our results suggest that while relatively greater rates of metabolic activity are associated with the early phase of the RCH induction, it can proceed under the anoxic condition, thereby suggesting its independence to aerobic respiration.

Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica.

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica.

Osmoregulation and salinity tolerance in the Antarctic midge, Belgica antarctica: seawater exposure confers enhanced tolerance to freezing and dehydration.

Summer storms along the Antarctic Peninsula can cause microhabitats of the terrestrial midge Belgica antarctica to become periodically inundated with seawater from tidal spray. As microhabitats dry, larvae may be exposed to increasing concentrations of seawater. Alternatively, as a result of melting snow or following rain, larvae may be immersed in freshwater for extended periods. The present study assessed the tolerance and physiological response of B. antarctica larvae to salinity exposure, and examined the effect of seawater acclimation on their subsequent tolerance of freezing, dehydration and heat shock. Midge larvae tolerated extended exposure to hyperosmotic seawater; nearly 50% of larvae survived a 10-day exposure to 1000 mOsm kg(-1) seawater and approximately 25% of larvae survived 6 days in 2000 mOsm kg(-1) seawater. Exposure to seawater drastically reduced larval body water content and increased hemolymph osmolality. By contrast, immersion in freshwater did not affect water content or hemolymph osmolality. Hyperosmotic seawater exposure, and the accompanying osmotic dehydration, resulted in a significant correlation between the rate of oxygen consumption and larval water content and induced the de novo synthesis and accumulation of several organic osmolytes. A 3-day exposure of larvae to hyperosmotic seawater increased freezing tolerance relative to freshwater-acclimated larvae. Even after rehydration, the freezing survival of larvae acclimated to seawater was greater than freshwater-acclimated larvae. Additionally, seawater exposure increased the subsequent tolerance of larvae to dehydration. Our results further illustrate the similarities between these related, yet distinct, forms of osmotic stress and add to the suite of physiological responses used by larvae to enhance survival in the harsh and unpredictable Antarctic environment.

Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration.

Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

Dehydration, rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, Belgica antarctica.

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.

Dehydration-induced cross tolerance of Belgica antarctica larvae to cold and heat is facilitated by trehalose accumulation.

Larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), are frequently exposed to dehydrating conditions on the Antarctic Peninsula. In this study, we examined how rates and levels of dehydration alter heat and cold tolerance and how these relate to levels of trehalose within the insect. When dehydrated, larvae tolerated cold and heat stress more effectively, although resistance to cold was more pronounced than heat resistance. Slow dehydration was more effective than rapid dehydration in increasing temperature tolerance. Severe dehydration (50% reduction in water content) caused a much greater increase in temperature tolerance than did mild dehydration (e.g. 10% water loss). Larvae severely dehydrated at a slow rate (98% RH) were more temperature tolerant than those dehydrated quickly (0 or 75% RH). These results indicate that the slower dehydration rate allows the larvae to more effectively respond to reduced water levels and that physiological adjustments to desiccation provide cross tolerance to cold and heat. Levels of trehalose increased during dehydration and are likely a major factor increasing subsequent cold and heat resistance. This hypothesis was also supported by experimental results showing that injection of trehalose enhanced resistance to temperature stress and dehydration. We conclude that changes in temperature tolerance in B. antarctica are linked to the rate and severity of dehydration and that trehalose elevation is a probable mechanism enhancing this form of cross tolerance.

Desiccation tolerance and drought acclimation in the Antarctic collembolan Cryptopygus antarcticus.

