Overwintering strategy of two weevils infesting three gorse species: When cold hardiness meets plant-insect interactions

The cold hardiness of two closely related weevil species, Exapion ulicis and E. lemovicinum was studied in relation to their life cycles. These two seed-eating weevils reproduce on Ulex plant species with different fruiting phenologies. E. ulicis lays eggs in spring and overwinters as an adult while E. lemovicinum lays eggs in autumn and overwinters as a larva. Adult weevils were collected in natural populations of Brittany (Western France) and characterized with morphological and molecular tools before experiments. We showed that both weevil species exhibited low supercooling points (SCPs) with mean seasonal values below −17 °C. Fresh mass, moisture content and sex were not correlated to supercooling ability. Weevils died upon freezing and the lower lethal temperatures (LLT) were within the range of SCP, indicating that both species are freezing intolerant. Comparison between species for SCP, LLT and survival to exposure at −8 °C in winter showed a higher cold resistance for E. ulicis than for E. lemovicinum. In addition, the seasonal evolution of cold hardiness differed depending on the species. These features suggest that response to cold of weevils is linked to their life cycles, and thus to the life history of their host plants.     

Effect of simulated fall heat waves on cold hardiness and winter survival of hemlock looper,Lambdina fiscellaria (Lepidoptera: Geometridae)

The hemlock looper (Lambdina fiscellaria) is an important pest of eastern Canadian forests. The ongoing climate warming could modify the seasonal ecology of this univoltine species that lays eggs at the end of summer and overwinters at this stage. Indeed, the increase in frequency and intensity of extreme climatic events such as fall heat waves could interfere with the winter metabolism of the hemlock looper. Moreover, the host plant quality, which influences the quantity of insect energetic reserves, the geographic origin of populations and the conditions prevailing during the cold acclimation period, could cause various responses of this pest to climate warming. The main objective of this study is to determine the impact of these factors on hemlock looper winter biology. In October 2010, hemlock looper eggs initially collected from two geographic areas in the province of Québec, and from parents reared on two host plants, were exposed to fall heat waves of different intensities during 5 consecutive days. Supercooling points and cryoprotectant levels were measured on eggs on four different dates in 2010–2011 and survival rate was measured in April 2011. Our results show that hemlock looper eggs have a very low supercooling point and high levels of trehalose, glucose and mannitol in September and November. However, there is no clear relationship between the concentration of these compounds and the decrease in supercooling points. Contents in trehalose, glucose and mannitol were significantly influenced by fall heat waves and by the origin of the population. Winter survival of eggs from the temperate population was negatively affected by strong heat waves while the boreal population was not affected. This study suggests that the metabolism and winter survival of temperate hemlock looper populations in Québec will be more affected by fall heat waves that will increase in frequency due to climate change, than boreal populations.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe

As an essential aspect of its invasive character in Europe, this study examined the cold hardiness of the harlequin ladybird Harmonia axyridis. This was done for field-collected populations in Belgium overwintering either in an unheated indoor or an outdoor hibernaculum. The supercooling point, lower lethal temperature and lower lethal time at 0 and −5 °C were determined. Possible seasonal changes were taken into account by monitoring the populations during each winter month. The supercooling point and lower lethal temperature remained relatively constant for the overwintering populations in the outdoor hibernaculum, ranging from −17.5 to −16.5 °C and −17.1 to −16.3 °C, respectively. In contrast, the supercooling point and lower lethal temperature of the population overwintering indoors clearly increased as the winter progressed, from −18.5 to −13.2 °C and −16.7 to −14.1 °C, respectively. A proportion of the individuals overwintering indoors could thus encounter problems surviving the winter due to premature activation at times when food is not available. The lower lethal time of field populations at 0 and −5 °C varied from 18 to 24 weeks and from 12 to 22 weeks, respectively. Morph type and sex had no influence on the cold hardiness of the overwintering adults. In addition, all cold tolerance parameters differed greatly between the laboratory population and field populations, implying that cold tolerance research based solely on laboratory populations may not be representative of field situations. We conclude from this study that the strong cold hardiness of H. axyridis in Europe may enable the species to establish in large parts of the continent.     

