Water balance in the sugarbeet root maggot Tetanops myopaeformis,during long‐term low‐temperature storage and after freezing

The sugarbeet root maggot Tetanops myopaeformis Röder (Diptera: Ulidiidae) can be stored in moist sand at 4–6 °C for up to 5 years and is freeze‐tolerant. The majority of stored larvae survive in a state of post‐diapause quiescence and the remainder are in a multi‐year diapause. The present study aims to determine larval water content and water loss rates in diapausing and low‐temperature stored larvae. Body water content ranges from 57% to 70.1%. Two distinct groupings of larvae are revealed based on dry weights. The first group consists of the diapausing larvae and larvae stored for 1 year. This group has significantly higher dry weights than the second grouping, which consists of the larvae stored for 2 and 3 years. There are no significant differences within each group. Larval water losses follow a first‐order kinetic relationship with time. Larvae stored for 2 years lose water at a significantly higher rate than diapausing larvae. Larvae exhibit no active water uptake at storage temperatures. A freezing event does not induce a significant decrease in wet weights, nor does it increase larval water loss rates. These results indicate that metabolic water and the microclimate during storage are key factors enabling the long‐term survival of T. myopaeformis larvae during low‐temperature storage, and may provide insights for maintaining other insect species under similar conditions.     

Cold hardiness adaptations of codling moth, cydia pomonella

The cold hardiness adaptations of natural and laboratory reared populations of the codling moth, Cydia pomonella, were examined. Hemolymph, gut, and whole body supercooling points (SCPs), 24-h LT50s, polyhydroxy alcohol concentrations, hemolymph freezing points, and hemolymph melting points were determined. Nondiapausing codling moth larvae do not have appreciable levels of ice nucleators in the hemolymph or gut. Whole body supercooling points were higher than hemolymph supercooling points. For nondiapausing larvae, LT50s were significantly higher than both the whole body and the hemolymph supercooling points, indicating the presence of chill sensitivity. As the larvae left the food source and spun a cocoon, both hemolymph and whole body SCPs decreased. Diapause destined larvae had significantly lower hemolymph SCPs than nondiapausing larvae, but whole body SCPs were not significantly different from nondiapausing larvae of the same age. The LT50s of diapause destined and diapausing larvae were significantly lower than that of nondiapausing larvae. Codling moths are freezing intolerant, with LT50s close to the average whole body supercooling point in diapause destined and diapausing larvae. The overwintering, diapausing larvae effectively supercool to avoid lethal freezing by removal of ice nucleators from the gut and body without appreciable increase of antifreeze agents such as polyols or antifreeze proteins.     

Seasonal and geographical variation in diapause and cold hardiness of the Asian corn borer,Ostrinia furnacalis

Asian corn borer, Ostrinia furnacalis (Guenée), is a key corn pest in the Asian‐Western Pacific countries. It overwinters as full‐grown larvae in plant stalks or in a spun‐silk covering located in the plant debris in the temperate regions of China. Supercooling point (SCP) and survival rate after low sub‐zero temperature treatment were assessed for field‐collected populations in the laboratory using a cool bath with a 1°C/min cooling rate until ?40°C. Mean SCPs were varied among geographical populations, with a significant decline from ?22.7°C of Haikou, the multivoltine tropical population in the south, to ?28.5°C of Gongzhuling, the univoltine temperate population in the northeast of China. In addition, there was more than 1°C difference in SCP between Gongzhuling univoltine and bivoltine populations that were from the same geographic origin. Mean SCPs of the Guangzhou population fluctuated over the year, with significantly lower SCPs in winter than in other seasons, which correlated with a significantly higher proportion of diapausing larvae in winter than in other seasons. Over 41% of overwintering larvae from the northeast population could withstand to be supercooled for a few minutes to the low sub‐zero temperature of ?40°C, but only 6.7% of their southern counterparts did so. The findings from this study suggest that O. furnacalis mostly takes advantage of freeze avoidance as diapausing larvae for overwintering in the southern region, whereas it exhibits freeze tolerance in diapause in the northeastern region.     

