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

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.

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.

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.

High temperature pulses decrease indirect chilling injury and elevate ATP levels in the flesh fly, Sarcophaga crassipalpis.

Indirect chilling injury commonly occurs during long-term exposure to low temperature in many organisms including insects. A previous study revealed increased rates of survival and reduced cold injury in flesh flies, Sarcophaga crassipalpis, that experienced an intermittent pulse of high temperature during a low-temperature regiment. We extended these studies by determining survival rates and ATP levels for flies that had undergone continuous long-term exposure at 0 degrees C versus those experiencing a 24-h warming pulse of either 15 or 20 degrees C. Survival among flies that had undergone a warming pulse was significantly greater than for flies that were maintained continuously at 0 degrees C. Furthermore, ATP levels of flies that had experienced a warming pulse were significantly higher than those of flies maintained at 0 degrees C. These data suggest that brief warming pulses during long-term cold storage allow regeneration of energy reserves that promote survival and reduce indirect chilling injury.

Increased dietary cholesterol enhances cold tolerance in Drosophila melanogaster.

For many years, non-freezing cold shock injury has been associated with damage to the cell membrane. In this study, we enhanced membrane cholesterol levels of Drosophila melanogaster by raising larvae on a cholesterol-augmented diet. Diet augmentation significantly increased the amount of cholesterol in the cell membranes of the adult flies (1.57+/-0.17 nmol per mg vs. 0.93+/-0.11 nmol per mg). Flies on the cholesterol-augmented diet exhibited a greater intrinsic cold tolerance: this group had a higher survival rate after a 2-h cold shock of -5 degree C than did the control group (71.0+/-6.6 percent vs 36.0+/-8.1 percent). Cholesterol-augmented flies also had a significantly greater capacity to rapidly cold-harden to -7 degree C (36.7+/-4.4 percent) compared to flies on a control diet (20.0+/-2.9 percent). These results suggest a mechanistic link between protection from chilling or cold shock injury and modifications to the cellular membrane.

Desiccation enhances rapid cold-hardening in the flesh fly Sarcophaga bullata: evidence for cross tolerance between rapid physiological responses.

Many insects use rapid cold-hardening (RCH), a physiological response to sub-lethal exposure to stressors, such as chilling and desiccation, to enhance their cold tolerance within minutes. Recently, drought-induced RCH, triggered by brief, mild desiccation, was described in larvae of the freeze-tolerant gall fly (Eurosta solidaginis). However, its prevalence and ecological significance in other insects is not known. Consequently, we used a freeze-intolerant model, the flesh fly, Sarcophaga bullata, to investigate the effects and mechanisms of drought-induced RCH. In addition, we investigated how drought- and cold-induced RCH interact by exposing flies to both desiccation and chilling. Desiccation for 3 h increased larval pupariation after cold shock from 28 to 40 %-the first example of drought-induced RCH in both a freeze-intolerant insect and in a non-overwintering life stage. We also found that desiccation and chilling together enhanced the cold hardiness of larvae and adults more than either did separately, suggesting that drought and cold trigger distinct physiological mechanisms that interact to afford greater cold tolerance. These results suggest that drought-induced RCH is a highly conserved response used by insects with diverse life history strategies. Furthermore, the protective interaction between drought- and cold-induced RCH suggests that, in nature, insects use multiple cues and physiological mechanisms to fine-tune their response to changing ambient conditions.

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.

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.

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.

Rapid cold-hardening protects Drosophila melanogaster from cold-induced apoptosis.

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to 7 degrees C for 2 h, whereas significantly more flies in the RCH group survived exposure to 7 degrees C for 2 h after a 2-h exposure to 5 degrees C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5 degrees C, 30 min), and frozen (20 degrees C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of approximately 150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.

Rapid cold-hardening increases membrane fluidity and cold tolerance of insect cells.

The rapid cold-hardening (RCH) response not only confers dramatic protection against cold-shock (non-freezing) injury, but also "instantaneously" enhances organismal performance. Since cold-shock injury is associated with damage to the cell membrane, we investigated the relationship between RCH and changes in cold tolerance and membrane fluidity at the cellular level. None of the adult flies (Sarcophaga bullata) in the cold-shocked treatment group survived direct transfer to -8 degrees C for 2 h; in contrast, 64.5% of flies in the RCH group survived exposure to -8 degrees C. Differences between the treatment groups also were reflected at the cellular level; only 21.3% of fat body cells in the cold-shocked group survived compared to 68.5% in the RCH group. Using 31P solid-state NMR spectroscopy, we determined that membrane fluidity increased concurrently with rapid cold-hardening of fat body cells. This result suggests that membrane characteristics may be modified very rapidly to protect cells against cold-shock injury.

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

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 degrees 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 degrees 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 approximately 24 nmol mg l(-1) protein) during this period. Freezing of larvae for 20 days at -20 degrees 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.

