Hardening trumps acclimation in improving cold tolerance of Drosophila melanogaster larvae

Abstract Chill‐susceptible insects are able to improve their survival of acute cold exposure over both the short term (i.e. hardening at a relatively severe temperature) and longer term (i.e. acclimation responses at milder temperatures over a longer time frame). However, the mechanistic overlap of these responses is not clear. Four larval stages of four different strains of Drosophila melanogaster are used to test whether low temperature acclimation (10 °C for 48 h) improves the acute cold tolerance (LT₉₀, ～2 h) of larvae, and whether acclimated larvae still show hardening responses after brief exposures to nonlethal cold or heat, or a combination of the two. Acclimation results in increased cold tolerance in three of four strains, with variation among instars. However, if acclimation is followed by hardening pre‐treatments, there is no improvement in acute cold survival. It is concluded that short‐term thermal responses (e.g. hardening) may be of more ecological relevance to short‐lived life stages such as larvae, and that the mechanisms of low temperature hardening and acclimation in D. melanogaster may be antagonistic, rather than complementary.     

Geographical and diapause-related cold tolerance in the blow fly,Calliphora vicina

Three geographical strains of the blow fly, Calliphora vicina, were tested for cold tolerance at 0 degrees, -4 degrees and -8 degrees C. Survival to eclosion after 1 to 18 days of cold exposure was greater for diapause-destined larvae than for nondiapause-destined larvae of the two northern strains (Nallikari, Finland 65 degrees N and Edinburgh, Scotland 55 degrees N) but not for the southernmost strain (Barga, Italy 44 degrees N) where no clear differences were apparent. Diapause-destined larvae of the Edinburgh strain were more cold tolerant than those from Nallikari, at both -4 degrees and -8 degrees C, a difference possibly attributable to the long-lasting snow cover in the more northern locality, which might insulate the overwintering soil microclimate. At 0 degrees C, however, Nallikari larvae were more cold tolerant than Edinburgh or Barga. This was also the case for nondiapause-destined larvae, indicating that cold tolerance may occur, in part, independently of the diapause programme. In all three strains diapausing larvae were more cold tolerant than same-age (nondiapausing) pupae. For Nallikari, but not Barga, wandering larvae from short-day exposed flies, therefore initially programmed for diapause, but diverted from the diapause pathway by larval breeding at 19 degrees C, were significantly more cold tolerant than nondiapause larvae from long-day parents, indicating some maternal regulation of larval cold tolerance. There was, however, no evidence for an additional cold hardiness in larvae acclimatised to cold by a gradual reduction of temperature.     

The influence of developmental stage on cold shock resistance and ability to cold-harden in Drosophila melanogaster

Thermal sensitivity and ability to rapidly cold- and heat-harden may change during ontogeny. This study reports how inherent cold tolerance and ability to rapidly cold-harden change across eight developmental stages in both genders of Drosophila melanogaster using a similar experimental approach for all stages. Inherent cold tolerance was estimated as LT50 by assaying cold shock survival over a wide range of temperatures (-16 to 5 degrees C). Rapid cold-hardening (RCH) was applied by cooling from 25 to 0 degrees C at -0.25 degrees C min(-1) followed by 1 h at 0 degrees C. Individuals were cold shocked either directly or after RCH to estimate the effect of RCH. We found large variation in cold tolerance among developmental stages and minor differences between genders. Eggs were most tolerant followed by adults, pupae and larvae. In the light of this and other studies it is suggested that there is a general pattern of stage specific thermal stress resistance in Drosophila. The capacity to rapidly cold-harden was found in both sexes of larval, pupal and adult stages, though some developmental stages showed negative or neutral effects of RCH which was probably due to the cost associated with the hardening treatment in these cold susceptible stages. The early presence of RCH indicates that the mechanisms behind hardening are not stage specific and that RCH may be an ecologically important trait in early stages of ontogeny.     

Rapid cold-hardening in a Karoo beetle, Afrinus sp.

Abstract.  In the insect rapid cold-hardening response, survival at subzero temperatures is greatly improved by a brief pre-exposure at a milder temperature. It is predicted that insects with minimal cold tolerance capabilities living in variable environments should use rapid cold-hardening to survive sudden cold snaps. This is tested in Afrinus sp., a beetle that lives in an exposed habitat on rock outcrops in the Karoo Desert, South Africa, where microclimate temperatures drop infrequently to below freezing. Afrinus sp. shows a significant rapid cold-hardening response: survival of a 2-h exposure to −6.5 °C is much improved after pre-exposure to −2 °C, to 0 °C with a 2-h return to the rearing temperature, and to 40 °C, but not after pre-exposure to 0 °C. Little is known about the mechanism of the rapid cold-hardening response, although the data suggest that rapid cold-hardening may be mediated via several different mechanisms.     