AMP-activated protein kinase and metabolic regulation in cold-hardy insects

Winter survival for many insects depends on cold hardiness adaptations as well as entry into a hypometabolic diapause state that minimizes energy expenditure. We investigated whether AMP-activated protein kinase (AMPK) could be involved in this adaptation in larvae of two cold-hardy insects, Eurosta solidaginis that is freeze tolerant and Epiblema scudderiana that uses a freeze avoidance strategy. AMPK activity was almost 2-fold higher in winter larvae (February) compared with animals collected in September. Immunoblotting revealed that phosphorylation of AMPK in the activation loop and phosphorylation of acetyl-CoA carboxylase (ACC), a key target of AMPK, were higher in Epiblema during midwinter whereas no seasonal change was seen in Eurosta. Immunoblotting also revealed a significant increase in ribosomal protein S6 phosphorylation in overwintering Epiblema larvae, and in both Eurosta and Epiblema, phosphorylation of eukaryotic initiation factor 4E-binding protein-1 dramatically increased in the winter. Pyruvate dehydrogenase (PDH) E1α subunit site 1 phosphorylation was 2-fold higher in extracts of Eurosta larvae collected in February versus September while PDH activity decreased by about 50% in Eurosta and 80% in February Eurosta larvae compared with animals collected in September. Glycogen phosphorylase phosphorylation was 3-fold higher in Epiblema larvae collected in February compared with September and also in these animals, triglyceride lipase activity increased by 70% during winter. Overall, our study suggests a re-sculpting of metabolism during insect diapause, which shifted to a more catabolic poise in freeze-avoiding overwintering Epiblema larvae, possibly involving AMPK.     

Effects of summer frost exposures on the cold tolerance strategy of a sub-Antarctic beetle

The sub-Antarctic beetle Hydromedion sparsutum (Coleoptera, Perimylopidae) is common locally on the island of South Georgia where sub-zero temperatures can be experienced in any month of the year. Larvae were known to be weakly freeze tolerant in summer with a mean supercooling point (SCP) around -4 degrees C and a lower lethal temperature of -10 degrees C (15min exposure). This study investigated the effects of successive freezing exposures on the SCP and subsequent survival of summer acclimatised larvae. The mean SCP of field fresh larvae was -4.2+/-0.2 degrees C with a range from -1.0 to -6.1 degrees C. When larvae were cooled to -6.5 degrees C on 10 occasions at intervals of 30min and one and four days, survival was 44, 70 and 68%, respectively. The 'end of experiment' SCP of larvae surviving 10 exposures at -6.5 degrees C showed distinct changes and patterns from the original field population depending on the interval between exposure. In the 30min interval group, most larvae froze between -6 and -8 degrees C, a depression of up to 6 degrees C from the original sample; all larvae were dead when cooling was continued below the SCP to -12 degrees C. In the one and four day interval groups, most larvae froze above -6 degrees C, showing no change as a result of the 10 exposures at -6.5 degrees C. As with the 30min interval group, some larvae froze below -6 degrees C, but with a wider range, and again, all were dead when cooled to -12 degrees C. However, in the one and four day interval groups, some larvae remained unfrozen when cooled to -12 degrees C, a depression of their individual SCP of at least 6 degrees C, and were alive 24h after cooling. In a further experiment, larvae were cooled to their individual SCP temperature at daily intervals on 10 occasions to ensure that every larva froze every day. Most larvae which showed a depression of their SCP of 2-4 degrees C from their day one value became moribund or died after six or seven freezing events. Survival was highest in larvae with SCPs of -2 to -3 degrees C on day one and which froze at this level on all 10 occasions. The results indicate that in larvae in which the SCP is lowered following sub-zero exposure, the depression of the SCP is greatest in individuals that do not actually freeze. Further, the data suggest that after successive frost exposures in early winter the larval population may become segregated into two sub-populations with different overwintering strategies. One group consists of larvae that freeze consistently in the temperature range from -1 to -3 degrees C and can survive multiple freeze-thaw cycles. A second group with lower initial SCPs (around -6 degrees C), or which fall to this level or lower (down to -12 degrees C) after freezing on one or more occasions, are less likely to freeze through extended supercooling, but more likely to die if freezing occurs.     

