Seasonal effects of dehydration on glucose mobilization in freeze-tolerant chorus frogs (Pseudacris triseriata) and freeze-intolerant toads (Bufo woodhousii and B. cognatus)

It has been hypothesized that freeze-tolerance in anurans evolved from a predisposition for dehydration tolerance. To test this hypothesis, we dehydrated summer/fall-collected and winter acclimated freeze-tolerant chorus frogs and dehydration-tolerant, but freeze-intolerant, Woodhouse's and Great Plains toads to 25% and 50% body water loss (BWL). Following treatments, we measured glucose, glycogen, and glycogen phosphorylase and glycogen synthetase (summer/fall only) activities in liver and leg muscle. Hepatic glucose levels were not significantly altered by dehydration in either summer/fall-collected frogs or toads. Conversely, winter acclimated frogs did show an increment (2.9-fold) in hepatic glucose with dehydration, accompanied by a reduction in hepatic glycogen levels. Winter acclimated toads did not mobilize hepatic glucose in response to dehydration. Further, hepatic glycogen and phosphorylase activities did not vary in any consistent manner with dehydration in winter toads. Mean leg muscle glucose values were elevated at 50% BWL relative to other treatments, significantly so compared to 25% BWL for summer/fall-collected frogs. The pattern of hepatic glucose mobilization with dehydration in winter frogs is consistent with that in other freeze-tolerant frog species, and provides additional support for the hypothesis that freezing tolerance evolved from a capacity for dehydration tolerance. However, the lack of hepatic glucose mobilization in response to dehydration in fall frogs suggests that a seasonal component to dehydration-induced regulation of glucose metabolism exists in chorus frogs. Furthermore, the absence of a dehydration-induced mobilization of hepatic glucose at both seasons in toads suggests that this dehydration response is not universal for terrestrial anurans.     

Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration.

Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.     

Second messenger and cAMP-dependent protein kinase responses to dehydration and anoxia stresses in frogs

The effects of whole body dehydration (up to 40% of total body water lost) or anoxia exposure (up to 2 days under N₂ gas) at 5 °C on tissue levels of adenosine 3′–5′ cyclic monophosphate (cAMP) and the percentage of cAMP-dependent protein kinase present as the free catalytic subunit (PKAc), as well as the levels of the protein kinase C (PKC) second messenger, inositol 1,4,5-trisphosphate (IP₃), were assessed in two anurans, the freeze-tolerant wood frog, Rana sylvatica, and the freeze-intolerant leopard frog, Rana pipiens. Dehydration of wood frogs resulted in a rapid elevation of liver cAMP and PKAc; cAMP was 3.4-fold greater than control values in animals that had lost 5% of total body water, whereas PKAc was elevated threefold in 20% dehydrated frogs. These results indicate protein kinase A mediation of the liver glycogenolysis and hyperglycemia that is induced by dehydration in this species. Skeletal muscle PKAc content also rose with dehydration but neither cAMP nor PKAc was affected by dehydration in leopard frog tissues. Anoxia exposure had different effects on signal transduction systems. PKAc was elevated after 1 h anoxia in R. sylvatica brain and was sustained over time but the enzyme was unaffected in other organs; by contrast, R. pipiens showed variable responses by PKAc to anoxia in three organs. Both species showed rapid (within 30 min) and large (3 to 7.8-fold) increases in IP₃ in liver of anoxic frogs that decreased slowly with continued anoxia. IP₃ also increased quickly in heart of anoxia-exposed wood frogs. This suggests that PKC may mediate various metabolic adjustments that promote hypoxia/anoxia resistance such as coordinating metabolic rate depression. A progressive rise in liver IP₃ during dehydration in wood frogs (reaching fourfold higher than controls in 40% dehydrated animals) may also mediate similar hypoxia resistance adaptations under this stress since anurans experience progressive hypoxia due to increased blood viscosity when water loss reaches high values. The patterns of second messenger and PKAc changes in wood frog liver during dehydration closely parallel the changes seen in these same parameters during natural freezing suggesting that the freeze tolerance of selected terrestrially hibernating anurans may have evolved out of various anuran mechanisms of dehydration resistance. Accepted: 2 January 1997     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

The regulation of heat shock proteins in response to dehydration in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45–1.65-fold) in the kidneys and lungs, and HSP40 (1.39–2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43–0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39–1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20–1.36-fold), but decreased in skeletal muscle (0.27–0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39–0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.     

Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog

Summary Wood frogs,Rana sylvatica, were sampled after freezing at –4°C (a short time course from 2 to 70 min after the appearance of the freezing exotherm) and thawing (20 h at 3°C after 70 min of freezing) and the regulation of liver glycolysis with respect to cryoprotectant glucose synthesis was examined. Within 5 min of the initiation of freezing, cryoprotectant concentrations in blood and liver had begun to increase. This was correlated with a rapid rise in the levels of hexose monophosphates in liver, including a 2.5 fold increase in glucose-6-P and 10 fold rise in fructose-6-P contents within the first 5 min post-exotherm. Contents of fructose-1,6-P₂, fructose-2,6-P₂, triose phosphates, P-enolpyruvate, and pyruvate did not significantly change over the course of freezing. Thawing sharply reduced the levels of hexose monophosphates in liver but raised P-enolpyruvate content by 2.3 fold. Changes in the contents of glycolytic intermediates over the freeze/thaw course are consistent with an inhibitory block of glycolysis at phosphofructokinase during freezing in order to facilitate a rapid glycogenolysis and production of cryoprotectant; during thawing, however, glycolysis appears to be inhibited at the level of pyruvate kinase.Possible regulatory control of cryoprotectant synthesis by covalent modification of liver glycolytic enzymes was examined. Glycogenolysis during freezing was facilitated by an increase in the percentage of glycogen phosphorylase in the activea (phosphorylated) form and also by an increase in the total amount (a+b) of enzyme expressed. For phosphofructokinase, kinetic changes as a result of freezing included a 40% reduction inK _m for fructose-6-P, a 60% decrease inK _a for fructose-2,6-P₂, and a 2 fold increase in I₅₀ for ATP. These changes imply a freezing-induced covalent modification of the enzyme but are not, apparently, the factors responsible for inhibition of glycolytic flux at the phosphofructokinase locus during glucose synthesis. Kinetic parameters of pyruvate kinase were not altered over the freeze/thaw course.     

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.     

Inhibition of chloroplastic respiration by osmotic dehydration

The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of ¹⁴CO₂ evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [¹⁴C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO₂ evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH₄Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction.     

Effect of freezing and dehydration on ion and cryoprotectant distribution and hemolymph volume in the goldenrod gall fly, Eurosta solidaginis

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at −5 or −20 °C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg⁻¹), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg⁺⁺ in the hemolymph were lower in larvae subjected to freezing (0.21 ± 0.01 μg mg⁻¹ dry mass) compared to controls (0.29 ± 0.01 μg mg⁻¹ dry mass), while intracellular levels of K⁺ were lower in groups exposed to low temperature (8.31 ± 0.21 μg mg⁻¹ dry mass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432 ± 25 mOsm kg⁻¹ and 549 ± 78 mOsm kg⁻¹ respectively. However, larvae subjected to dehydration and freezing at −20 °C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.     

影响蝴蝶兰类原球茎耐脱水性的主要因素

为探讨蝴蝶兰(Phalaenopsis spp.)类原球茎(protocorm-like body,PLB)耐脱水性的主要影响因素,对PLB的平均粒重、含水率、脱水相对湿度、时间、温度、光周期与耐脱水性的关系进行了研究.结果表明,PLB的平均粒重与脱水后失水率、含水率、相对电导率、成活率呈显著或极显著相关.在较高湿度下...     

Post-translational Regulation of Hexokinase Function and Protein Stability in the Aestivating Frog <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis endure substantial dehydration which can impose hypoxic stress due to impaired blood flow. Tissues may increase reliance on anaerobic glycolysis for energy production making the regulation of hexokinase (HK) important. We investigated the enzymatic properties and phosphorylation state of purified HK from the muscle of control and dehydrated (30 % total body water lost) frogs. Bioinformatic tools were also applied to analyze the structural implication of HK phosphorylation in silico. HK from the muscle of dehydrated frogs showed a significantly higher V_max (3.4-fold) and K_m for glucose (2.4-fold) compared with control HK but the K_m for ATP was unaltered. HK from dehydrated frogs also showed greater phosphoserine content (20 % increase) and lower phosphothreonine (22 % decrease) content compared to control HK. Control HK had a higher melting temperature (T_m = 61.9 °C) than from dehydrated (T_m = 54.2 °C) frogs when thermostability was tested using differential scanning fluorimetry. In silico phosphorylation of a Xenopus HK caused alterations in active site binding, corroborating phosphorylation as the probable mechanism for kinetic regulation. Physiological consequences of dehydration-induced HK phosphorylation appear to facilitate glycolytic metabolism in hypoxic situations. Augmented HK function increases the ability of Xenopus to overcome compromised oxidative phosphorylation associated with ischemia during dehydration.     

Osmotic and metabolic responses to dehydration and urea-loading in a dormant,terrestrially hibernating frog

Physiological responses to dehydration in amphibians are reasonably well documented, although little work has addressed this problem in hibernating animals. We investigated osmotic and metabolic responses to experimental manipulation of hydration state in the wood frog (Rana sylvatica), a terrestrial hibernator that encounters low environmental water potential during autumn and winter. In winter-conditioned frogs, plasma osmolality varied inversely with body water content (range 69–79%, fresh mass) primarily due to increases in sodium and chloride concentrations, as well as accumulation of glucose and urea. Decreased hydration was accompanied by a marked reduction in the resting rate of oxygen consumption, which was inversely correlated with plasma osmolality and urea concentration. In a separate experiment, resting rates of oxygen consumption in fully hydrated frogs receiving injections of saline or saline containing urea did not differ initially; however, upon dehydration, metabolic rates decreased sooner in the urea-loaded frogs than in control frogs. Our findings suggest an important role for urea, acting in concert with dehydration, in the metabolic regulation and energy conservation of hibernating R. sylvatica.     

