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.

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

BackgroundTracing the reliance of insect cold tolerance traits on latitudinally and locally varying environmental conditions, and their associations with different morphological traits and molecular mechanisms, is essential for understanding the processes involved in adaptation. We explored these issues in non-diapausing females of two closely-related species, Drosophila montana and Drosophila flavomontana, originating from diverse climatic locations across several latitudes on the western coast and the Rocky Mountains of North America. Moreover, we traced the impact of one of the key regulators of circadian behavioural rhythms, vrille, on fly cold tolerance and cold acclimation ability by silencing this gene with RNA interference in D. montana.ResultsWe performed principal component analysis (PCA) on variables representing bioclimatic conditions on 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 in North-South direction based on summer and annual mean temperatures and growing season length. Cold tolerance tests showed D. montana to be more cold-tolerant than D. flavomontana, as could be expected by species distributions, and chill coma resistance (CTmin) of this species showed association with latitudinally varying temperatures (PC2). Chill coma recovery time (CCRT) showed cold tolerance of both species to increase towards northern latitudes, and in D. flavomontana this trait was also associated with PC1. D. flavomontana flies were darkest in coastal populations and in the northern parts of the Rocky Mountains, but colouration showed no linkage with fly cold tolerance. Body size (measured as weight) decreased towards cold environments in both species, but large size correlated with fast CCRT among D. montana individuals. Finally, silencing of vrille suggested this gene to play an essential role in CTmin and cold acclimation, but not in CCRT.ConclusionsOur 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 functional circadian clock gene vrille is essential both for short- and long-term cold acclimation elucidating connection between circadian clock and cold tolerance.