Rapid Cold Hardening Research Articles

Combined transcriptomic and metabolomic approach uncovers molecular mechanisms of cold tolerance in a temperate flesh fly

The ability to respond rapidly to changes in temperature is critical for insects and other ectotherms living in variable environments. In a physiological process termed rapid cold-hardening (RCH), exposure to nonlethal low temperature allows many insects to significantly increase their cold tolerance in a matter of minutes to hours. Additionally, there are rapid changes in gene expression and cell physiology during recovery from cold injury, and we hypothesize that RCH may modulate some of these processes during recovery. In this study, we used a combination of transcriptomics and metabolomics to examine the molecular mechanisms of RCH and cold shock recovery in the flesh fly, Sarcophaga bullata. Surprisingly, out of ∼15,000 expressed sequence tags (ESTs) measured, no transcripts were upregulated during RCH, and likewise RCH had a minimal effect on the transcript signature during recovery from cold shock. However, during recovery from cold shock, we observed differential expression of ∼1,400 ESTs, including a number of heat shock proteins, cytoskeletal components, and genes from several cell signaling pathways. In the metabolome, RCH had a slight yet significant effect on several metabolic pathways, while cold shock resulted in dramatic increases in gluconeogenesis, amino acid synthesis, and cryoprotective polyol synthesis. Several biochemical pathways showed congruence at both the transcript and metabolite levels, indicating that coordinated changes in gene expression and metabolism contribute to recovery from cold shock. Thus, while RCH had very minor effects on gene expression, recovery from cold shock elicits sweeping changes in gene expression and metabolism along numerous cell signaling and biochemical pathways.

Pre-adapted to the maritime Antarctic? – Rapid cold hardening of the midge, Eretmoptera murphyi

During the 1960s, the midge, Eretmoptera murphyi, was transferred from sub-Antarctic South Georgia (55oS 37oW) where it is endemic to a single location on maritime Antarctic Signy Island (60oS 45oW). Its distribution has since expanded considerably, suggesting that it is pre-adapted to the more severe conditions further south. To test one aspect of the level of its pre-adaptation, the rapid cold hardening (RCH) response in this species was investigated. When juvenile (L1–L2) and mature (L3–L4) larvae of E. murphyi were directly exposed to progressively lower temperatures for 8h, they exhibited Discriminating Temperatures (DTemp, temperature at which there is 10–20% survival of exposed individuals) of −11.5 and −12.5°C, respectively. The mean SCP was above −7.5°C in both larval groups, confirming the finding of previous studies that this species is freeze-tolerant. Following gradual cooling (0.2°Cmin−1), survival was significantly greater at the DTemp in both larval groups. The response was strong, lowering the lower lethal temperature (LLT) by up to 6.5°C and maintaining survival above 80% for at least 22h at the DTemp. RCH was also exhibited during the cooling phase of an ecologically relevant thermoperiodic cycle (+4°C to −3°C). Mechanistically, the response did not affect freezing, with no alteration in the supercooling point (SCP) found following gradual cooling, and was not induced while the organism was in a frozen state. These results are discussed in light of E. murphyi’s pre-adaptation to conditions on Signy Island and its potential to colonize regions further south in the maritime Antarctic.

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

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.

Cold tolerance and cold‐induced modulation of gene expression in two Drosophila virilis group species with different distributions

The importance of high and low temperature tolerance in adaptation to changing environmental conditions has evoked new interest in modulations in gene expression and metabolism linked with stress tolerance. We investigated the effects of rapid cold hardening and cold acclimatization on the chill coma recovery times of two Drosophila virilis group species, Drosophila montana and D. virilis, with different distributions and utilized a candidate gene approach to trace changes in their gene expression during and after the cold treatments. The study showed that cold acclimatization clearly decreases chill coma recovery times in both species, whereas rapid cold hardening did not have a significant effect. Microarray analysis revealed several genes showing expression changes during different stages of cold response. Amongst the 219 genes studied, two genes showed rather consistent expression changes: hsr-omega, which was up-regulated in both study species during cold acclimatization, and Eip71CD, which was down-regulated in nearly all of the cold treatments. In addition, 29 genes showed expression changes that were more treatment- and/or species specific. Overall, different stages of cold response elicited changes mainly in genes involved in heat shock response, circadian rhythm and metabolism.

