Mads Kuhlmann Andersen

The ability to withstand cold is arguably one of the most important traits defining the biogeographical distribution of insects. For most species, those classified as chill susceptible, cold tolerance is limited by the ability to maintain vital physiological functions at low temperature. However, there is currently no unified understanding of how physiological failure develops during cold stress or how this process explains variation in cold sensitivity within and among species. Although disruption of ion homeostasis has been implicated in chilling injury, it remains unclear whether it represents a common physiological limit across cold stress intensities and degrees of chill tolerance. Here, we tested whether ion balance disruption represents a unifying physiological syndrome of chill susceptibility by simultaneously quantifying cold mortality and hemolymph [K⁺] in Drosophila . We examined this relationship across i) a broad range of cold stress intensities, ii) 25 species spanning extensive variation in cold sensitivity, biogeography and phylogeny, and iii) acclimation groups of D. melanogaster developed between 12 and 27°C. Across all comparisons, mortality was associated with the same cold-stress response, characterized by progressive hyperkalemia and mortality following a ~150% increase in hemolymph [K⁺] above baseline ([K⁺] _crit ). In contrast, adaptive and plastic variation in cold sensitivity was explained by differences in the critical temperature at which ion homeostasis failed (T _c ). Extending this framework to published data from other insect orders revealed a remarkably similar [K⁺] _crit , suggesting that ionoregulatory collapse represents a conserved physiological constraint defining insect cold survival.

Chill susceptible insects such as Drosophila are vulnerable to progressive disruption of homeostasis during cold stress, and low‐temperature exposure is a key factor affecting their physiology and distribution. Comparative studies of cold tolerance traditionally use single‐condition assays for interspecific comparisons, but the emergence of thermal death time (TDT) models offers a comprehensive framework to assess cold tolerance across different stress intensities and durations. Here we construct TDT curves for six Drosophila species, spanning boreal to tropical habitats, using Lt ₅₀ estimates across a range of stressful low temperatures (Lt ₅₀ —time to reach 50% mortality—ranging from ~20 min to 2 days). For all species, the TDT curves provided good fits to the log(Lt ₅₀ ) vs. temperature data ( R ² = 0.87–0.99). TDT curves from all species had steep slopes demonstrating that cold injury rate has a high thermal sensitivity (high Q ₁₀ ) such that small changes in temperature have profound effects on survival duration. The similarity of TDT slopes among most species indicates that a similar physiological dysfunction could underlie cold injury across most Drosophila species. Further, additive accumulation of cold‐induced injury in split‐dose experiments suggests that acute and moderate cold damage could represent the same physiological failure occurring at different rates. TDT curve intercepts (species‐specific tolerance thresholds) differed markedly between boreal, temperate and tropical species and correlated strongly with their habitat temperature. Data from the present study and meta‐analysis of published data find that basal species cold tolerance increases by ~0.45°C per°C lowering of the species winter temperature. When also considering cold acclimation cues in cold climates, we argue that the experienced level of cold stress intensity is largely similar across environments inhabited by the Drosophila genus. This reaffirms that cold tolerance is an important trait shaping the fundamental niche of both boreal and tropical species. Overall, the TDT analysis of Drosophila at low temperature provides a powerful and predictive tool for quantifying insect cold resistance. This approach enables detailed cross‐species comparisons that allow for both ecological and physiological inference. On a general level, we argue that TDT analysis offers relevant approximations of insect cold resistance that could help predict insect responses to climatic change. Read the free Plain Language Summary for this article on the Journal blog.

Chill-susceptible insects such as Drosophila are vulnerable to progressive disruption of ion and water homeostasis during cold stress, and low temperature exposure is a key factor affecting their physiology and distribution. Comparative studies of cold tolerance traditionally use simple or single-condition assays for interspecific comparisons, but the emergence of thermal death time (TDT) models offers a comprehensive framework to assess cold tolerance across different stress intensities and durations. Here we construct TDT curves for six Drosophila species, spanning boreal to tropical habitats, using Lt ₅₀ estimates across a range of stressful low temperatures (Lt ₅₀ ranging from ~ 20 min to 2 days). For all species, the TDT curves provided good fits to the log(Lt ₅₀ ) vs. temperature data (R ² = 0.87 - 0.99). TDT curves from all species had steep slopes demonstrating that cold injury rate has a high thermal sensitivity such that small changes in temperature have profound effects on survival duration. The interspecific similarity of TDT slopes indicates that a conserved physiological dysfunction underlies cold injury across species. Further, additive accumulation of cold-induced injury in split-dose experiments suggests that acute and moderate cold damage represent the same underlying physiological dysfunction occurring at different rates. The TDT curve intercepts (species-specific tolerance thresholds) differed markedly between boreal, temperate, and tropical species and correlated strongly with their habitat temperature. Data from the present study and meta-analysis of published data find that the inherent species cold tolerance decreases by ~ 0.45 °C for each °C colder the winter environment of the species is. When also considering the cold acclimation cues in cold climates we argue the experienced level of cold stress intensity is similar across environments inhabited by the Drosophila genus. This suggests that cold tolerance is important in shaping the fundamental niche of both boreal and tropical species. Overall, the TDT analysis of Drosophila at low temperature provides a powerful and predictive tool for quantifying insect cold tolerance. This approach enables detailed cross-species comparisons that allows for both ecological and physiological inference. Thus, TDT curves offer relevant approximations of insect cold resistance that could help predict insect responses to climatic change.