Cold tolerance of the overwintering larval instars of light brown apple moth Epiphyas postvittana

The light brown apple moth, Epiphyas postvittana, a leafroller native to southeastern Australia was discovered in California in 2006. The highly polyphagous nature of this pest adds to the importance of being able to predict the potential distribution of this invader across the North American continent. The spread of ectothermic species that lack winter diapause, such as E. postvittana, can be limited by their ability to tolerate cold temperature extremes. In this study we examined the cold hardiness of 4th to 6th instar E. postvittana, the only life stages known to overwinter in California, through a combination of supercooling point (SCP) and mortality at low temperatures. Our results showed that the mean SCP for E. postvittana ranged from −14.1 °C for 6th instars to −16.0 °C for 4th instars. Lethal time leading to 50% mortality (LT₅₀) for the three instars combined were 2.5 h at −10.5 °C, 41 h at −6.5 °C and 198 h at −0.9 °C. At 3 °C, the LT₅₀ of 4th instars was significantly lower at 775 h than that for 5th and 6th instars combined at 1029 h. The cold hardiness characteristics of later-instar E. postvittana larvae were comparable to those of pink bollworm, Pectinophora gossypiella, a diapausing invasive with a geographic distribution restricted to southern California. Slightly greater cold hardiness is shown by the indigenous non-diapausing leafroller Argyrotaenia franciscana, which is restricted to the Pacific Coast of North America. We therefore conclude that the moderate cold hardiness of E. postvittana will substantially limit its spread into northern temperate regions of North America.     

Pre-freeze mortality in three species of aphids from sub-Antarctic Marion Island

Understanding the mechanisms by which aphids survive low temperature is fundamental in forecasting the risk of pest outbreaks. Aphids are chill susceptible and die at a temperature close to that at which a small exothermal event is produced. This event, which can be identified using differential scanning calorimetry (DSC), normally occurs at a higher temperature than the supercooling point (SCP) and has been termed a pre-freeze event (PFE). However, it is not known what causes the PFE or whether it signifies the death of the aphid. These questions are addressed here by using a sensitive DSC to quantify the PFE and SCP and to relate these thermal events to the lower lethal temperature (LT₅₀) of sub-Antarctic aphids acclimated to low temperatures. PFEs were observed in each of the 3 species of aphids examined. They occurred over a narrower temperature range and at a higher temperature range than the SCP (−8.2 to −13.8 and −5.6 to −29.8 °C, respectively). Increased acclimation temperature resulted in increased SCPs in Myzus ascalonicus but not in Rhopalosiphum padi. The LT₅₀ reduced by approximately 1 °C from −9.3 to −10.5 °C with reduced acclimation temperature (10–0 °C). The LT₅₀ was close to the temperature at which the PFE occurred but statistically significantly higher than either the PFE or the SCP. In the majority of cases the PFE exotherm occurred well before the main exotherm produced by the bulk of the insect’s body water freezing (SCP). However, in a few cases it occurred at the same temperature or before the super-cooling point making the term, pre-freeze event (PFE), rather misleading. The possible origins of the PFE are discussed.     

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.     

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.     

Parental size and environmental conditions affect egg capsule production by Nassarius reticulatus (Linnaeus 1758) (Gastropoda: Nassariidae)

In shallow coastal habitats scavenging netted whelks Nassarius reticulatus attached egg capsules to the stipes of red algae Chondrus crispus and occasionally on Furcellaria lumbricalis and Plumaria plumose. In the laboratory egg capsules were laid on aquaria sides and lids by individuals ≥ 21 mm shell length. Larger size classes produced more egg capsules and spawned over a longer period and in doing so partitioned less energy into shell growth. Large netted whelks (25-28.9 mm) produced larger capsules which contained significantly more and larger eggs than those produced by smaller individuals (21-24.9 mm). Egg capsule production continued throughout the year by regularly fed N. reticulatus held at ambient seawater temperatures. Egg production increased in the spring and summer with peak production during June (15 °C), decreased between August and October and resumed again during the winter (November to February at ∼ 7 °C). During the summer (15-16 °C) egg capsules were smaller and contained smaller eggs than those deposited during the winter (7-10 °C), although the number of eggs · capsule⁻¹ was similar. Enforced food limitation reduced the number and size of the egg capsules, the number and size of eggs produced · female⁻¹ and the duration of the breeding period. Hatching success of N. reticulatus egg capsules was high (95%) even at winter seawater temperatures (11-8.5 °C) and the duration of embryonic development was fastest between 15 and 17.5 °C.     