In vivo and in vitro rapid cold-hardening protects cells from cold-shock injury in the flesh fly.

The capacity to undergo rapid the cold-hardening response (RCH) has been documented in diverse groups of insects and functions to protect against non-freezing cold injury and to preserve physiological performance in response to environmental cooling. The RCH response is remarkable for the rapidity of its induction; however the mechanism by which insects perceive cold and transduce this input at the cellular level has received little attention. To test the hypothesis that cells from isolated tissues can undergo RCH in response to cold, we assessed cell viability in four tissues that had undergone either RCH (0 degree C, 2 h followed by -8 degrees C, 2 h) or cold-shock (-8 degrees C, 2 h) both in vivo and in vitro from the adult flesh fly Sarcophaga crassipalpis (Diptera: Sarcophagidae) using fluorescent probes. Adult flies showed a significantly higher survival rate in the RCH group than in the cold-shocked group. Similarly, in all tissues tested, both in vivo and in vitro, RCH significantly improved cell survival compared with the respective cold-shocked groups. To our knowledge this is the first report to demonstrate that isolated cells and tissues from insects can undergo RCH. These results indicate that insect cells are capable of cold-sensing without neuroendocrine mediation; direct induction at the cellular level also helps to explain the swiftness of the RCH response.

Effect of age, diet, diapause and juvenile hormone on oogenesis and the amount of vitellogenin and vitellin in the twospotted stink bug, Perillus bioculatus (Heteroptera: pentatomidae).

Vitellogenic oocytes from Perillus bioculatus have two native vitellins, Vt1 and Vt2, with molecular masses of 553 and 228 kDa, respectively. The hemolymph contains a major vitellogenin, Vg, with a molecular mass of 528 kDa that consists of three apoproteins with masses of 177, 84 and 59 kDa, respectively. Antibodies to purified Vt2 reacted with ovary extracts, egg extracts and female hemolymph, but not with male hemolymph in immunodiffusion tests. Western blots showed that anti Vt2 reacted with both Vt1, Vt2 and with Vg. Vitellogenesis starts at an ovarian score of 12 at 2.4 days after emergence. The first cycle of egg development is completed in ovaries with a score of 112 at 7.7 days. During this 5.3 day period, the ovaries of a single female incorporated 1833 &mgr;g of protein to form vitellin. Vitellogenin levels start to increase in females 2.5 days after emergence and reached 17.8 &mgr;g/&mgr;l by 5.5 days. After 5.5 days vitellogenin levels fluctuated between 9.7 and 19.9 &mgr;g/&mgr;l. Most diapausing females contained no ovarian follicles in the vitellarium and their hemolymph contained less than 1 &mgr;g/&mgr;l of vitellogenin. Treating diapausing females with 1 &mgr;g of JH III increased vitellogenin levels over 120-fold. Insects maintained on a liver-based artificial diet had lower vitellogenin levels than the controls at all sample times and did not show an increase in vitellogenin concentration until 11.5 days. Treating insects on the artificial diet with 10 &mgr;g of JH III elevated vitellogenin levels to about a fourth of that found in prey-fed insects of a comparable age. This suggests that females fed the artificial diet have low levels of essential materials needed for vitellogenin production.

Characterization of a new Bacillus thuringiensis isolate highly active against Cochylis hospes.

A new bacterial isolate, 00-50-5, from sunflower head extracts was identified as Bacillus thuringiensis (Bt) according to its morphology. Bt isolate 00-50-5 was highly active against the banded sunflower moth (BSM), Cochylis hospes Walsingham. A sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 4-15% gradient gel of whole strain protein of 00-50-5 revealed six proteins with molecular masses (Mr) of 133, 80, 60, 27, 15, and 14 kDa. SDS-PAGE of pH 4.2-precipitated proteins (PP) or activated proteins formed by adding the BSM larval gut protease at 1:50 (wt/wt, protease/PP) showed five bands, including two major proteins of Mr 60 kDa and 27 kDa, and three small peptides of Mr 15, 13, and 7 kDa. The BSM larval gut protease was able to completely digest the proteins when present at a high ratio (10:1, wt/wt, protease/PP). The 60- and 27-kDa proteins could be digested by subtilisin Carlsberg at ratios of 1:50 or 1:1 (wt/wt, protease/PP), but neither BSM larval gut protease nor trypsin was effective at the same ratios. Three small peptides of Mr 15, 13, and 7 kDa were digested by the gut protease at a ratio of 1:1 (wt/wt). The N-terminal sequence of 1-31 amino acid residues for the 27-kDa protein showed 96.7% homology to a 31-amino acid fragment from camelysin, a protease from B. cereus, indicating that the 27-kDa protein may be a camelysin and a novel active protein against BSM.