Clara Garfiel Byrge

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.

Cold tolerance is a key determinant of poleward colonization in insects. However, the physiological basis underlying interspecific differences in cold tolerance is not fully understood. Here, we analyzed cold tolerance and metabolomic profiles in warm- and cold-acclimated phenotypes of 43 Drosophila species representing a latitudinal gradient from the tropics to the boreal zone. We found a strong positive correlation between cold tolerance and climatic variables associated with habitat seasonality and temperature. Including the effects of cold acclimation, we found most species have similar ‘safety margins’, measured as the difference between the average environmental temperature and the lower lethal temperature. Searching for metabolomic signatures of cold tolerance, we found that the warm-acclimated flies of cold-hardy species had moderately but significantly higher constitutive signals of putative cryoprotectants such as trehalose, glucose, glycerol and mannitol/sorbitol. Cold acclimation (and the transition to a winter dormant phenotype) resulted in a strong accumulation of myo-inositol, which occurred only in species of the virilis group. Other temperate and boreal species either showed only moderate, idiosyncratic accumulations of sugars/polyols and free amino acids, or did not accumulate any ‘classical’ cryoprotectant at all. Thus, our results suggest that the colonization of boreal regions by Drosophila does not necessarily depend on the seasonal accumulation of classical cryoprotectants. In contrast, virtually all cold-acclimated species showed a significant increase in products of phospholipid catabolism, suggesting that remodeling of biological membranes is a clear and ubiquitous signature of cold acclimation in Drosophila.

Zebrafish have become a widely used vertebrate model in physiology and reliable measures of their metabolic rate are needed. We have developed a 3D-printed respirometer and swim tunnel system and used it for obtaining accurate measurement of standard metabolic rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish under rest and maximal exercise, respectively. We compared a slow (stepwise) protocol to a fast (continuous) protocol for determining MMR. The fast protocol yielded slightly (but not significantly) higher oxygen consumption rates than the slow protocol and the data, in contrast to the slow protocol, followed a normal distribution. These findings point to the fast protocol as a fast and reliable method for obtaining accurate values of MMR in zebrafish. We make the 3D drawings for printing the system available to researchers, to help streamline the field of metabolic research in zebrafish and other smaller fish species.