Synchrotron X-Ray Visualisation of Ice Formation in Insects during Lethal and Non-Lethal Freezing

Although the biochemical correlates of freeze tolerance in insects are becoming well-known, the process of ice formation in vivo is subject to speculation. We used synchrotron x-rays to directly visualise real-time ice formation at 3.3 Hz in intact insects. We observed freezing in diapausing 3^rd instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above −14°C, and non-diapausing 3^rd instar larvae of C. amoena and Drosophila melanogaster (Diptera: Drosophilidae), neither of which survive freezing. Freezing was readily observed in all larvae, and on one occasion the gut was seen to freeze separately from the haemocoel. There were no apparent qualitative differences in ice formation between freeze tolerant and non-freeze tolerant larvae. The time to complete freezing was positively related to temperature of nucleation (supercooling point, SCP), and SCP declined with decreasing body size, although this relationship was less strong in diapausing C. amoena. Nucleation generally occurred at a contact point with the thermocouple or chamber wall in non-diapausing larvae, but at random in diapausing larvae, suggesting that the latter have some control over ice nucleation. There were no apparent differences between freeze tolerant and non-freeze tolerant larvae in tracheal displacement or distension of the body during freezing, although there was markedly more distension in D. melanogaster than in C. amoena regardless of diapause state. We conclude that although control of ice nucleation appears to be important in freeze tolerant individuals, the physical ice formation process itself does not differ among larvae that can and cannot survive freezing. This suggests that a focus on cellular and biochemical mechanisms is appropriate and may reveal the primary adaptations allowing freeze tolerance in insects.     

Cold hardiness of larvae of the southwestern corn borer, Diatraea grandiosella

The cold-hardening capacity of field-collected larvae from southeast Missouri and laboratory-reared larvae of the southwestern corn borer, Diatraea grandiosella Dyar, was examined. Supercooling points of non-diapause and diapause larvae collected from maize plants grown in Missouri (36°30 N lat.) were ca.-7.0°C. The hemolymph melting points of diapause field larvae (-0.8°C) were significantly lower than those of non-diapause larvae collected in July (-0.5°C). The supercooling points of hemolymph from non-diapause and diapause field larvae ranged randomly from-10° to-18°C. Supercooling points of non-diapause laboratory larvae increased from-13° to-10°C prior to pupation, whereas those of diapause larvae increased similarly before the onset of diapause, but then decreased during diapause to ca.-17°C. No change in supercooling points or capacity to survive in the presence of ice was observed in diapause laboratory larvae acclimated at 4°C for 63 days. Laboratory and field larvae began to freeze at ca.-1.5°C in the presence of ice, but survived to several degrees below their melting points. The high supercooling points of field larvae appeared to be due to the presence of an environmental ice-nucleator. Although data for laboratory larvae indicate sufficiently low supercooling points to permit winter survival in southeastern Missouri, considerable larval mortality occurs in the field due to inoculative freezing and the presence of an ice-nucleator.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Freeze tolerance in larvae of the winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae): a contrast to adult strategy for survival at low temperatures

The winter-active Diamesa mendotae Muttkowski (Diptera: Chironomidae) is freeze intolerant in the adult stage with a low mean supercooling point (SCP) of ~−20 °C. However, cold-hardiness strategies for immatures of this species are unknown. In this study, we measured SCP values for D. mendotae larvae, pupae and adults using surface-contact thermometry. In addition, the lower lethal temperature (LLT) was determined for the larval stage. The mean SCPs for larvae (−7.4 °C) and pupae (−9.1 °C) were relatively high compared to adults (−19.7 °C). Our results indicate that the larvae of D. mendotae are freeze tolerant with a LLT₉₉ (−25.4 °C), ~−10 °C lower than their minimum SCP (−15.6 °C). Freeze tolerance in these larvae may be a strategy to provide protection from short-term exposures to ice crystals or to permit diapause within frozen substrates. The change in cold-hardiness strategy from freeze tolerant to freeze intolerant between the larval and adult stages of this species is likely a result of the different habitats occupied by these two life stages.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