The sea urchin Lytechinus variegatus lives close to the upper thermal limit for early development in a tropical lagoon

Thermal tolerance shapes organisms' physiological performance and limits their biogeographic ranges. Tropical terrestrial organisms are thought to live very near their upper thermal tolerance limits, and such small thermal safety factors put them at risk from global warming. However, little is known about the thermal tolerances of tropical marine invertebrates, how they vary across different life stages, and how these limits relate to environmental conditions. We tested the tolerance to acute heat stress of five life stages of the tropical sea urchin Lytechinus variegatus collected in the Bahía Almirante, Bocas del Toro, Panama. We also investigated the impact of chronic heat stress on larval development. Fertilization, cleavage, morula development, and 4‐armed larvae tolerated 2‐h exposures to elevated temperatures between 28–32°C. Average critical temperatures (LT₅₀) were lower for initiation of cleavage (33.5°C) and development to morula (32.5°C) than they were for fertilization (34.4°C) or for 4‐armed larvae (34.1°C). LT₅₀ was even higher (34.8°C) for adults exposed to similar acute thermal stress, suggesting that thermal limits measured for adults may not be directly applied to the whole life history. During chronic exposure, larvae had significantly lower survival and reduced growth when reared at temperatures above 30.5°C and did not survive chronic exposures at or above 32.3°C. Environmental monitoring at and near our collection site shows that L. variegatus may already experience temperatures at which larval growth and survival are reduced during the warmest months of the year. A published local climate model further suggests that such damaging warm temperatures will be reached throughout the Bahía Almirante by 2084. Our results highlight that tropical marine invertebrates likely have small thermal safety factors during some stages in their life cycles, and that shallow‐water populations are at particular risk of near future warming.     

Thermal death kinetics of fifth-instar Plodia interpunctella (Lepidoptera: Pyralidae)

Heat treatments have been suggested as alternatives to chemical fumigants for control of postharvest insects in dried fruits and nuts. Conventional forced hot air treatments heat product too slowly to be practical, but radio frequency treatments are capable of more rapid product heating. While developing radio frequency heat treatments for dried fruits and nuts, the heat tolerance of nondiapausing and diapausing fifth-instar larvae of the Indianmeal moth, Plodia interpunctella (Hübner), was determined using a heating block system developed by Washington State University. Both a 0.5th order kinetic model and a classical empirical model were used to estimate lethal exposure times for temperatures of 44-52 degrees C for nondiapausing fifth-instar larvae. We obtained 95% mortality at exposures suitable for practical radio frequency treatments (< or = 5 min) with temperatures of 50 and 52 degrees C. Diapausing larvae were significantly more tolerant than nondiapausing larvae at the lowest treatment temperature and shortest exposure, but differences were not significant at more extreme temperature-time combinations. Previous studies showed that fifth-instar larvae of the navel orangeworm, Amyelois transitella (Walker), were more heat tolerant than either diapausing or nondiapausing Indianmeal moth larvae. Consequently, efficacious treatments for navel orangeworm would also control Indianmeal moth.     

The evolution of cold tolerance in Drosophila larvae

Temperature is a primary determinant of insect and other ectotherm distribution and activity. Physiological and behavioral adaptations allow many insects to survive at subzero temperatures, yet the evolutionary influences on insect cold tolerance are unclear. Supercooling points, basal cold tolerance, cold-tolerance strategy, and inducible cold tolerance from rapid cold-hardening or acclimation were measured in a phylogenetically independent context in larvae of 27 phylogenetically diverse Drosophila species acquired from stock collections. Supercooling capacity is attributed primarily to physical factors, such as dry mass and water mass. Species of the obscura group were more resistant to acute cold tolerance than species of other groups within the genus, and plasticity in cold tolerance is constrained by phylogeny rather than by basal cold tolerance. The more cold-tolerant freeze-avoiding species appear to have arisen multiple times in Drosophila and are distinct from chill-susceptible species, which likely indicate the ancestral state. A phylogenetic influence is apparent on several measures of cold tolerance, which show considerable interspecific variation and indicate varying physiological mechanisms among Drosophila species when temperature limits are met.     

