Metabolism and bound water in overwintering insects

The freezing-tolerant gall fly larva, Eurosta solidaginis, provides an excellent model system for the study of metabolic adaptation and metabolic control for lowtemperature survival during overwintering. Low-temperature acclimation of the larvae results in dramatic alterations in metabolic flux producing a sequential synthesis of two cryoprotectants, glycerol at warmer temperatures followed by sorbitol when larvae are exposed to 5 °C. Regulation of metabolism in the larvae appears to exploit temperature change, temperature effects on enzyme kinetics, and temperature/modulator interactions with enzymes producing the alterations in metabolic flux leading to differential polyol synthesis. For instance, temperature/modulator effects on phosphofructokinase appear to be the major factor halting carbon flow into glycerol synthesis at low temperatures and diverting flux instead into the pathway of sorbitol synthesis. Alterations in the cellular content of bound water and the metabolic pools of free versus bound soluble metabolites may also have important regulatory consequences for low-temperature metabolism. Bound water content of the larvae increases with low-temperature acclimation and is attributable to changes in water binding by both low-molecular-weight (polyols) and highmolecular-weight (proteins, glycogen) subcellular components. A restrictive effect of high bound water content may be one factor causing the strong depression of metabolic activity seen in the larvae as a result of extracellular freezing. In addition, bound water may have a more subtle effect in determining the relative pool sizes of bound versus free metabolites in the cell. ³¹P-NMR studies of whole larvae show that the content of free phosphorylated intermediates in the cell diminishes with decreasing temperatures despite a measured constancy in the total pool size of these intermediates. An increase in the content of bound metabolites with low temperature may restrict metabolism by limiting the availability of substrates and effectors of enzyme reactions.     

Reversible phosphorylation regulation of NADPH-linked polyol dehydrogenase in the freeze-avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH‐dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K_m for glyceraldehyde in 5°C‐acclimated larvae was 7.0 mM and doubled in ? 15°C‐exposed larvae. This change suggested that PDH is regulated by a state‐dependent covalent modification. Indeed, high and low K_m forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K_m glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K_m of the ? 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from ? 15°C‐acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the ? 15°C enzyme. These experiments established that PDH is regulated by state‐dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months. © 2011 Wiley Periodicals, Inc.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: Winter profiles of a natural population

Larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) utilize a freeze-avoidance strategy for winter survival. Cold-hardiness adaptations of an outdoor population of the species were profiled over the 1984–1985 winter. Over the autumn months supercooling points of the larvae dropped from −13.9±2.3°C to −37.8±2.8°C (the lowest winter temperature recorded was −26°C), water content of the larvae decreased from 57.2±1.2 to 24.8±1.6% of fresh weight, and glycerol content of the larvae rose to an average of 2030 μmol/g wet weight or 18.7% of fresh weight. All parameters stabilized over the mid-winter months. Glycerol production was largely accounted for by the loss of stored glycogen while lipid and protein reserves remained nearly constant over the winter months. Supercooling-point depression and glycerol systhesis both appeared to be initiated after the first overnight exposures to subzero temperatures. Highest rates of glycerol production, about 60 μmol g⁻¹ d⁻¹, were achieved with mean daily temperatures of about 0°C and subzero nights. Glycerol content was rapidly cleared in the spring but only 20% of the resulting carbon was restored as glycogen.     

AMP-activated protein kinase and metabolic regulation in cold-hardy insects

Winter survival for many insects depends on cold hardiness adaptations as well as entry into a hypometabolic diapause state that minimizes energy expenditure. We investigated whether AMP-activated protein kinase (AMPK) could be involved in this adaptation in larvae of two cold-hardy insects, Eurosta solidaginis that is freeze tolerant and Epiblema scudderiana that uses a freeze avoidance strategy. AMPK activity was almost 2-fold higher in winter larvae (February) compared with animals collected in September. Immunoblotting revealed that phosphorylation of AMPK in the activation loop and phosphorylation of acetyl-CoA carboxylase (ACC), a key target of AMPK, were higher in Epiblema during midwinter whereas no seasonal change was seen in Eurosta. Immunoblotting also revealed a significant increase in ribosomal protein S6 phosphorylation in overwintering Epiblema larvae, and in both Eurosta and Epiblema, phosphorylation of eukaryotic initiation factor 4E-binding protein-1 dramatically increased in the winter. Pyruvate dehydrogenase (PDH) E1α subunit site 1 phosphorylation was 2-fold higher in extracts of Eurosta larvae collected in February versus September while PDH activity decreased by about 50% in Eurosta and 80% in February Eurosta larvae compared with animals collected in September. Glycogen phosphorylase phosphorylation was 3-fold higher in Epiblema larvae collected in February compared with September and also in these animals, triglyceride lipase activity increased by 70% during winter. Overall, our study suggests a re-sculpting of metabolism during insect diapause, which shifted to a more catabolic poise in freeze-avoiding overwintering Epiblema larvae, possibly involving AMPK.     

Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: Laboratory investigations of temperature cues in the regulation of cold hardiness

Laboratory manipulations of ambient temperature were used to investigate the role of temperature in triggering or modulating cold-hardiness adaptations, supercooling-point depression and cryoprotectant accumulation, in larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens), a freeze-intolerant species. Low temperature strongly facilitated cryoprotectant synthesis; larvae subjected to a 1°C per day decrease in temperature showed a major increase in the rate of glycerol synthesis when temperature fell below 5°C with highest rates of synthesis, greater than 90 μmol g⁻¹ d⁻¹, at temperatures between 0 and −10°C. Conversely, abrupt rewarming of larvae from −18 to 23°C in mid-November stimulated a rapid loss of glycerol (from a starting level of 1763 ± 278 μmol/g wet weight) with a half time of only 1.5 days. Supercooling-point depression was not keyed to ambient temperature but appeared to be an endogenous event occurring over the same time interval in laboratory animals held at warm or cold temperatures, as well as in outdoor animals. Rewarming of cold-adapted larvae in November resulted in only a small rise in supercooling point (and did not break diapause) but rewarming in February resulted in a 19°C increase in supercooling point in 4 days, followed rapidly by pupation.     

Glucose‐6‐phosphate dehydrogenase is posttranslationally regulated in the larvae of the freeze‐tolerant gall fly,Eurosta solidaginis,in response to freezing

The freeze‐tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose‐6‐phosphate dehydrogenase (G6PDH) mediates the rate‐limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5°C) and frozen (?15°C) E. solidaginis larvae by a single‐step chromatography method utilizing 2′,5′‐ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in K_m for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.     

Fructose-1,6-bisphosphatase from a cold-hardy insect: Control of cryoprotectant glycerol catabolism

Fructose 1,6-bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 ± 6.5 kD and a subunit molecular weight of 37.6 ± 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer at 22°C and rose to 7.31 at 5°C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 ± 4.1 kJ/mol^?1. K_m values for FBPase decreased from 4.7 ± 0.34 μM at 22°C to 1.3 ± 0.05 μM at 5°C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6-bisphosphate (F2,6P₂), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCI. Inhibition by AMP and F2,6P₂ increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FBPase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I₅₀ values for AMP and F2,6P₂ increase at 22°C (compared with 5°C), high glycerol levels override F2,6P₂ inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. © 1995 Wiley-Liss, Inc.     

松针瘿蚊越冬幼虫体内酶活性的时序变化

昆虫的越冬耐寒过程与糖酵解、磷酸己糖途径和抗冻保护性物质合成等一些中间代谢有关的酶有关。该文对松针瘿蚊Thecodiplosis japonensis老熟幼虫1998/1999越冬期间体内上述代谢酶活性的变化进行了研究。越冬期间体内糖原磷酸化酶活性明显地增加,糖酵解有关的酶（己糖激酶、乳酸脱氢酶和醛缩酶）活性较低,以保证更多的碳源（糖原）转化成海藻糖。越冬期间,体内葡萄糖-6-磷酸脱氢酶活性增高所产生的还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH),可为细胞在亚低温状态下发挥正常功能以及体内抗冻保护性物质的合成提供还原动力,同时通过调节体内海藻糖酶活性来维持越冬期间较高含量的海藻糖和移除春季体内累积的过多的海藻糖。     

Purification and characterization of protein phosphatase-1 from two cold-hardy goldenrod gall insects.

The catalytic subunit of protein phosphatase-1 (PP-1) was purified to homogeneity from final instar larvae (the overwintering stage) of freeze avoiding (Epiblema scudderiana) and freeze tolerant (Eurosta solidaginis) cold-hardy insects. Arrhenius plots showed that activity of PP-1 from both species was strongly suppressed at low temperature. Acidic shifts in pH optima and increased inhibition by okadaic acid were also observed when the enzymes were assayed at 4 degrees C compared with 24 degrees C. The data identify multiple ways by which PP-1 can be inhibited at low temperature and this inhibition appears to be key to sustaining high glycogen phosphorylase activity in support of polyol synthesis at low temperatures.     