The availability of water is recognized as the most important determinant of the distribution and activity of terrestrial organisms within the maritime Antarctic. Within this environment, arthropods may be challenged by drought stress during both the austral summer, due to increased temperature, wind, insolation, and extended periods of reduced precipitation, and the winter, as a result of vapor pressure gradients between the surrounding icy environment and the body fluids. The purpose of the present study was to assess the desiccation tolerance of the Antarctic springtail, Cryptopygus antarcticus, under ecologically-relevant conditions characteristic of both summer and winter along the Antarctic Peninsula. In addition, this study examined the physiological changes and effects of mild drought acclimation on the subsequent desiccation tolerance of C. antarcticus. The collembolans possessed little resistance to water loss under dry air, as the rate of water loss was >20% h(-1) at 0% relative humidity (RH) and 4 degrees C. Even under ecologically-relevant desiccating conditions, the springtails lost water at all relative humidities below saturation (100% RH). However, slow dehydration at high RH dramatically increased the desiccation tolerance of C. antarcticus, as the springtails tolerated a greater loss of body water. Relative to animals maintained at 100% RH, a mild drought acclimation at 98.2% RH significantly increased subsequent desiccation tolerance. Drought acclimation was accompanied by the synthesis and accumulation of several sugars and polyols that could function to stabilize membranes and proteins during dehydration. Drought acclimation may permit C. antarcticus to maintain activity and thereby allow sufficient time to utilize behavioral strategies to reduce water loss during periods of reduced moisture availability. The springtails were also susceptible to desiccation at subzero temperatures in equilibrium with the vapor pressure of ice; they lost approximately 40% of their total body water over 28 d when cooled to -3.0 degrees C. The concentration of solutes in the remaining body fluids as a result of dehydration, together with the synthesis of several osmolytes, dramatically increased the body fluid osmotic pressure. This increase corresponded to a depression of the melting point to approximately -2.2 degrees C, and may therefore allow C. antarcticus to survive much of the Antarctic winter in a cryoprotectively dehydrated state.

High resistance to oxidative damage in the Antarctic midge Belgica antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins.

Intense ultraviolet radiation, coupled with frequent bouts of freezing-thawing and anoxia, have the potential to generate high levels of oxidative stress in Antarctic organisms. In this study, we examined mechanisms used by the Antarctic midge, Belgica antarctica, to counter oxidative stress. We cloned genes encoding two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), and showed that SOD mRNA was expressed continuously and at very high levels in larvae, but not in adults, while Cat mRNA was expressed in both larvae and adults but at a somewhat reduced level. SOD mRNA was expressed at even higher levels in larvae that were exposed to direct sunlight. Catalase, a small heat shock protein, Hsp70 and Hsp90 mRNAs were also strongly upregulated in response to sunlight. Total antioxidant capacity of the adults was higher than that of the larvae, but levels in both stages of the midge were much higher than observed in a freeze-tolerant, temperate zone insect, the gall fly Eurosta solidaginis. Assays to measure oxidative damage (lipid peroxidation TBARS and carbonyl proteins) demonstrated that the Antarctic midge is highly resistant to oxidative stress.

Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium.

In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH in B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at -5 degrees C significantly increased organismal freezing tolerance at both -15 degrees C and -20 degrees C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20 degrees C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay, thus indicating that cold-sensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at -5 degrees C and those frozen at -5 degrees C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response.

Aquaporins play a role in desiccation and freeze tolerance in larvae of the goldenrod gall fly, Eurosta solidaginis.

Survival 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.

Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge, Belgica antarctica.

The midge, Belgica antarctica Jacobs, is subjected to numerous environmental stressors during its 2-year life cycle on the Antarctic Peninsula, and in response it has evolved a suite of behavioral, physiological, and life-cycle modifications to counter these stressors, but thus far only a limited number of biochemical adaptations have been identified. In this study, we use a metabolomics approach to obtain a broad overview of changes in energy metabolism, amino acids, and polyols in response to three of the midge's major stresses: heat, freezing, and desiccation. Using GC-MS analysis, a total of 75 compounds were identified. Desiccation (50% water loss) elicited the greatest physiological response (as determined by principal components analysis) when compared to untreated controls, with many elevated metabolites from pathways of central carbohydrate metabolism and a decrease in free amino acids. When larvae were frozen (6h at -10 degrees C), alanine and aspartate increased as well as urea. Freezing also increased three polyols (glycerol, mannitol, erythritol), while desiccation increased only two polyols (glycerol, erythritol). Heating the midges for 1h at 30 degrees C elevated alpha-ketoglutarate and putrescine while suppressing glycerol, glucose, and serine levels. Freezing and desiccation elicited elevation of four shared metabolites, whereas no shared metabolites were elevated by heat. All three treatments resulted in a reduction in serine, potentially identifying this amino acid as a marker for stress in this species. A number of metabolic changes, especially those in the sugar and polyol pools, are adaptations that have potential to enhance survival during both cold and desiccation.