Long-Term Cold Acclimation Extends Survival Time at 0°C and Modifies the Metabolomic Profiles of the Larvae of the Fruit Fly Drosophila melanogaster

Background Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately −5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum.Principal FindingsWe found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt50) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt50 (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines.ConclusionThird-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring.

FITNESS COSTS OF RAPID COLD-HARDENING IN CERATITIS CAPITATA

Rapid cold-hardening (RCH) is a unique form of phenotypic plasticity which confers survival advantages at low temperature. The fitness costs of RCH are generally poorly elucidated and are important to understanding the evolution of plastic physiology. This study examined whether RCH responses, induced by ecologically relevant diel temperature fluctuations, carry metabolic, survival, or fecundity costs. We predicted that potential costs in RCH would be manifested as differences in metabolic rate, fecundity, or survival in flies which have hardened versus those which have not, or flies that have experienced more RCH events would show greater costs than those which have experienced fewer events. One group of flies cooled to 10°C for 2 h for 11 consecutive days experienced daily RCH (Hardened), whereas the other group exposed to 15°C for the same 2-h period each day formed a Control group. Hardened flies had higher survival at -5°C for 2 h than control flies (69 ± 9% vs. 44 ± 19%, P = 0.04). Hardened flies showed no metabolic or fecundity costs, but had reduced average survival (P = 0.0403). Thus, a major cost to repeated low temperature exposures in Ceratitis capitata is through direct mortality caused by chilling injury, although this appears not to be a direct cost of RCH.

Enhancement of supercooling capacity and survival by cold acclimation, rapid cold and heat hardening in Spodoptera exigua

Insects can increase their resistance to cold stress by prior exposure to non-lethal cold temperatures. Here, we investigated the supercooling capacity and survival of eggs, 3rd and 5th instar larvae, and pupae of Spodoptera exigua (Lepidoptera: Noctuidae) during CA, and responses to various pre-treatment protocols, including constant temperatures, thermoperiods, and RCH, RHH, RCH+RHH and RHH+RCH combined with thermoperiods. Only acclimated eggs demonstrated a significant decrease in SCP, from −20.7±0.3 to −22.9±0.3°C, among all experimental groups compared to non-acclimated stages. Survival increased by 17.5% for eggs, 40.0% and 13.3% for 3rd and 5th instar larvae, and by 20.0% for pupae after CA. Compared to controls, survival of eggs under the conditions of thermoperiod (5:15°C), thermoperiod (5:15°C)+RHH, and thermoperiod (5:15, 10:20, and 15:25°C)+RCH significantly increased. In addition, survival of 3rd and 5th instar larvae and pupae increased under the conditions of thermoperiod (5:15°C) and thermoperiod (5:15°C)+RCH, possibly due to the induction of heat shock proteins or cryoprotectants. However, the pre-treatments of thermoperiod+RCH+RHH and thermoperiod+RHH+RCH did not significantly enhance survival of any developmental stage. These adaptive responses may allow S. exigua to enhance supercooling capacity and survival in response to seasonal or unexpected diurnal decreases in environmental temperatures.

Rapid cold hardening increases cold and chilling tolerances more than acclimation in the adults of the sycamore lace bug, Corythucha ciliata (Say) (Hemiptera: Tingidae)

The sycamore lace bug, Corythucha ciliata is a new, invasive pest of Platanus trees in China. Although C. ciliata is often subjected to acute low temperatures in early winter and spring in northern and eastern China, the cold tolerance of C. ciliata has not been well studied. The objectives of this study were to determine whether adults of C. ciliata are capable of rapid cold hardening (RCH), and to compare the benefits of RCH vs. cold acclimation (ACC) in the laboratory. When the adult females incubated at 26 °C were transferred directly to the discriminating temperature (−12 °C) for 2 h, survival was only 22%. However, exposure to 0 °C for 4 h before transfer to −12 °C for 2 h induced RCH, i.e., increased survival to 68%. RCH could also be induced by gradual cooling of the insects at rates between 0.1 and 0.25 °C min −1. The protection against cold shock obtained through RCH at 0 °C for 4 h was lost within 1 h if the adults were returned to 26 °C before exposure to −12 °C. Survival at both −12 and −5 °C was greater for RCH-treated than for ACC-treated adults (for ACC, adults were kept at 15 °C for 5 days), and the lethal temperature (2 h exposure) was lower for RCH-treated than for ACC-treated adults. The results suggest that RCH may help C. ciliata survive the acute low temperatures that often occur in early winter and early spring in northern and eastern China.