Protective agents: Regulation of synthesis

The exogenous cues to overwintering adaptations vary not just between components of hardening but between species. One species, P. brevicornis, initiates glycerol synthesis in response to 0 °C exposures while a second species, E. solidaginis, increases glycerol levels not in response to temperature but in apparent association with changes in total body mass. This species maintains a constant annual percentage of water while occupying a hibernaculum that dries considerably. During overwintering, E. solidaginis losses approximately 50% of its total body mass. In addition to the changes described, this species (northern populations) increases the amount of water bound to both protein and low-molecular-weight compounds during hardening. The increase in binding exceeds threefold between 25 and ?30 °C (0.193 to 0.633 g/g dry wt) (29). These data do not unequivocally demonstrate the existence of a hydration trigger to glycerol synthesis but are adequate to put forth such a hypothesis. A decrease in total bulk water levels due to both wet weight loss and increases in bound water may provide conditions of functionally reduced intracellular metabolic water. Since polyol production necessitates the disruption of carbon flow between glucose-6-phosphate and pyruvate, one or more enzymes may be sensitive to water reductions. Pyruvate kinase is sensitive to available water levels. Inhibition of this enzyme would likely cause a shunting of carbon metabolism to glycerol production. This hypothesis becomes attractive in light of the observation that in a variety of species, glycerol accumulations have been correlated with dehydration and hyperosmotic conditions. A common adaptative mechanism may exist in response to apparently different environmental perturbations.     

ENZYMES OF FATTY ACID METABOLISM IN OVERWINTERING MATURE LARVAE OF THE PINE NEEDLE GALL MIDGE,THECODIPLOSIS JAPONENSIS*

Abstract In this study, overwintering larvae of pine needle gall midge, Thmodiplosis jaHnensis, were sampled at various dates in the winter of 1997 and profiles of some enzymes of fatty acid metabolism were studied. During overwintering, a decrease in total lipids in T. japonensis larvae suggested the use of fat reserves to maintain basal metabolism. Activities of two enzymes associated with fatty acid synthesis, i. e. malic enzyme and ATP‐dependent citrate lyase, decreased from December to mid‐January, then increased from the end of February, indicating a reduced potential for fatty acid synthesis during the winter. Enzymes for fatty acid oxidation, as indicated by the activities of hydroxyacyl‐CoA dehydrogenase, carnitine‐palmitoyl transferase and acetoacetyl‐CoA thiolase, showed different profiles. The potential for ketone body metabolism, as measured by p‐hydroxybutyrate dehydrogenase activity, decreased in the course of winter, indicating that ketone body as a metabolic fuel during overwintering is not important.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

The ontogeny of cold tolerance in the gall fly, Eurosta solidagensis

The lower lethal temperature of many insects indicates an overwintering flexibility as a result of either extensive supercooling or production of cryoprotectants. Ontogenetically, the gall fly (Eurosta solidagensis) utilizes both means of seasonal cryoprotection. All stages except third instar larvae demonstrate supercooling points well below the lowest temperature normally experienced by that particular stage. The third instar larvae exhibit a high supercooling point but are well protected by a cryoprotectant system consisting of glycerol, sorbitol, and trehalose. Glycerol is accumulated, possibly from triglyceride sources, during early autumn and reaches plateau levels (0·6 M) by early winter. Sorbitol synthesis is delayed until freezing exposures and reaches a plateau with glycerol at 0·3 M. It is not until mid-winter that peak trehalose levels are reached (300 mg %). All cryoprotectant levels are a reflection of haemolymph concentrations.Laboratory acclimation experiments further quantify these results. Trehalose synthesis is time and temperature dependent and appears to be affected by developmental processes.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.     

In Vivo Assessment of Cold Adaptation in Insect Larvae by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Background

Temperatures below the freezing point of water and the ensuing ice crystal formation pose serious challenges to cell structure and function. Consequently, species living in seasonally cold environments have evolved a multitude of strategies to reorganize their cellular architecture and metabolism, and the underlying mechanisms are crucial to our understanding of life. In multicellular organisms, and poikilotherm animals in particular, our knowledge about these processes is almost exclusively due to invasive studies, thereby limiting the range of conclusions that can be drawn about intact living systems.

Methodology

Given that non-destructive techniques like ¹H Magnetic Resonance (MR) imaging and spectroscopy have proven useful for in vivo investigations of a wide range of biological systems, we aimed at evaluating their potential to observe cold adaptations in living insect larvae. Specifically, we chose two cold-hardy insect species that frequently serve as cryobiological model systems–the freeze-avoiding gall moth Epiblema scudderiana and the freeze-tolerant gall fly Eurosta solidaginis.

Results

In vivo MR images were acquired from autumn-collected larvae at temperatures between 0°C and about −70°C and at spatial resolutions down to 27 µm. These images revealed three-dimensional (3D) larval anatomy at a level of detail currently not in reach of other in vivo techniques. Furthermore, they allowed visualization of the 3D distribution of the remaining liquid water and of the endogenous cryoprotectants at subzero temperatures, and temperature-weighted images of these distributions could be derived. Finally, individual fat body cells and their nuclei could be identified in intact frozen Eurosta larvae.

Conclusions

These findings suggest that high resolution MR techniques provide for interesting methodological options in comparative cryobiological investigations, especially in vivo.     

VARIABLE SELECTION ON EUROSTA'S GALL SIZE. II. A PATH ANALYSIS OF THE ECOLOGICAL FACTORS BEHIND SELECTION

We examined phenotypic selection exerted by natural enemies on the gall-making fly Eurosta solidaginis in an extensive field study of 16 populations, spanning four generations. Gall-makers that induce small galls are vulnerable to the attack of Eurytoma gigantea. This imposes upward directional selection on gall size. Insectivorous birds, predominantly the downy woodpecker, are more likely to attack larvae that induce large galls than small ones, and this imposes downward directional selection. We used path analysis to explore the relative contributions of these natural enemies to the net directional selection on gall size. The path models further examined several ecological factors that influence selection intensity through their effects on parasitoid and bird attack rates. Net directional selection varied more strongly with E. gigantea attack than bird attack. Competitive interactions among birds and the three parasitoid species, including E. gigantea, were evidenced by low winter bird attack rates in fields where a high proportion of galls contained the overwintering parasitoids. Eurytoma gigantea attack was heavier in fields where mean gall size was small and bird attack heavier in fields where mean gall size was large. Neither birds nor E. gigantea showed simple density-dependent attack. Data suggested a form of frequency-dependent attack by birds but not by E. gigantea.     

Changes in abundance of aquaporin-like proteins occurs concomitantly with seasonal acquisition of freeze tolerance in the goldenrod gall fly, Eurosta solidaginis

The accumulation of cryoprotectants and the redistribution of water between body compartments play central roles in the capacity of insects to survive freezing. Aquaporins (AQPs) allow for rapid redistribution of water and small solutes (e.g. glycerol) across the cell membrane and were recently implicated in promoting freeze tolerance. Here, we examined whether aquaporin-like protein abundance correlated with the seasonal acquisition of freezing tolerance in the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae). Through the autumn, larvae became tolerant of freezing at progressively lower temperatures and accumulated the cryoprotectant glycerol. Furthermore, larvae significantly increased the abundance of membrane-bound aquaporin and aquaglyceroporin-like proteins from July through January. Acute exposure of larvae to cold and desiccation resulted in upregulation of the AQP3-like proteins in October, suggesting that their abundance is regulated by environmental cues. The seasonal increase in abundance of both putative aquaporins and aquaglyceroporins supports the hypothesis that these proteins are closely tied to the seasonal acquisition of freeze tolerance, functioning to permit cells to quickly lose water and take-up glycerol during extracellular ice formation, as well as reestablish water and glycerol concentrations upon thawing.     

Responses of protein phosphatases and cAMP-dependent protein kinase in a freeze-avoiding insect, Epiblema scudderiana

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of winter cold hardiness and show multiple metabolic adaptations for subzero survival including accumulation of large amounts of glycerol as a colligative antifreeze. Induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. Changes in the activities of several signaling enzymes including cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) were monitored over the winter and during experimental exposures of larvae to subzero temperatures (-4 degrees C, a temperature that triggers rapid glycerol synthesis, or -20 degrees C, a common midwinter ambient temperature) or anoxia. A strong increase in the amount of active PP1 in the latter part of the winter may be responsible for shutting off glycogenolysis once glycerol levels are maximized. There appears to be a limited role for PKA in overwintering but PP2A and PP2C activities rose when larvae were exposed to -20 degrees C and PTP activities rose significantly over the winter months and also in response to laboratory subzero (-20 degrees C) and anoxia exposures. The strong responses by PTPs suggest that these may be involved in cell cycle and growth arrest during winter diapause.     

Environmental triggers to cryoprotectant modulation in separate populations of the gall fly, Eurost a solidaginis (Fitch)

Northern and southern populations of the gall fly Eurosta solidaginis utilize quantitatively distinct adaptive strategies when exposed to a laboratory simulation of winter temperatures. Both populations are freezing tolerant and rely in part on the temperature-dependent accumulation of glycerol and sorbitol, and static but elevated levels of trehalose for protection. The accumulation triggers are time-temperature dependent. For northern and southern populations, exposure to 5°C for periods exceeding 24 hr, but less than 5 days or 5°C with a gradual reduction (1°C/day) results in the accumulation of sorbitol at 1.5 μg/mg/day. Glycerol levels remain essentially constant between 10° and ?25°C in each population. However, the concentration of glycerol in the Minnesota population is 3–4 times greater that that of the Texas specimens. Haemolymph melting points varied quantitatively with changing cryoprotectant levels. No significant difference was noted between the supercooling points of each population. This suggests that ice-nucleator levels were comparable throughout the exposure period.