Physiological and biochemical analysis of overwintering and cold tolerance in two Central European populations of the spruce bark beetle, Ips typographus

Overwintering success is one of the key aspects affecting the development and outbreaks of the spruce bark beetle, Ips typographus (L.) populations. This paper brings detailed analysis of cold tolerance, and its influence on overwintering success, in two Central European populations of I. typographus during two cold seasons. Evidence for a supercooling strategy in overwintering adults is provided. The lower lethal temperature corresponds well to the supercooling point that ranges between −20 and −22 °C during winter months. The supercooled state is stabilized by the absence of internal ice nucleators and by seasonal accumulation of a mixture of sugars and polyols up to the sum concentration of 900 mM. The cryoprotective function of accumulated metabolites is probably based on increasing the osmolality and viscosity of supercooled body fluids and decreasing the relative proportion of water molecules available for lethal formation of ice nuclei. No activity of thermal hysteresis factors (stabilizers of supercooled state) was detected in hemolymph. Lethal times for 50% mortality (Lts50) in the supercooled state at −5, −10 or −15 °C are weeks (autumn, spring) or even months (winter), suggesting relatively little mortality caused by chill injury. Lts50 at −15 °C are significantly shorter in moist (6.9 days) than in dry (>42 days) microenvironment because there is higher probability of external ice nucleation and occurrence of lethal freezing in the moist situation.     

Cold-hardiness of Dermacentor marginatus (Acari: Ixodidae)

The cold-hardiness of Dermacentor marginatus using laboratory-reared offspring of ticks collected in Germany was characterized. Investigations of unfed stages revealed that adult ticks suffered 50% mortality at –10°C after 4–5 months, but larvae and nymphs suffered mortality within few days, whereas –15°C was lethal for all stages within a very short period. Larval hatch and moulting of engorged larvae and nymphs did not occur at 10°C. Embryonic development of eggs with larval hatch was considerably reduced by exposure of eggs to 10°C. Engorged females did not lay eggs at 10°C, the oviposition capability, however, persisted over 6 months at 10°C, 5 months at 5°C, 3 months at 0°C and 2 months at –10°C without substantial decrease of the oviposition capacity or reduction of viable eggs. These results present evidence that unfed adult ticks are the ecoepidemiologically most effective stages, which are capable to tolerate long and extremely cold winters without substantial impairment of the population density. It is also considered that engorged females interrupt their oviposition at low and subzero temperatures delaying it for months and so contribute in bypassing winter conditions. None of the stages survived supercooling indicating that D. marginatus is freeze intolerant. Mean supercooling point (SCP) ranged between –26°C in eggs and –12, 6°C in engorged females. Compared with eggs, the SCP of the other stages was significantly higher. In conclusion, the SCP is considered to have no predictive value in the context with cold-hardiness.     

Effects of ice-seeding temperature and intracellular trehalose contents on survival of frozen Saccharomyces cerevisiae cells

Freezing tolerance is an important characteristic for baker’s yeast, Saccharomyces cerevisiae, as it is used to make frozen dough. The ability of yeast cells to survive freezing is thought to depend on various factors. The purpose of this work was to study the viability of yeast cells during the freezing process. We examined factors potentially affecting their survival, including the growth phase, ice-seeding temperature, intracellular trehalose content, freezing period, and duration of supercooling. The results showed that the ice-seeding temperature significantly affected cell viability. In the stationary phase, trehalose accumulation did not affect the viability of yeast cells after brief freezing, although it did significantly affect the viability after prolonged freezing. In the log phase, the ice-seeding temperature was more important for cell survival than the presence of trehalose during prolonged freezing. The importance of increasing the extracellular ice-seeding temperature was verified by comparing frozen yeast survival rates in a freezing test with ice-seeding temperatures of −5 °C and −15 °C. We also found that the cell survival rates began to increase at 3 h of supercooling. The yeast cells may adapt to subzero temperatures and/or acquire tolerance to freezing stress during the supercooling.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Egg production and early development of Thysanoessa inermis and Euphausia pacifica (Crustacea: Euphausiacea) in the northern Gulf of Alaska