Host‐mediated shift in the cold tolerance of an invasive insect

While many insects cannot survive the formation of ice within their bodies, a few species can. On the evolutionary continuum from freeze‐intolerant (i.e., freeze‐avoidant) to freeze‐tolerant insects, intermediates likely exist that can withstand some ice formation, but not enough to be considered fully freeze tolerant. Theory suggests that freeze tolerance should be favored over freeze avoidance among individuals that have low relative fitness before exposure to cold. For phytophagous insects, numerous studies have shown that host (or nutrition) can affect fitness and cold‐tolerance strategy, respectively, but no research has investigated whether changes in fitness caused by different hosts of polyphagous species could lead to systematic changes in cold‐tolerance strategy. We tested this relationship with the invasive, polyphagous moth, Epiphyas postvittana (Walker). Host affected components of fitness, such as larval survivorship rates, pupal mass, and immature developmental times. Host species also caused a dramatic change in survival of late‐instar larvae after the onset of freezing—from less than 8% to nearly 80%. The degree of survival after the onset of freezing was inversely correlated with components of fitness in the absence of cold exposure. Our research is the first empirical evidence of an evolutionary mechanism that may drive changes in cold‐tolerance strategies. Additionally, characterizing the effects of host plants on insect cold tolerance will enhance forecasts of invasive species dynamics, especially under climate change.     

Cold tolerance, overwintering and establishment potential of Thrips palmi

The cold tolerance and overwintering survival of the quarantine regulated pest and virus vector, Thrips palmi (Karny) (Thysanoptera: Thripidae), is examined and discussed in terms of its establishment potential in the U.K. Thrips palmi adults and first‐instar larvae have a wide distribution of supercooling points (SCPs) but show ‘pre‐freeze’ mortality as a result of both acute and chronic exposures to temperatures above the SCP range. Thrips palmi did not develop enhanced cold tolerance in response to cues previously shown to cold‐acclimate other thrips species. The acute cold tolerance of T. palmi is higher than that of the recently established and biologically similar species, Frankliniella occidentalis, which is thought to be capable only of very limited winter survival outdoors in the U.K. However, the more ecologically meaningful chronic assays reveal the opposite pattern. If introduced to the U.K., overwintering of T. palmi would thus be largely restricted to protected environments, as its cold tolerance is insufficient to permit outdoor survival for a complete winter. This assertion was demonstrated by caged populations that died out after as little as 25 days in outdoor winter conditions in Yorkshire, U.K. The reversal of relative tolerance of the two species when considering chronic and acute assays suggests that these forms of low temperature mortality have different physiological bases, and emphasizes the need to use both forms of assay in assessments of overwintering capacity.     

Thermal survival limits of young and mature larvae of a cold stenothermal chironomid from the Alps (Diamesinae: Pseudodiamesa branickii [Nowicki, 1873])

The threats posed by climate change make it important to expand knowledge concerning cold and heat tolerance in stenothermal species from habitats potentially threatened by temperature changes. Thermal limits and basal metabolism variations were investigated in Pseudodiamesa branickii (Diptera: Chironomidae) under thermal stress between ‐20 and 37 °C. Supercooling point (SCP), lower (LLTs) and upper lethal temperatures (ULTs), and oxygen consumption rate were measured in overwintering young (1st and 2nd instar) and mature (3rd and 4th instar) larvae from an Alpine glacier‐fed stream. Both young and mature larvae were freezing tolerant (SCPs = ‐7.1 °C and ‐6.4 °C, respectively; LLT₁₀₀ <SCP and > ‐20 °C) and thermotolerant (ULT₅₀ = 31.7 ± 0.4, 32.5 ± 0.3, respectively). However, ontogenetic differences in acute tolerance were observed. The LLT₅₀ calculated for the young larvae (= ‐7.4 °C) was almost equal to their SCP (= ‐7.1 °C) and the overlapping of the proportion of mortality curve with the CPIF curve highlighted that the young larvae are borderline between freezing tolerance and freezing avoidance. Furthermore, a lower ULT₁₀₀ in the young larvae (of ca. 1 °C), suggests that they are less thermotolerant than mature larvae. Finally, young larvae exhibit a higher oxygen consumption rate (mgO₂/gAFDM/h) at any temperature tested and are overall less resistant to oxygen depletion compared to mature larvae at ≥10 °C. These findings suggest that mature larvae enter into a dormant state by lowering their basal metabolism until environmental conditions improve in order to save energy for life cycle completion during stressful conditions.     

