Geographic variation in larval cold tolerance and exposure across the invasion front of a widely established forest insect

As the global climate changes, high and low temperature extremes can drive changes in species distributions. Across the range of a species, thermal tolerance can experience plasticity and may undergo selection, shaping resilience to temperature stress. In this study, we measured variation in the lower thermal tolerance of early instar larvae of an invasive forest insect, Lymantria dispar dispar L. (Lepidoptera: Erebidae), using populations sourced from the climatically diverse invasion of the Eastern United States. In two chill coma recovery experiments, we recorded recovery time following a period of exposure to a non-lethal cold temperature. A third experiment quantified growth responses after chill coma recovery to evaluate sublethal effects. Our results indicate that cold tolerance is linked to regional climate, with individuals from cold climate populations recovering faster from chill coma. While this geographic gradient is seen in many species, detecting this pattern is notable for an introduced species founded from a single point-source introduction. We demonstrate that the cold temperatures used in our experiments occur in nature from cold snaps after spring hatching, but negative impacts to growth and survival appear low. We expect that population differences in cold temperature performance manifest more from differences in temperature-dependent growth than acute exposure. Evaluating intraspecific variation in cold tolerance increases our understanding of the role of climatic gradients on the physiology of an invasive species, and contributes to tools for predicting further expansion.

A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance

The thermotolerance–plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint exists.

Genetic Variability, Population Differentiation, and Correlations for Thermal Tolerance Indices in the Minute Wasp, Trichogramma cacoeciae

Temperature is a main driver of the ecology and evolution of ectotherms. In particular, the ability to move at sub-lethal low temperatures can be described through three thermal tolerance indices—critical thermal minimum (CTmin), chill coma temperature (CCT), and activity recovery (AR). Although these indices have proven relevant for inter-specific comparisons, little is known about their intraspecific variability as well as possible genetic correlations between them. We thus investigated these two topics (intraspecific variability and genetic correlations between thermal tolerance indices) using the minute wasp, Trichogramma cacoeciae. Strains from T. cacoeciae were sampled across three geographic regions in France—two bioclimatic zones along a sharp altitudinal cline in a Mediterranean context (meso-Mediterranean at low elevations and supra-Mediterranean at higher elevations) and a more northwestern area characterized by continental or mountainous climates. Our results evidenced a significant effect of both the longitude and the severity of the cold during winter months on CCT. Results were however counter-intuitive since the strains from the two bioclimatic zones characterized by more severe winters (northwestern area and supra-Mediterranean) exhibited opposite patterns. In addition, a strong positive correlation was observed between CCT and CTmin. Neither strain differentiation nor the covariations between traits seem to be linked with the molecular diversity observed on the part of the mitochondrial marker COI.

A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance

The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint truly exists.

Effect of starvation on the cold tolerance of adult Drosophila suzukii (Diptera: Drosophilidae)

The spotted wing drosophila, Drosophila suzukii, is an invasive pest in Europe and North America. Access to resources may be challenging in late fall, winter and early spring and flies may suffer from food deprivation along with cold stress in these periods. Whereas a plethora of studies have been performed on the overwintering capacity of D. suzukii, the effects of starvation on the fly's cold tolerance have not been addressed. In the present study, young D. suzukii adults (reared at 25°C, LD 12:12 h) were deprived of food for various periods (0, 12, 24 and 36 h), after which chill coma recovery time, critical thermal minimum, as well as acute and chronic cold tolerance were assessed. Additionally, the body composition of adults (body mass, water content, total lipid, glycerol, triglycerides, glucose and proteins) before and after starvation periods was analysed to confirm that starvation had detectable effects. Starved adults had a lower body mass, and both lipid and carbohydrate levels decreased with starvation time. Starvation slightly increased critical thermal minimum and affected chill coma recovery time; however, these changes were not gradual with starvation duration. Starvation promoted acute cold tolerance in both sexes. This effect appeared faster in males than in females. Food deprivation also led to enhanced survival to chronic cold stress. Short-term starvation was thus associated with significant changes in body composition in D. suzukii, and these alterations could alter some ecologically relevant traits related to cold tolerance, particularly in females. Our results suggest that food deprivation during short time (<36 h) can promote cold tolerance (especially survival after a cold stress) of D. suzukii flies. Future studies should address the ecological significance of these findings as short food deprivation may occur in the fields on many occasions and seasons.

Multiple paths to cold tolerance: the role of environmental cues, morphological traits and the circadian clock gene vrille

Background Tracing the association between insect cold tolerance and latitudinally and locally varying environmental conditions, as well as key morphological traits and molecular mechanisms, is essential for understanding the processes involved in adaptation. We explored these issues in two closely-related species, Drosophila montana and Drosophila flavomontana, originating from diverse climatic locations across several latitudes on the coastal and mountainous regions of North America. We also investigated the association between sequence variation in one of the key circadian clock genes, vrille, and cold tolerance in both species. Finally, we studied the impact of vrille on fly cold tolerance and cold acclimation ability by silencing it with RNA interference in D. montana. Results We performed a principal component analysis (PCA) on variables representing bioclimatic conditions on the study sites and used latitude as a proxy of photoperiod. PC1 separated the mountainous continental sites from the coastal ones based on temperature variability and precipitation, while PC2 arranged the sites based on summer and annual mean temperatures. Cold tolerance tests showed D. montana to be more cold-tolerant than D. flavomontana and chill coma resistance (CTmin) of this species showed an association with PC2. Chill coma recovery time (CCRT) of both species improved towards northern latitudes, and in D. flavomontana this trait was also associated with PC1. D. flavomontana flies were darkest in the coast and in the northern mountainous populations, but coloration showed no linkage with cold tolerance. Body size decreased towards cold environments in both species, but only within D. montana populations largest flies showed fastest recovery from cold. Finally, both the sequence analysis and RNAi study on vrille suggested this gene to play an essential role in D. montana cold resistance and acclimation, but not in recovery time. Conclusions Our study demonstrates the complexity of insect cold tolerance and emphasizes the need to trace its association with multiple environmental variables and morphological traits to identify potential agents of natural selection. It also shows that a circadian clock gene vrille is essential both for short- and long-term cold acclimation, potentially elucidating the connection between circadian clock system and cold tolerance.

Chill coma onset and recovery fail to reveal true variation in thermal performance among populations of Drosophila melanogaster

Species from colder climates tend to be more chill tolerant regardless of the chill tolerance trait measured, but for Drosophila melanogaster, population-level differences in chill tolerance among populations are not always found when a single trait is measured in the laboratory. We measured chill coma onset temperature, chill coma recovery time, and survival after chronic cold exposure in replicate lines derived from multiple paired African and European D. melanogaster populations. The populations in our study were previously found to differ in chronic cold survival ability, which is believed to have evolved independently in each population pair; however, they did not differ in chill coma onset temperature and chill coma recovery time in a manner that reflected their geographic origins, even though these traits are known to vary with origin latitude among Drosophila species and are among the most common metrics of thermal tolerance in insects. While it is common practice to measure only one chill tolerance trait when comparing chill tolerance among insect populations, our results emphasise the importance of measuring more than one thermal tolerance trait to minimize the risk of missing real adaptive variation in insect thermal tolerance.

A neuroendocrine pathway modulating osmotic stress in Drosophila

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.