Limited plasticity of low temperature tolerance in an Australian cantharid beetle Chauliognathus lugubris

Spatial and/or taxonomic bias in thermal tolerance and plasticity data can severely impact projections of climate change responses and limit the understanding of the evolution of thermal performance curves. Thus, further data from under‐represented groups and geographical locations are important for synthesizing and predicting the physiological responses of insects to climate variability. For example, the magnitude of rapid cold‐hardening (RCH) and seasonal acclimatization of low temperature tolerance are typically poorly documented for nondipteran species from the southern Hemisphere. Moreover, few studies assess RCH responses under different acclimation regimes. To address this paucity of data, the low temperature survival, RCH and acclimation ability of Chauliognathus lugubris (F.) are assessed from an adult aggregation collected in Armidale, New South Wales, Australia. Beetles are acclimated to either 27 or 20 °C for 1 week and then tested for their ability to survive cold shock or rapidly cold‐harden. There is no effect of acclimation on low temperature survival (mean survival range at −5.4 °C for 2 h: 4–52% in 27 and 20 °C acclimation groups). In addition, beetles show no significant improvement in survival after acute thermal pretreatments. In conclusion, these data suggest a generally poor acclimation potential of low temperature survival and no RCH responses in adult Australian cantharid beetles, which is accordance with what might be expected given the microclimate experienced, their ability for behavioural regulation and the life history of the species.

Thermal tolerance of Frankliniella occidentalis: Effects of temperature, exposure time, and gender

Western flower thrips (WFT), Frankliniella occidentalis, is an invasive worldwide pest that causes great economic loss. Temperature plays an important role in shaping insect distributions. In this study, we report the survival rates of 3-day-old WFT exposed to rapid changes in extreme high and low temperatures. Temperature, exposure time, and gender all significantly affected the survival of adult WFT, with the lowest survival associated with more extreme temperatures and/or longer exposures. The temperature required to kill 50% of exposed WFT individuals (LT 50) decreased with extended exposure time, but females were more tolerant to extreme temperature than males. Investigation of rapid cold or heat hardening suggested that a short prior exposure to a sub-lethal low or high temperature increased WFT survival during a subsequent exposure to a lethal temperature. Tolerance of extreme temperatures and an ability to undergo rapid hardening are of great ecological relevance in determining the geographic distribution of WFT, allowing it to survive better in temporary bouts of extreme temperature stress. Our findings provide useful information on the environmental limits on the distribution of WFT, which have implications for control of this pest.

Rapid Cold Hardening and Expression of Heat Shock Protein Genes in the B-BiotypeBemisia tabaci

This paper describes the rapid cold hardening processes of the sweetpotato whitefly, Bemisia tabaci (Gennadius). It was found that all developmental stages of B. tabaci have the capacity of rapid cold hardening and the length of time required to induce maximal cold hardiness at 0 °C varies with stage. There was only 18.3% survival when adult whiteflies were transferred directly from 26 °C to -8.5 °C for 2 h. However, exposure to 0 °C for 1 h before transfer to -8.5 °C increased the survival to 81.2%. The whiteflies show "prefreeze" mortality when they were exposed to temperatures above the supercooling point (SCP), although the range of SCP of whiteflies is -26 °C to -29 °C. The rapid cold hardening had no effect on SCP and reduced the lower lethal temperature of adults from -9 °C to -11 °C. Rapid cold-hardened adults had a similar lifespan as the control group but deposited fewer eggs than nonhardened individuals. The expression profiles during cold hardening and recovery from this process revealed that HSP90 did not respond to cold stress. However, HSP70 and HSP20 were significantly induced by cold with different temporal expression patterns. These results suggest that the rapid cold hardening response is possibly advantageous to whiteflies that are often exposed to drastic temperature fluctuations in spring or autumn in northern China, and the expression of HSP70 and HSP20 may be associated with the cold tolerance of B. tabaci.

Rapid cold-hardening response in Ophraella communa LeSage (Coleoptera: Chrysomelidae), a biological control agent of Ambrosia artemisiifolia L.