Effects of diapause and cold-acclimation on the avoidance of freezing injury in fat body tissue of the rice stem borer, Chilo suppressalis Walker

Overwintering freeze-tolerant larvae of Chilo suppressalis can survive at -25 degrees C, but non-diapausing larvae cannot. We reported earlier that to prevent intracellular freezing, which causes death in overwintering larvae of the Saigoku ecotype distributed in southwestern Japan, water leaves and glycerol enters fat body cells through water channels during freezing. However, it is still unclear how diapause and low-temperature exposure are related to the acquisition of freeze tolerance. We compared the extent of tissue damage, accumulation of glycerol, and transport of glycerol and water in fat body tissues between cold-acclimated and non-acclimated non-diapausing and diapausing larvae. The tissue from cold-acclimated diapausing larvae could survive only when frozen in Grace's insect medium with 0.25 M glycerol at -20 degrees C. The protection provided by glycerol was offset by mercuric chloride, which is a water-channel inhibitor. Fat body tissue isolated from non-acclimated diapausing larvae was injured by freezing even though glycerol was added to the medium, but the level of freezing injury was significantly lower than in non-diapausing larvae. Radiotracer assays in cold-acclimated diapausing larvae showed that during freezing, water left the cells into the medium and glycerol entered the cells from the medium at the same time. Therefore, in Saigoku ecotype larvae of the rice stem borer, both diapause and cold-acclimation are essential to accumulate glycerol and activate aquaporin for the avoidance of freezing injury.     

The Siberian timberman Acanthocinus aedilis: a freeze-tolerant beetle with low supercooling points

Larvae of the Siberian timberman beetle Acanthocinus aedilis display a number of unique features, which may have important implications for the field of cold hardiness in general. Their supercooling points are scattered over a wide temperature range, and some individuals have supercooling points in the low range of other longhorn beetles. However, they differ from other longhorn beetles in being tolerant to freezing, and in the frozen state they tolerate cooling to below −37°C. In this respect they also differ from the European timberman beetles, which have moderate supercooling capacity and die if they freeze. The combination of freezing tolerance and low supercooling points is unusual and shows that freezing at a high subzero temperature is not an absolute requirement for freezing tolerance. Like other longhorn beetles, but in contrast to other freeze-tolerant insects, the larvae of the Siberian timberman have a low cuticular water permeability and can thus stay supercooled for long periods without a great water loss. This suggests that a major function of the extracellular ice nucleators of some freeze-tolerant insects may be to prevent intolerable water loss in insects with high cuticular water permeability, rather than to create a protective extracellular freezing as has generally been assumed. The freezing tolerance of the Siberian timberman larvae is likely to be an adaptation to the extreme winter cold of Siberia.     

Identification of tissues showing the lowest tolerance to freezing in larvae of the rice stem borer, Chilo suppressalis

Abstract.  Even though overwintering larvae of the rice stem borer, Chilo suppressalis , are freeze-tolerant, they cannot survive below −30 °C. Furthermore, nondiapausing larvae cannot survive freezing. However, the cause of death due to freezing is unclear. To identify the cause of death by freezing in larvae, those tissues most injured by low temperatures are identified using the vital stain trypan blue. In overwintering larvae, the midgut of dead larvae stains blue, and remarkable colour density differences between dead and surviving larvae are observed in the midgut. In nondiapausing larvae incubated at −10 °C for several hours, the fat body of dead larvae is strongly stained. Furthermore, increases in mortality with treatment time correspond with increases in the area of the fat body stained. Sterile nondiapausing larvae with lower supercooling points, below −20 °C, do not freeze at −10 °C and survive the treatment. However, all the larvae die when subjected to inoculative freezing at −10 °C, and the fat body stains blue. These results suggest that the midgut in overwintering larvae and the fat body in nondiapausing larvae have the lowest tolerance to freezing.     

Age‐dependent changes in tolerance to cold and accumulation of cryoprotectants in overwintering and non‐overwintering larvae of European corn borer Ostrinia nubilalis

Abstract The age‐dependent cold hardiness profile of Ostrinia nubilalis is compared between nondiapausing and diapausing larvae, as well as with field‐collected larvae. The results suggest that both cold tolerance and accumulation of cryoprotectants depends upon the age of O. nubilalis larva. Late fifth‐instar nondiapausing larvae are more cold tolerant than younger fifth‐instars because they show enhanced ability to withstand sub‐zero temperatures. No appreciable difference is observed between the experimental groups of diapausing larvae as far as their supercooling ability and tolerance at sub‐zero temperatures above the supercooling point. In general, both field‐collected and diapausing larvae are more cold tolerant than nondiapausing larvae, indicating a direct link between diapause and cold hardiness. The age of diapausing larvae affects the ability to accumulate glycerol. Glycerol levels of 45‐day‐old diapausing larvae are significantly higher (2.7‐fold) compared with 90‐day‐old diapausing larvae. Moreover, diapausing larvae display a five‐ to 13‐fold higher glycerol content compared with nondiapausing larvae. There is a trend for an age‐dependent cold hardiness profile in O. nubilalis and further tests that could demonstrate a causal relationship between age and cold tolerance are needed.     