Early life history patterns were studied in the dominant euphausiids from the northern Gulf of Alaska (GOA) in 2001-2004. Gravid females of Thysanoessa inermis were observed in April and May. Brood size varied from 10 to 1021 eggs with an average of 138 ± 19 (95% CI) eggs female^− 1. Most gravid females started to release eggs within the first 2 days of incubation. The average number of eggs released per female was similar in incubation Day 1 and 2, but significantly smaller on Day 3 and 4. About 25% of the females were continuously releasing eggs over 3 days rather than producing a single distinctive brood. In contrast, gravid females of Euphausia pacifica were observed from early July through October. Most gravid females released eggs on the first day of observation, while only 2% of females produced eggs repeatedly. Brood size varied from 20 to 246 eggs with an average of 102 ± 12 (95% CI) eggs female^− 1. The relationship between E. pacifica brood size and ambient chlorophyll-a concentration was sigmoidal (r² = 0.73), with food saturated brood size of 144 ± 14(SE, P < 0.001) eggs, and half-saturation occurring at 0.46 ± 0.02(SE, P < 0.001) mg chlorophyll-a m^− 3. The average interbrood interval of E. pacifica reared at 12 °C and satiated food conditions in the laboratory was ∼ 8 days, suggesting their potential individual fecundity in the GOA was 1148-1530 eggs per spawning season. Hatching and early development (from egg to furcilia stage) was studied under 5 °C, 8 °C and 12 °C. Hatching was nearly synchronous and lasted 3-6 h, depending on incubation temperature. Development times from egg to the first furcilia stage ranged between 20 and 33 days for T. inermis, and 15 and 45 days for E. pacifica at 12 °C and 5 °C, respectively.     

Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately − 40 °C, with some individuals supercooling to as low as − 58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to − 100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of − 58 to − 70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.     

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4 °C, 12 °C, 21 °C, and 37 °C, the activation energy for water transport was determined to be E_a = 51 ± 3 kJ/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130 °C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below −15 °C.     

Low temperature survival in different life stages of the Iberian slug, Arion lusitanicus

The slug Arion lusitanicus Mabille (Gastropoda: Pulmonata: Arionidae) is an invasive species which has spread to most parts of Europe. The area of origin is unknown, but A. lusitanicus seems to cope well with the local conditions in the countries to which it has migrated. It spreads rapidly, occurs often in high densities and has become a serious pest in most European countries. Therefore there is an urgent need for better knowledge of the ecophysiology of A. lusitanicus, such as the influence of climatic conditions, in order to develop prognostic models and strategies for novel pest management practises.The aim of our study was to investigate the influence of subzero temperatures in relation to winter survival. A. lusitanicus is shown to be freeze-tolerant in some life stages. Most juveniles and some adult slugs survived being frozen at −1.3 °C for 3 days, but none of the slugs survived freezing at −3 °C. The eggs survived subzero temperatures (down to −2 °C) probably by supercooling. Juveniles and adults may also survive in a supercooled state (down to −3 °C) but are generally poor supercoolers. Therefore, the winter survival of A. lusitanicus depends to a high degree on migration to habitats protected from low winter temperatures, e.g. under plant litter, buried in the soil or in compost heaps.     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

Survival of freezing by hydrated tardigrades inhabiting terrestrial and freshwater habitats

The seasonality and unpredictability of environmental conditions at high altitudes and latitudes govern the life cycle patterns of organisms, giving rise to stresses that cause death or development of specific adaptations. Ice formation is a major variable affecting the survival of both freshwater fauna and fauna inhabiting lichens, mosses and leaf litter. Tardigrades occupy a wide range of niches in marine, freshwater and terrestrial environments. The highest number of species is found in terrestrial habitats thanks to their ability to enter anhydrobiosis and cryobiosis. The cryobiotic ability of tardigrade species from polar regions is well known. Consequently, we focused our research on the ability to survive freezing in the active hydrated state using seven tardigrade species differing in phylogenetic position and collected at various altitudes and from different habitats in a temperate area. Specimens were cooled at different cooling rates (from 0.31 °C min⁻¹ to 3.26 °C min⁻¹). Even though the final survival and the time required by animals to recover to active life were both inversely related to the cooling rate, highly significant interspecific differences were found. Species survival ability ranged from excellent to none. Species living in xeric habitats withstood freezing better than those living in hygrophilous habitats, while true limnic species did not exhibit any cryobiotic ability. The ability to withstand freezing seems linked to the anhydrobiotic ability. The differences in cryptobiotic performance among tardigrade species seem more influenced by selective pressures linked to local adaptation to habitat characteristics than by phylogenetic relationships.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.