The mountain pine beetle (Dendroctonus ponderosae) is an eruptive bark beetle that overwinters as a freeze-avoidant larvae under the bark of pine hosts. In recent years, D. ponderosae has undergone a climate change-driven range expansion into previously unsuitable habitats with historically more severe winter conditions. Dendroctonus ponderosae overwinters in a non-feeding dormant phase, and energy use is important to post-overwintering fitness. Little is known about how D. ponderosae balances energy supply and demand during overwintering. We quantified shifts in energy reserve (supply) and Complex I activity (as an index of demand) in D. ponderosae during natural overwintering and simulated early winter onset. We collected D. ponderosae larvae from infested lodgepole pine in the autumn (October), winter (January) and spring (April), and sampled a portion of these animals. During autumn and winter, another set of larvae were subjected to either mild overwintering conditions at 6°C or an experimental cold stress of stepwise decreases in temperature to test how an early onset of cold conditions influences the energetic status of overwintering individuals. Dendroctonus ponderosae larvae exposed to natural winter conditions accumulated lipids and proteins early in overwintering, which were then available for later use. Early exposure to cold stress in the autumn before full winter acclimatization, however, depleted energy reserves. These findings suggest that the timing and regulation of seasonal acclimatization in D. ponderosae have important implications for energy use that can influence subsequent fitness, and thus warming of the overwintering period may facilitate early winter feeding and enhance energy gain of D. ponderosae larvae, which could further exacerbate the spread and impact of this pest.

The ability to tolerate low temperature is among the most important traits defining the functional niche of insects, and it is clear that cold tolerance of most insects is intimately linked to their ability to defend membrane potential (Vm). Failure to maintain membrane polarization results in the loss of neuromuscular function and may ultimately initiate cell death and organismal injury. Prolonged cold exposure challenges membrane polarization through loss of transmembrane ion balance; however, the insect muscle Vm is also dependent on a strong and temperature-dependent electrogenic effect driven by Na+/K+-ATPase activity. In the present study, we investigated the electrogenic contribution of the Na+/K+-ATPase at benign (20°C) and low (0°C) temperature in ten Drosophila species representing a broad spectrum of chill tolerance. We found that the electrogenic effect of the Na+/K+-ATPase contributes a considerable component of the muscle Vm in all ten species at 20°C. This electrogenic contribution is reduced significantly at 0°C in the chill-sensitive species, while tolerant species retain their electrogenic effect at low temperature. Thus, the initial cold-induced muscle depolarization, which is a hallmark of chill-sensitive insects, is largely caused by the loss of Na+/K+-ATPase-dependent electrogenic polarization. We hypothesized that maintenance of Na+/K+-ATPase activity in the cold would be energetically costly, but in contrast to our hypothesis we found no evidence for major energetic costs in the species that maintain membrane polarization at low temperature. On the basis of these observations, we discuss how other adaptations at the protein or membrane level could explain the observed intraspecific differences.

Thermal acclimation induces variation in the temperatures leading to spreading depolarization at the critical thermal limits in invertebrates, but mechanistic inferences based on electrophysiology might be skewed by thermodynamic effects. Here, we quantify the thermal dependence of spreading depolarization parameters in fruit flies, use it to infer mechanisms, and provide perspectives for future comparative research. In addition, we propose Drosophila as a model system to understand this event in vertebrates, including humans.

The ability to tolerate low temperature is among the most important traits defining the functional niche of insects and it clear that cold tolerance of most insects is intimately linked to their ability to defend membrane potential (V _m ). Failure to maintain membrane polarization results in loss of neuromuscular function and may ultimately initiate cell death and organismal injury. Prolonged cold exposure challenges membrane polarization through loss of transmembrane ion balance; however, the insect muscle V _m is also dependent on a strong and temperature-dependent electrogenic effect driven by Na ⁺ /K ⁺ -ATPase activity. In the present study we investigate the electrogenic contribution of the Na ⁺ /K ⁺ -ATPase at benign (20°C) and low (0°C) temperature in ten Drosophila species representing a broad spectrum of chill tolerance. We find that the electrogenic effect of the Na ⁺ /K ⁺ -ATPase contributes a considerable component of the muscle V _m in all ten species at 20°C. This electrogenic contribution is reduced significantly at 0°C in the chill sensitive species, while tolerant species retain their electrogenic effect at low temperature. Thus, the initial cold-induced muscle depolarization, that is a hallmark of chill sensitive insects, is largely caused by loss of Na ⁺ /K ⁺ -ATPase-dependent electrogenic polarization. We hypothesized that maintenance of Na ⁺ /K ⁺ -ATPase activity in the cold would be energetically costly, but in contrast to our hypothesis we find no evidence for major energetic costs in the species that maintain membrane polarization at low temperature. On the basis of these observations we discuss how other adaptations at the protein or membrane level could explain the observed intraspecific differences.

Acclimation and evolutionary adaptation can produce phenotypic changes that allow organisms to cope with challenges. Determining the relative contributions and the underlying mechanisms driving phenotypic shifts from acclimation and adaptation is of central importance to understanding animal responses to change. Rates of evolution have traditionally been considered slow relative to ecological processes that shape biodiversity. Many organisms nonetheless show patterns of genetic variation that suggest that adaptation may act sufficiently fast to allow continuous change in phenotypes in response to environmental change (called ‘adaptive tracking’). In Drosophila, both plastic and evolved differences in chill tolerance are associated with ionoregulation. Here, we combine an acclimation experiment, field collections along a well-characterized latitudinal cline, and a replicated field experiment to assess the concordance in the direction, magnitude, and potential mechanisms of acclimation and adaptation on chill coma recovery and elemental (Na and K) stoichiometry in both sexes of Drosophila melanogaster. Acclimation strongly shaped chill coma recovery, spatial adaptation produced comparatively modest effects, and temporal adaptation had no significant effect. Leveraging knowledge on the mechanisms underlying variation in chill tolerance traits, we find that relationships between elemental stoichiometry and chill coma recovery in the context of acclimation may differ from those that are associated with spatial adaptive change.