Cryoprotective dehydration and the resistance to inoculative freezing in the Antarctic midge, Belgica antarctica.

During 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 degrees C in an environment at equilibrium with the vapor pressure of ice reduced larval water content by approximately 40% and depressed the body fluid melting point more than threefold to -2.6 degrees 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 insect's 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.

Mechanisms to reduce dehydration stress in larvae of the Antarctic midge, Belgica antarctica.

The Antarctic midge, Belgica antarctica, is exposed to frequent periods of dehydration during its prolonged larval development in the cold and dry Antarctic environment. In this study, we determined the water requirements of the larvae and the mechanisms it exploits to reduce the stress of drying. Larvae lost water at an exceptionally high rate (>10%/h) and tolerated losing a high portion (>70%) of their water content. Larvae were unable to absorb water from subsaturated water vapor (< or = 0.98 a(v)) to replenish their water stores, thus this midge relies exclusively on the intake of liquid water to increase its pool of body water and maintain water balance. To reduce dehydration stress, the midge employed a variety of mechanisms. Behaviorally, the larvae suppressed water loss by clustering. In response to slow dehydration, glycerol concentration increased 2-fold and trehalose concentration increased 3-fold, responses that are known to decrease the rate of water loss and increase dehydration tolerance. No changes in the mass of cuticular lipids occurred in response to desiccation, but the observed shift to longer hydrocarbons likely contributes to reduced water loss as the larvae dehydrate. As the larvae dehydrated, their oxygen consumption rate dropped, resulting in a reduction of water loss by respiration. Lastly, one bout of slow dehydration also enhanced the larva's ability to survive subsequent dehydration, suggesting that the larvae have the capacity for drought acclimation. Thus, these hydrophilic midge larvae prevent dehydration by multiple mechanisms that collectively reduce the water loss rate and increase dehydration tolerance.

Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect.

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 up-regulate 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.

The effects of temperature and inter-individual variation on the locomotor performance of juvenile turtles.

The effects of temperature on aquatic and terrestrial locomotor performance, including measures of burst speed, endurance, and righting response, the inter-individual correlation between measures of locomotor performance, and the temporal repeatability of performance were assessed in juvenile western painted turtles, Chrysemys picta bellii. Locomotor performance increased as temperature increased, with Q(10) values ranging from 1.33 to 1.98 for burst speed and 2.28 to 2.76 for endurance measures. Righting response performance also increased with temperature. Aquatic and terrestrial measures of locomotor performance were highly correlated; however, righting response was not correlated with any other measure of performance. Measures of terrestrial locomotor performance were highly repeatable over the entire 30-week study period, whereas aquatic locomotor performance was only repeatable through week 12. The righting response was repeatable over a 6-week study period. Both the interindividual variation and temperature effects on locomotor performance likely influences the survival of turtles, especially juveniles, by affecting the length of time turtles are exposed to potential predators and their ability to escape.

Rapid cold-hardening increases the freezing tolerance of the Antarctic midge Belgica antarctica.

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 degrees C for 24 h; by contrast, nearly all cold-acclimated larvae survived -10 degrees C, and a significant number could survive -15 degrees 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 degrees C, were transferred to -5 degrees C for 1 h prior to exposures to -10, -15 or -20 degrees C. RCH significantly increased survival of summer-acclimatized larvae frozen at -10 degrees 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 cold-acclimated larvae, RCH significantly increased freeze tolerance to both -15 and -20 degrees C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at -20 degrees C for 24 h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance.