No abstract

SUMMARYTo avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroidae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. DAFPs and associated thermal hysteresis activity (THA) have been shown previously in hemolymph and midgut fluid, but the presence of DAFPs has not been explored in primary urine, a potentially important site that can contain endogenous icenucleating compounds that could induce freezing. A maximum mean THA of 2.65±0.33°C was observed in primary urine of wintercollected D. canadensis larvae. THA in primary urine increased significantly through autumn, peaked in the winter and decreased through spring to levels of 0.2-0.3°C in summer, in a pattern similar to that of hemolymph and midgut fluid. THA was also found in hindgut fluid and excreted rectal fluid, suggesting that these larvae not only concentrate AFPs in the hindgut, but also excrete AFPs from the rectal cavity. Based on dafp transcripts isolated from Malpighian tubule epithelia, cDNAs were cloned and sequenced, identifying the presence of transcripts encoding 24 DAFP isoforms. Six of these Malpighian tubule DAFPs were known previously, but 18 are new. We also provide functional evidence that DAFPs can inhibit ice nucleators present in insect primary urine. This is potentially critical because D. canadensis larvae die if frozen, and therefore ice formation in any body fluid, including the urine, would be lethal.

Freeze tolerant Cope’s gray treefrog, Hyla chrysoscelis, accumulates glycerol during a process of cold‐acclimation in anticipation of freezing. Glycerol acts as a cryoprotectant to control osmotic gradients formed by extracellular ice crystals during freezing. The aquaglyceroporin, HC‐3, is abundantly expressed in the membrane of erythrocytes from H. chrysoscelis, where it facilitates osmotically driven transmembrane water flux and glycerol diffusion, both important in freeze tolerance. We hypothesize that HC‐3 functions to moderate the dynamic cell shape changes that occur during the freeze/thaw process as a result of hypotonicity induced cell volume expansion. Cultured erythrocytes subjected to moderate hypotonic stress (70 mOsM) in the absence of 0.3 mM HgCl2, underwent a series of shape changes: they swelled, initially elongated, then became swollen and round, before returning to an elongated state. Erythrocytes treated with an HC‐3 morpholino to knockdown HC‐3 expression become spherical and remained spherical in response to hypotonic challenge. Within 600 seconds of the initiation of the hypotonic challenge, 80% of control cells succumbed to hypotonicity‐induced cell lysis, whereas 70% of the morpholino‐treated cells remained intact. These data indicate that HC‐3 functions in regulating erythrocyte cell volume in response to hypotonic challenge, implicating a role for HC‐3 in cellular freeze tolerance. Grant Funding Source: This research was supported by NSF Research Grant IOS‐1121457.

Cope's gray treefrog, Hyla chrysoscelis is a freeze‐tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC‐3, an ortholog of mammalian aquaporin 3, functions as an aquaglyceroporin to facilitate transmembrane flux of water and glycerol. HC‐3 protein is in higher abundance and is preferentially localized to the plasma membrane in RBCs from cold‐acclimated treefrogs as compared to warm‐acclimated animals. The objective of this study is to determine the signal for HC‐3 membrane translocation that occurs during the cold‐acclimation period. After 48 hrs, cultured erythrocytes from H. chrysoscelis were exposed to cAMP (1 uM; 30 minutes), vasopressin (10 IU; 30 minutes), or epinephrine (1 uM; 60 minutes), isolated from the culture media, and fixed on microscope slides. Fluorescent immunocytochemistry showed enhanced HC‐3 membrane localization in cells exposed to epinephrine and cAMP as compared to controls. These data indicate HC‐3 translocation and membrane localization is enhanced in a cAMP‐dependent pathway. This research was supported by NSF Research Grant IOS‐1121457 and the UD University Honors Program.

Cope’s gray treefrog Hyla chrysoscelis, accumulates and distributes glycerol as a cryoprotectant in anticipation of freezing. Transmembrane glycerol and water flux in H. chysoscelis erythrocytes likely occurs through HC‐3, an ortholog of mammalian aquaporin 3. HC‐3 protein is in higher abundance and is preferentially localized to the plasma membrane in RBCs from cold‐acclimated treefrogs as compared to warm‐acclimated animals. It is hypothesized that neuroendocrine agonists via receptor mediated second messenger pathways integrate signals derived from fasting, dehydration, diurnal, and/or temperature changes during cold‐acclimation to regulate HC‐3 expression as part of the mechanism of freeze tolerance. In this study, cultured H. chrysoscelis erythrocytes were exposed to 1 uM epinephrine for 30 and 60 minutes. Native HC‐3 expression increased 3 fold at 30 minutes and 5.5‐fold at 60 minutes relative to controls, whereas glycosylated HC‐3 expression increased by 1.1‐fold at 30 minutes and by 2 ‐fold at 60 minutes relative when exposed to epinephrine. Moreover, epinephrine treatment resulted in membrane localization as compared to cytosolic distribution in control cells. Erythrocytes pre‐treated with Calphostin C, a PKC inhibitor, showed no HC‐3 membrane localization, and native HC‐3 expression was reduced by 66% relative to controls and 94% relative to epinephrine‐treated cells. Thus, epinephrine begins a PKC‐dependent mechanism that results in an increase in HC‐3 abundance, HC‐3 membrane localization, and enhanced glycosylation in erythrocytes. These regulatory mechanisms are consistent with the in vivo regulation of HC‐3 expression observed in erythrocytes from cold‐acclimated treefrogs.