Shu-Xia Yi

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

SUMMARY 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.

Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 degrees C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide. Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20-30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.

Calcium signaling mediates cold sensing in insect tissues

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

Mild desiccation rapidly increases freeze tolerance of the goldenrod gall fly, Eurosta solidaginis : evidence for drought-induced rapid cold-hardening

SUMMARY Overwintering insects may experience extreme cold and desiccation stress. Both freezing and desiccation require cells to tolerate osmotic challenge as solutes become concentrated in the hemolymph. Not surprisingly, physiological responses to low temperature and desiccation share common features and may confer cross-tolerance against these stresses. Freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae), experience extremely dry and cold conditions in winter. To determine whether mild desiccation can improve freeze tolerance at organismal and cellular levels, we assessed survival, hemolymph osmolality and glycerol levels of control and desiccated larvae. Larvae that lost only 6–10% of their body mass, in as little as 6 h, had markedly higher levels of freeze tolerance. Mild, rapid desiccation increased freezing tolerance at −15°C in September-collected larvae (33.3±6.7 to 73.3±12%) and at −20°C in October-collected larvae (16.7±6.7 to 46.7±3.3%). Similarly, 6 h of desiccation improved in vivo survival by 17–43% in fat body, Malpighian tubule, salivary gland and tracheal cells at −20°C. Desiccation also enhanced intrinsic levels of cold tolerance in midgut cells frozen ex vivo (38.7±4.6 to 89.2±5.5%). Whereas hemolymph osmolality increased significantly with desiccation treatment from 544±16 to 720±26 mOsm, glycerol levels did not differ between control and desiccated groups. The rapidity with which a mild desiccation stress increased freeze tolerance closely resembles the rapid cold-hardening response, which occurs during brief sub-lethal chilling, and suggests that drought stress can induce rapid cold-hardening.

Rapid cold-hardening blocks cold-induced apoptosis by inhibiting the activation of pro-caspases in the flesh fly Sarcophaga crassipalpis

Apoptosis plays important roles in the selective elimination of sub-lethally damaged cells due to various environmental stresses. The rapid cold-hardening (RCH) response protects insects from the otherwise lethal consequences of injury due to cold-shock. We recently demonstrated that cold shock induces apoptotic cell death in insects and that RCH functions to specifically block cold-shock-induced apoptosis. In the present study we used isolated fat body, midgut, muscle, and Malpighian tubules from adult flesh flies Sarcophaga crassipalpis to test the following hypotheses: (1) cold-induced apoptosis varies among different tissues and (2) RCH blocks the apoptotic pathway by preventing the activation of pro-caspases. Cold-shock induced substantial amounts of apoptotic cell death that matched with tissue damage as determined using vital dyes. RCH treatment significantly reduced apoptotic cell death in all tested tissues. Caspase-3 (executioner) activity was 2–3 times higher in the cold- and heat-shocked groups than in control and RCH groups. Likewise, the activity of caspase-9 (initiator) showed a similar trend as for caspase-3 in all tissues but midgut. In addition, cold-shock and heat-shock treatments also increased caspase-2 activity 2–3 folds in both soluble and membrane fractions of fat body and muscle extracts compared to controls.

Changes in gut and malpighian tubule transport during seasonal acclimatization and freezing in the gall fly Eurosta solidaginis

SUMMARY Since few studies have examined cold tolerance at the organ level in insects, our primary objective was to characterize the functional responses of the gut and Malpighian tubules (MT) to seasonal acclimatization, chilling and freezing in larvae of the goldenrod gall fly Eurosta solidaginis Fitch (Diptera, Tephritidae). From September to December, hemolymph osmolality (455-926 mOsmol kg l-1) and freezing tolerance increased markedly in field-collected larvae. Chlorophenol Red was readily transported into the lumen of the foregut, the posterior portion of the midgut, the ureter, the proximal region of the anterior pair of MT, and entire posterior pair of MT. Ouabain and KCN inhibited transport of Chlorophenol Red in the gut and MT. Transport was readily detected at 0°C and the rate of transport was directly related to temperature. The rate of fluid transport by the MT decreased steadily from a monthly high in September (10.7±0.8 nl min-1 for the anterior pair; 12.7±1.0 nl min-1 for the posterior pair) until secretion was no longer detectable in December; this decrease parallels entry into diapause for this species. Even in larvae that died following freezing for 40 days at -20°C, individual organ function was retained to a limited extent. Through the autumn, cholesterol concentrations in the hemolymph increased nearly fourfold. In contrast, the ratio of cholesterol to protein content (nmol mg l-1) in the MT membrane remained relatively constant (22∼24 nmol mg l-1 protein) during this period. Freezing of larvae for 20 days at -20°C caused a significant decrease in cholesterol levels in the hemolymph and the MT membranes compared to unfrozen controls. These results suggest that cholesterol plays a role in seasonal cold hardening and freeze tolerance in insects.

Cold-hardening during long-term acclimation in a freeze-tolerant woolly bear caterpillar, Pyrrharctia isabella.

ABSTRACT The banded woolly bear caterpillar, Pyrrharctia isabella (Lepidoptera: Erebidae), overwinters in leaf litter and survives freezing under natural conditions. Following 18 weeks of cold acclimation at 5°C, all caterpillars could survive 1 week of continuous freezing at −20°C or seven cycles of freezing–thawing at −20°C, but none survived freezing at −80°C. Field-collected caterpillars had a temperature of crystallization of −7.7±0.5°C that decreased significantly to −9.5±0.6°C after 12 weeks of acclimation at 5°C. Hemolymph levels of free proline, total amino acids and proteins reached a peak during the first 4 weeks of acclimation; concomitantly, hemolymph osmolality increased markedly during this interval (from 364 to 1282 mosmol kg−1). In contrast, hemolymph pH decreased during the first 4 weeks of acclimation before this trend reversed and pH values gradually returned to initial values. However, pH reached its peak value following 1 week at −20°C, but decreased after longer periods of freezing. During cold acclimation, cholesterol levels decreased in the hemolymph and the membrane fraction of fat body but not in other tissues. Lethal freezing at −80°C reduced cell survival in foregut tissue and caused leakage of free proline, total amino acids and proteins from tissues into the hemolymph. The addition of glycerol to the bathing medium reduced freezing injury in fat body cells, as evidenced by reduced leakage of amino acids and proteins. Summary: After an extended period of cold acclimation, woolly bear caterpillars survived freezing at −20°C. Cold-hardening markedly increased hemolymph osmolality, as well as hemolymph levels of free proline, total amino acids and proteins.