Cold hardiness and overwintering strategy of the pink maize stalk borer,Sesamia nonagrioides Lef (lepidoptera,noctuidae)

The cold-hardening capacity of larvae of the pink maize stalk borer,Sesamia nonagrioides Lef., was examined. Supercooling points (SCPs) of field collected diapausing larvae from south-east and south-west France and non-diapausing and diapausing laboratory-reared larvae did not differ and ranged between –5 and –8°C. Thus, this insect possesses sufficient supercooling ability to avoid freezing over its normal environmental temperature ranges. Despite this, we found thatSesamia presents paradoxical cold reactions. Mortality of cold acclimated diapausing larvae after short-term exposure to temperatures above the SCP is high, supporting the view thatSesamia is cold-sensitive. On the other hand,Sesamia could survive freezing for at least 24 h to temperatures close to the SCP. This ability does not seem to be related to haemolymph trehalose, the sole cold-accumulated compound detected by gas chromatography and mass spectrometry. Despite the mildness of the winter 1990–1991, only 5% of the field population survive and pupate in April. The main part of the population died from November to January, the period during which larvae were mainly located in the part of the corn stem above the ground and experienced air temperatures. After January, all surviving larvae were excuusively located in the root, 10 cm below the soil, where they experienced milder temperatures than air. They exhibited a constant low rate of mortality, possibly independent of the cold. In their current distribution area, survival of overwintering larvae ofS. nonagrioides is only related to the microclimate of the overwintering site and freezing tolerance capacity seems to be irrelevant. This study allows us to propose a non-pollutant pest control method based on the behavioral strategy of this insect.     

Geographic variation in winter hardiness of a common agricultural pest,Leptinotarsa decemlineata,the Colorado potato beetle

Successful latitudinal expansions into temperate climates depend largely upon the evolution of novel adaptive traits or the presence of pre-adaptive or exapted mechanisms for survival in seasonal climates. Geographic comparisons of ancestral (pre-expansion) and derived (post-expansion) populations provide a useful framework for understanding the evolutionary conditions that facilitate geographic expansions. Using a common agricultural pest, the Colorado Potato Beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) as a model, we conducted a regional comparison of cold hardiness and overwintering success among ancestral (southern Mexico) and derived (Vermont and Kansas, USA) L. decemlineata populations. In order to determine if ancestral and derived beetle populations vary physiologically for cold hardiness, we compared supercooling points (SCPs) of three geographic populations of L. decemlineata. We also tested if ancestral and derived beetle populations differed in their overwintering behavior and success by performing an overwintering field experiment. Ancestral and derived populations did not express different physiological responses (i.e. SCPs) to freezing temperatures. However, ancestral and derived populations responded differently to the onset of winter conditions and displayed dissimilar overwintering behaviors. The majority of ancestral beetles failed to initiate diapause and dug upward within experimental mesocosms. Differences in overwintering behavior also resulted in significant variation in overwintering success as derived populations displayed higher overwintering survivorship when compared with ancestral populations. Given our results, it is evident that research exploring the interaction of the ecological factors and evolutionary processes is necessary to fully realize the dynamics of biological invasions.     

Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sarcophaga crassipalpis

ABSTRACT. Supercooling points (SCP) and low temperature tolerance were determined for larval, pupal and adult stages of Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). No stage tolerates tissue-freezing. Ontogenetic changes in SCP profiles are similar for comparable developmental stages of diapause and non-diapause groups. Feeding larvae have SCPs near -7°C which decrease to -11°C in the postfeeding wandering phase of the final larval instar. The lowest SCPs are recorded for pupae at -23°C. The capacity to survive at -17°C varies with age of the diapausing pupae: 10-day-old pupae are less cold tolerant than pupae that have been in diapause for 45–80 days. Although the SCP of non-diapausing pupae is as low as in diapausing pupae, non-diapausing pupae are extremely sensitive to low temperature exposure and do not survive to adult eclosion when exposed to -17°C for as little as 20 min. The use of hexane to break pupal diapause has no effect on SCPs or low temperature tolerance.