Effects of dehydration on thermoregulatory behavior and thermal tolerance limits of Rana catesbeiana ()

Predicting the effects of high environmental temperatures and drought on populations requires understanding how these conditions will influence the thermoregulatory behavior and thermal tolerance of organisms. Ectotherms show proportional (fine-tuned) and all-or-none (abrupt) responses to avoid overheating. Scattered evidence suggests that dehydration alters these behavioral responses and thermal tolerance, but these effects have not been evaluated in an integrative manner. We examined the effects of hydration level on the behavioral thermoregulation and behavioral and physiological thermal limits of the “bullfrog” (Rana catesbeiana), a well-studied and important invasive species. To examine the effects of dehydration on proportional responses, we compared the Preferred Body Temperatures (PBT) of frogs with restricted and unrestricted access to water. To assess the effect of dehydration on all-or-none responses, we measured and compared the Voluntary Thermal Maximum (VTMax) at different hydration levels (100%, 90%, 80% of body weight at complete hydration). Finally, to understand the effect of dehydration on physiological thermal tolerance, we measured the Critical Thermal Maximum (CTMax) of frogs at matched hydration levels. PBT, VTMax, and CTMax all decreased in response to higher dehydration levels. However, bullfrogs changed their PBT more than their VTMax or CTMax in response to dehydration. Moreover, some severely dehydrated individuals did not exhibit a VTMax response. We discuss the implications of our results in the context of plasticity of thermoregulatory responses and thermal limits, and its potential application to mechanistic modeling.     

Effects of dehydration on water relations and survival of lumbricid earthworm egg capsules

Earthworm egg capsules of five species were compared with regard to survival and water relations upon exposure to controlled dehydration at 20°C. Cocoons of the investigated species all contained about 3.5 g water·g^-1 dry weight when fully hydrated. Approximately 18% of this does not readily freeze upon cooling to -40°C and is referred to as osmotically inactive water. Cocoons exposed to desiccation lose a large proportion of the osmotically active water over 1–4 days until water in the cocoon fluids has equilibrated with surrounding water vapour. The amount of osmotically inactive water, on the other hand, is only reduced by 10–20%. Dendrobaena octaedra was the species most tolerant to drought, its tolerance limit coinciding with loss of practically all osmotically active water. For the five species investigated, there seemed not to be any clear correlation between drought tolerance and microhabitat. Previous investigations have suggested a very close relation between tolerance to dehydration and to subzero temperatures in overwintering earthworm cocoons. Survival at a given level of dehydration at room temperature is less than at temperatures below 0°C, and the tolerance of room temperature dehydration is not closely correlated with cold hardiness across the range of the species studied.Abbreviations dw dry weight - DSC differential scanning calorimetry - fw _pd fresh weight of partially dehydrated cocoons - OAW osmotically active water - OIW osmotically inactive water - Osm osmolality - water potential - R universal gas constant - T absolute temperature - V specific volume of water     

Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog,Neobatrachus wilsmorei

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6–12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na⁺ and K⁺ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na⁺ and K⁺ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K⁺ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K⁺ did not increase above that which can be accounted for by dehydration, indicating that K⁺ gradients were maintained.Abbreviations bm body mass - DPM disintegrations per minute - dw dry weight - MR metabolic rate - vO₂ rate of oxygen consumption - ww wet weight     

Contributions of the kidneys and intestines to water conservation,and plasma levels of antidiuretic hormone,during dehydration in house sparrows (Passer domesticus)

Summary The contributions of the kidneys, the small intestine and the lower intestine (rectum plus cloaca) to water conservation during dehydration in unanaesthetized, unrestrained house sparrows (Passer domesticus) were assessed. Thirty hours of acute dehydration resulted in a 12% loss in body mass and a significant increase in plasma osmolality. Glomerular filtration rate declined by 55%, from 7.7 to 3.5 ml/h, and urine flow rate delined by more than 80%, from 0.2 to 0.03 ml/h. These changes are likely attributable to a large increase in plasma levels of arginine vasotocin during dehydration, from <26 pg/ml in hydrated birds to greater than 200 pg/ml after 30 h dehydration. Flow of water from the ileum to the lower intestine was reduced during dehydration, primarily because of a reduced flow of dry matter (with no significant reduction in water content). The rate of water loss in the excreta declined from 0.2 ml/h in hydrated birds to 0.04 ml/h in dehydrated birds. The rate of water reabsorption in the lower intestine (equal to the rate of water loss in the excreta minus the combined rates of inflow into the lower intestine from the urine and the ileal contents) slightly exceeded the rate of water flow from the ileum in both hydrated and dehydrated birds. We suggest that much of the water reabsorbed in the lower intestine of hydrated birds derives from the urine, but that primarily water from ileal contents is reabsorbed in dehydrated birds. That is, urine undergoes significant post-renal modification in hydrated but not dehydrated house sparrows.     

Cryoprotectants and Extreme Freeze Tolerance in a Subarctic Population of the Wood Frog

Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml^-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml^-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml^-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg^-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.     

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.