Physiological and molecular mechanisms associated with cross tolerance between hypoxia and low temperature in Thaumatotibia leucotreta

Biochemical adaptations allow insects to withstand exposures to hypoxia and/or hypothermia. Exposure to hypoxia may interact either synergistically or antagonistically with standard low temperature stress responses yet this has not been systematically researched and no clear mechanism has been identified to date. Using larvae of false codling moth Thaumatotibia leucotreta, a pest of southern Africa, we investigated the physiological and molecular responses to hypoxia or temperature stress pre-treatments, followed by a standard low temperature exposure. Survival rates were significantly influenced by pre-treatment conditions, although T. leucotreta shows relatively high basal resistance to various stressors (4% variation in larval survival across all pre-treatments). Results showed that mild pre-treatments with chilling and hypoxia increased resistance to low temperatures and that these responses were correlated with increased membrane fluidity (increased UFA:SFA) and/or alterations in heat shock protein 70 (HSP70); while general mechanical stress (shaking) and heat (2 h at 35 °C) do not elicit cross tolerance (no change in survival or molecular responses). We therefore found support for some limited cold hardening and cross tolerance responses. Given that combined exposure to hypoxia and low temperature is used to sterilize commodities in post-harvest pest management programs, researchers can now exploit these mechanisms involved in cross tolerance to develop more targeted control methods.     

RAPID COLD HARDENING PROVIDING HIGHER COLD TOLERANCE THAN COLD ACCLIMATION IN THE PINE NEEDLE GALL MIDGE THECODIPLOSIS JAPO‐NENSIS LARVAE*

Abstract The responses of overwintering larvae of the pine needle gall midge Thecodiplosis japonensis Uchida et Inouye to rapid cold hardening and cold acclimation were studied. A rapid cold hardening response is found in the 3rd instar larvae of T. japonensis. When overwintering larvae are transferred directly from 27°C to ‐ 15°C for 3 h, there is only 17.9% survival, whereas exposure to 4°C for 2 h prior to transfer to ‐ 15°C increases survival to 40.0%. The acquired cold tolerance is transient and is rapidly lost (after 15 min at 27°C). Rapid cold hardening is more effective in maintaining larval survival than cold acclimation. Different mechanisms are suggested to regulate the insect's cold hardiness under rapid cold hardening and cold acclimation.     

Diapause induction in Trichogramma embryophagum Htg. (Hym., Trichogrammatidae): the dynamics of thermosensitivity

It is known that the low temperature is the most important factor inducing the pre-pupal diapause in Trichogramma species. The position of the thermosensitive period over the life cycle and temporal variation of the degree of responsiveness were investigated in T. embryophagum Htg. by transferring pre-imaginal stages between 'neutral' temperature of 15°C and 'diapause-inducing' temperature of 10°C. Our experiments showed that 6 days long exposure at 10°C significantly increased the percentage of diapausing pre-pupae when started during rather large part of development: from embryo up to early pre-pupa. The highest thermosensitivity was recorded during the embryo and the larval stages, with some decrease during the hatching period. Treatments with shorter cold exposures (2–3 days) gave similar results. Even 24 h long exposure at 10°C increased the percentage of diapausing pre-pupae when applied during egg or early larval stage. Being started at the same stage of development, longer cold exposures caused stronger increase in the percentage of diapausing individuals. The experiments did not reveal any significant daily changes in thermosensitivity: at 12 : 12 h light : dark, larvae subjected to the low temperature during six photophases showed practically the same percentage of diapausing individuals as those subjected to the low temperature during six scotophases, and as those subjected to the 3 days long uninterrupted cold exposure. Hence, in natural conditions even occasional short-term cold periods could be accumulated.     

Dehydration-induced cross tolerance of Belgica antarctica larvae to cold and heat is facilitated by trehalose accumulation

Larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), are frequently exposed to dehydrating conditions on the Antarctic Peninsula. In this study, we examined how rates and levels of dehydration alter heat and cold tolerance and how these relate to levels of trehalose within the insect. When dehydrated, larvae tolerated cold and heat stress more effectively, although resistance to cold was more pronounced than heat resistance. Slow dehydration was more effective than rapid dehydration in increasing temperature tolerance. Severe dehydration (50% reduction in water content) caused a much greater increase in temperature tolerance than did mild dehydration (e.g. 10% water loss). Larvae severely dehydrated at a slow rate (98% RH) were more temperature tolerant than those dehydrated quickly (0 or 75% RH). These results indicate that the slower dehydration rate allows the larvae to more effectively respond to reduced water levels and that physiological adjustments to desiccation provide cross tolerance to cold and heat. Levels of trehalose increased during dehydration and are likely a major factor increasing subsequent cold and heat resistance. This hypothesis was also supported by experimental results showing that injection of trehalose enhanced resistance to temperature stress and dehydration. We conclude that changes in temperature tolerance in B. antarctica are linked to the rate and severity of dehydration and that trehalose elevation is a probable mechanism enhancing this form of cross tolerance.     

Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis

Developmental patterns of low-temperature tolerance and glycerol production were determined for larval, pupal and adult stages of the flesh fly Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). Both diapause and non-diapause-destined flies were reared at relatively high temperatures, 20° or 25°C, prior to testing. Cold tolerance was greatest for diapause pupae aged 12–35 days after pupariation. Among non-diapause-destined flies, pupae exhibited a greater level of low temperature tolerance than larvae or adults. Although diapause pupae were more tolerant than non-diapause pupae maximal cold tolerance was not attained in either group until 10 days after pupariation. Non-diapause-destined feeding and wandering larvae had higher glycerol levels than larvae destined for diapause. During the first 6 weeks after pupariation glycerol titres increased steadily in diapause pupae. Rapid loss of glycerol is associated with the termination of pupal diapause.     

Effect of cold acclimation and rapid cold-hardening on the survival of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) under cold stress

Spodoptera frugiperda is a highly invasive pest species that recently invaded Africa and Asia causing severe economic losses, primarily related to corn and rice crops. Temperature is one of the most important environmental factors that influence the invasion of pests into new habitats. However, little is known regarding the thermal tolerance characteristics of invasive S. frugiperda. Thus, we investigated the response of four developmental stages of S. frugiperda (i.e., eggs, third and sixth instar larvae, and pupae) to cold acclimation (CA) and rapid cold-hardening (RCH). All individuals suffered high mortality with 24-h temperature treatments at <?5°C and >35 °C. The CA treatment significantly increased the survival rate of the eggs and third instar larvae, although it did not affect the sixth instar larvae and it decreased the pupation rate. The RCH treatment at 5 °C for 5 h or 2 °C for 2 h increased the cold tolerance capabilities of the third and sixth instar larvae, respectively. Thus, the larval stage appears to be crucial for the cold tolerance of S. frugiperda. Our findings improve the current understanding of the cold tolerance characteristics of S. frugiperda and indicate its potential for survival in the newly invaded temperate regions of Asia.     

Thermal death kinetics of red flour beetle (Coleoptera: Tenebrionidae)

While developing radio frequency heat treatments for dried fruits and nuts, we used a heating block system developed by Washington State University to identify the most heat-tolerant life stage of red flour beetle, Tribolium castaneum (Herbst), and to determine its thermal death kinetics. Using a heating rate of 15 degrees C/min to approximate the rapid heating of radio frequency treatments, the relative heat tolerance of red flour beetle stages was found to be older larvae > pupae and adults > eggs and younger larvae. Lethal exposure times for temperatures of 48, 50, and 52 degrees C for the most heat-tolerant larval stage were estimated using a 0.5th order kinetic model. Exposures needed for 95% mortality at 48 degrees C were too long to be practical (67 min), but increasing treatment temperatures to 50 and 52 degrees C resulted in more useful exposure times of 8 and 1.3 min, respectively. Red flour beetle was more sensitive to changes in treatment temperature than previously studied moth species, resulting in red flour beetle being the most heat-tolerant species at 48 degrees C, but navel orangeworm, Amyelois transitella (Walker), being most heat tolerant at 50 and 52 degrees C. Consequently, efficacious treatments for navel orangeworm at 50-52 degrees C also would control red flour beetle.     

Effect of temperature on development, overwintering and establishment potential of Franklinothrips vespiformis in the UK

This study investigated the effect of temperature on the development and overwintering potential of the predatory thrips Franklinothrips vespiformis (Crawford) (Thysanoptera: Aeolothripidae), a biological control agent used against glasshouse pests in continental Europe and Israel. Developmental rates increased linearly with rearing temperatures. It was estimated that 304.9 degree days, above a lower threshold temperature of 11.9 °C, were required for F. vespiformis to complete development from egg to adult eclosion. The effect of low temperatures (–5, 0, and 5 °C) was examined on adult female and larval survival. Subsequent reproductive and developmental attributes of survivors were also investigated. Lethal time experiments indicated that larval stages are more cold tolerant than adult F. vespiformis females. Surviving larvae increased their developmental times to adults with decreasing temperature and increasing exposure periods and second instars were significantly more successful than first instars in reaching adulthood. Surviving adult females decreased their oviposition rate with decreasing temperature and increasing exposure periods, and exposures to low temperatures affected the number of viable eggs produced. The results are discussed in the context of overwintering and establishment potential of F. vespiformis in the UK in the event of introducing the predatory thrips as a biological control agent against glasshouse pests.     

Cold tolerance and cold hardening strategy of the Japanese pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae)

The Japanese pine sawyer, Monochamus alternatus , is an important pine forest pest and vector transmitting the pine wilt nematode that causes pine wilt disease. Low temperatures in autumn, winter and spring often differentially affect mortality of M. alternatus larvae. In this paper, we mainly compared the differences of mortality and cold hardening of larvae from different seasons, based on supercooling point (SCP) and cumulative probability of individuals freezing (CPIF). The cold hardening of the larvae from autumn, winter and spring seasons were largely different. Correlations between mortality and CPIF of autumn and spring larvae were highest on day 1/4, and gradually decreased with prolonged exposure duration. This beetle's death mainly resulted from freezing in short exposure duration. However, the correlation between mortality and CPIF of winter larvae increased gradually with the prolonged exposure duration. Death did not mainly result from freezing in long exposure duration. Autumn larvae are more susceptible and adaptable than winter and spring larvae. Winter larvae have a slight freeze-tolerance trend. Our research showed that M. alternatus came into complex cold-hardening strategies under natural selection. Freeze avoidance is the primary strategy; with prolonged exposure duration to above SCP or < 0 °C, chill tolerance is more important; this is followed by freeze tolerance during harsh winters.     

The effects of selection for cold tolerance on cross-tolerance to other environmental stressors in Drosophila melanogaster

Abstract  Cross tolerance, whereby tolerance to one environmental stress is correlated with tolerance to other stressors, is thought to be widespread in insects. We used lines of Drosophila melanogaster Meigen (Diptera: Drosophilidae) selected for survival at a 1-h exposure to −5°C to examine the extent to which this selection results in increased tolerance to other stresses, including high and low temperatures, desiccation and starvation. While selection improved tolerance to acute cold exposure and survival at −5°C, there was little effect of selection regime on tolerance to other stressors. There was no correlation between tolerances to any of the stressors, suggesting different mechanisms of tolerance. This supports arguments that correlations between stress tolerances during selection experiments with D. melanogaster may be coincidental. The magnitude of heat-hardening was apparently constrained by basal tolerance among lines, but the magnitude of the rapid cold-hardening response was not correlated with basal cold tolerance, implying that the relationship between inducible and basal tolerances differs at high and low temperatures.     

Upper lethal temperature limits of the common furniture beetle Anobium punctatum (Coleoptera: Anobiidae)

The susceptibility of the egg, larval and adult stages of Anobium punctatum De Geer (Coleoptera: Anobiidae) to heat (46–54°C, 25–30% RH) was investigated. The larval stage was found to be most tolerant to heat. Very short exposure (5 min) of the larvae to temperatures of 52°C and above led to 100% mortality of the larvae. Treatment for 1.5 and 1 h at 47° and 48°C, respectively, led to 100% mortality. At 46°C, longer treatment time (2.5 h) was necessary. Exposure to 45°C for 3.5 h did not lead to immediate death of the larvae. The actual mortality is assumed to be greater than the results actually reported, as most of the larvae had not resumed normal activity at the end of the two-week observation period. The egg and adult stages were more sensitive to heat than the larvae; shorter exposure times were sufficient to obtain 100% mortality.     

Evolution of thermotolerance in seasonal environments: the effects of annual temperature variation and life-history timing in Wyeomyia smithii

In organisms with complex life cycles, the adaptive value of thermotolerance depends on life-history timing and seasonal temperature profiles. We illustrate this concept by examining variation in annual thermal environments and thermal acclimation among four geographic populations of the pitcher plant mosquito. Only diapausing larvae experience winter, whereas both postdiapause and nondiapause adults occur only during the growing season. Thus, adults experience transient cold stress primarily during the spring. We show that adult cold tolerance (chill coma recovery) is enhanced in spring-like conditions via thermal acclimation but is unaffected by diapause state. Moreover, adult mosquitoes from northern populations were more cold tolerant than those from southern populations largely because acclimation responses were steeper in the north. In contrast to cold tolerance, there was no significant acclimation of heat tolerance (heat knockdown), and no significant differences in heat tolerance between northern and southern populations. Field temperature data show that because of evolved differences in diapause timing, adult exposure to cold stress is remarkably consistent across geography. This suggests that geographic variation in cold tolerance may not be the result of direct selection on adults. Our results illustrate the importance of the interplay between phenological and thermal adaptation for understanding variation along climatic gradients.