In China, an unintentionally introduced beetle Ophraella communa shows good control efficiency against invasive common ragweed, Ambrosia artemisiifolia. With the aim of understanding the capacity of O. communa to overcome sub-zero low temperature rapidly, the longevity, survival and fecundity of the beetle adults were studied at conditions of different rapid cold-hardening in the laboratory. The results showed that approximately 20% female or male individuals still survive at −12°C for 2 h, thus the discriminating temperature for rapid cold-hardening of both female and male beetles is −12°C. The survival, longevity and fecundity of adult beetles were significantly affected by rapid cold-hardening treatments. Compared to the control, the survival rates of adult beetles experiencing 2 h of rapid cold-hardening at 2, 5 and 8°C prior to exposure to its discriminating temperature (−12°C) were significantly increased. Adult longevity and fecundity were significantly higher after pre-treatment at 2, 5 and 8°C for 2 h than those of the control. Both longest longevity and maximum fecundity of adult beetles were observed at the rapid cold-hardening of 2°C. Our experiment implies that the cold-tolerance ability of O. communa can be rapidly increased after rapid cold-hardening treatments, which reveals an important ecological significance for population establishment and expansion of O. communa in the invaded areas of common ragweed.

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.

The oatmeal nematode Panagrellus redivivus survives moderately low temperatures by freezing tolerance and cryoprotective dehydration

The cold tolerance abilities of only a few nematode species have been determined. This study shows that the oatmeal nematode, Panagrellus redivivus, has modest cold tolerance with a 50% survival temperature (S (50)) of -2.5°C after cooling at 0.5°Cmin(-1) and freezing for 1h. It can survive low temperatures by freezing tolerance and cryoprotective dehydration; although freezing tolerance appears to be the dominant strategy. Freezing survival is enhanced by low temperature acclimation (7days at 5°C), with the S (50) being lowered by a small but significant amount (0.42°C). There is no cold shock or rapid cold hardening response under the conditions tested. Cryoprotective dehydration enhances the ability to survive freezing (the S (50) is lowered by 0.55°C, compared to the control, after 4h freezing at -1°C) and this effect is in addition to that produced by acclimation. Breeding from survivors of a freezing stress did not enhance the ability to survive freezing. The cold tolerance abilities of this nematode are modest, but sufficient to enable it to survive in the cold temperate environments it inhabits.

Survival rate and expression of Heat-shock protein 70 and Frost genes after temperature stress in Drosophila melanogaster lines that are selected for recovery time from temperature coma

In this study, we investigated the physiological mechanisms underlying temperature tolerance using Drosophila melanogaster lines with rapid, intermediate, or slow recovery from heat or chill coma that were established by artificial selection or by free recombination without selection. Specifically, we focused on the relationships among their recovery from heat or chill coma, survival after severe heat or cold, and survival enhanced by rapid cold hardening (RCH) or heat hardening. The recovery time from heat coma was not related to the survival rate after severe heat. The line with rapid recovery from chill coma showed a higher survival rate after severe cold exposure, and therefore the same mechanisms are likely to underlie these phenotypes. The recovery time from chill coma and survival rate after severe cold were unrelated to RCH-enhanced survival. We also examined the expression of two genes, Heat-shock protein 70 (Hsp70) and Frost, in these lines to understand the contribution of these stress-inducible genes to intraspecific variation in recovery from temperature coma. The line showing rapid recovery from heat coma did not exhibit higher expression of Hsp70 and Frost. In addition, Hsp70 and Frost transcription levels were not correlated with the recovery time from chill coma. Thus, Hsp70 and Frost transcriptional regulation was not involved in the intraspecific variation in recovery from temperature coma.

Phenotypic plasticity of thermal tolerance contributes to the invasion potential of Mediterranean fruit flies (Ceratitis capitata)

1. The invasion success of Ceratitis capitata probably stems from physiological, morphological, and behavioural adaptations that enable them to survive in different habitats. However, it is generally poorly understood if variation in acute thermal tolerance and its phenotypic plasticity might be important in facilitating survival of C. capitata upon introduction to novel environments.2. Here, by comparison of widely distributed C. capitata with a narrowly distributed congener, C. rosa, we show that both species have similar levels of survival to acute high and low temperature exposures under common rearing conditions. However, these species differ dramatically in the time‐course of plastic responses to acute low temperature treatments.3. The range of temperatures that induce rapid cold hardening (RCH) are similar for both species. However, C. capitata has two distinct advantages over C. rosa. First, at 5°C C. capitata develops RCH significantly faster than C. rosa. Second, C. capitata maintains a RCH response longer than C. rosa (8 vs. 0.5 h).4. A simple population survival model, based on the estimated time‐course of RCH responses determined for both species, was undertaken to simulate time to extinction for both species introduced into a similar thermally variable environment. The model showed that time to extinction is greater for C. capitata than for C. rosa, especially in habitats where temperatures frequently drop below 10°C.5. Thus, variation in RCH responses may translate into significant variation in survival upon introduction to novel thermal habitats for C. capitata, particularly in cooler and more thermally variable geographic regions, and may contribute to their ongoing invasion success relative to other, more geographically constrained Ceratitis species.

Rapid Cold Hardening in Drosophila requires MAPK signaling

Increasing evidence supports the hypothesis that rapid cold hardening (RCH) protects insects in nature. However, the cellular/biochemical mechanisms of RCH remain unclear. In Sarcophaga crassipalpis RCH involves accumulation of glycerol and Mitogen Activated Protein Kinase (MAPK) signaling pathways. For Drosophila melanogaster RCH correlates with changes in membrane lipid composition, but does not appear to involve the production of low‐molecular weight cryoprotectants. The present study utilized a pharmacological approach to determine whether RCH in D. melanogaster involves MAPK signaling. Rearing D. melanogaster on yeast paste containing the MAPK inhibitor SB203580 for 2 days following eclosion impaired their ability to rapidly cold harden (evaluated as resistance to mortality caused by cold shock injury), in a dose dependent manner. These data suggest that MAPK signaling is important to RCH in D. melanogaster.

Cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera

The cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera, was examined using measures of total body water content, supercooling point and mortality for a range of sub-zero temperature exposure regimes. The supercooling points for starved and fed nymphs were −13.1 ± 0.9 and −12.6 ± 1.6 °C, and freezing caused complete mortality. Above these temperatures, nymphs were cold tolerant to different degrees based on whether they were starved or given access to food and water for 24 h prior to exposure. The rate of cooling also had a significant effect on mortality. Very rapid cooling to −7 °C caused 84 and 87% mortality for starved and fed nymphs respectively, but this significantly decreased for starved nymphs if temperature declined by more ecologically realistic rates of 0.5 and 0.1 °C min −1. These results are indicative of a rapid cold hardening response and are discussed in terms of the likely effects of cold nights and frost on first-instar nymphal survival in the field.

Responses of the bed bug, Cimex lectularius, to temperature extremes and dehydration: levels of tolerance, rapid cold hardening and expression of heat shock proteins

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately -20 degrees C, all were killed by a direct 1 h exposure to -16 degrees C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4 degrees C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1-h exposure to 0 degrees C improved their subsequent tolerance of -14 and -16 degrees C. In response to heat stress, fewer than 20% of the bugs survived a 1-h exposure to 46 degrees C, and nearly all were killed at 48 degrees C. Dehydration, heat acclimation at 30 degrees C for 2 weeks and rapid heat hardening at 37 degrees C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.

Low-temperature tolerance of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa

(1) The low-temperature tolerance of false codling moth (FCM) Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant aspect of this pest's population dynamics and has implications for post-harvest control and sterile insect technique programs. (2) Here, we report results of experiments examining the effects of time, temperature and their interactions on low-temperature tolerance in adult FCM. In addition, using a variety of thermal pre-treatments, we examine the potential for hardening responses over several hours to improve low-temperature tolerance in FCM. (3) Lower lethal temperature assays showed significant effects of time, temperature and significant interactions between time and temperature on survival ( p<0.0001 in all cases). The temperature at which the probability of survival of 50% of the FCM population after 2 h of exposure was −4.5 °C, which varied significantly to −0.5 °C in 10 h. Gender and early adult age did not affect low-temperature tolerance of FCM. (4) Using a range of non-lethal, low- and high-temperature pre-treatments, FCM survival could not be increased ( p>0.84) and thus limited evidence for rapid cold hardening was found. These results are discussed with respect to microclimate temperatures in typical FCM environments and have implications for understanding population dynamics in this species and the diversity of low-temperature responses of insects.