Uncovering the benefits of fluctuating thermal regimes on cold tolerance of drosophila flies by combined metabolomic and lipidomic approach.

When exposed to constant low temperatures (CLTs), insects often suffer from cumulative physiological injuries that can severely compromise their fitness and survival. Yet, mortality can be considerably lowered when the cold stress period is interrupted by periodic warm interruption(s), referred to as fluctuating thermal regimes, FTRs. In this study, we have shown that FTRs strongly promoted cold tolerance of Drosophila melanogaster adults. We then assessed whether this marked phenotypic shift was associated with detectable physiological changes, such as synthesis of cryoprotectants and/or membrane remodeling. To test these hypotheses, we conducted two different time-series Omics analyzes in adult flies submitted to CLTs vs. FTRs: metabolomics (GC/MS) and lipidomics (LC/ESI/MS) targeting membrane phospholipids. We observed increasing levels in several polyhydric alcohols (arabitol, erythritol, sorbitol, mannitol, glycerol), sugars (fructose, mannose) and amino acids (serine, alanine, glutamine) in flies under CLT. Prolonged exposure to low temperature was also associated with a marked deviation of metabolic homeostasis and warm interruptions as short as 2h were sufficient to periodically return the metabolic system to functionality. Lipidomics revealed an increased relative proportion of phosphatidylethanolamines and a shortening of fatty acyl chains in flies exposed to cold, likely to compensate for the ordering effect of low temperature on membranes. We found a remarkable correspondence in the time-course of changes between the metabolic and phospholipids networks, both suggesting a fast homeostatic regeneration during warm intervals under FTRs. In consequence, we suggest that periodic opportunities to restore system-wide homeostasis contribute to promote cold tolerance under FTRs.

Effects of fluctuating thermal regimes on cold survival and life history traits of the spotted wing Drosophila (Drosophila suzukii).

Drosophila suzukii is an invasive pest causing severe damages to a large panel of cultivated crops. To facilitate its biocontrol with strategies such as sterile or incompatible insect techniques, D. suzukii must be mass-produced and then stored and transported under low temperature. Prolonged cold exposure induces chill injuries that can be mitigated if the cold period is interrupted with short warming intervals, referred to as fluctuating thermal regimes (FTR). In this study, we tested how to optimally use FTR to extend the shelf life of D. suzukii under cold storage. Several FTR parameters were assessed: temperature (15, 20, 25 °C), duration (0.5, 1, 2, 3 h), and frequency (every 12, 24, 36, 48 h) of warming intervals, in two wild-type lines and in two developmental stages (pupae and adults). Generally, FTR improved cold storage tolerance with respect to constant low temperatures (CLT). Cold mortality was lower when recovery temperature was 20 °C or higher, when duration was 2 h per day or longer, and when warming interruptions occurred frequently (every 12 or 24 h). Applying an optimized FTR protocol to adults greatly reduced cold mortality over long-term storage (up to 130 d). Consequences of FTR on fitness-related traits were also investigated. For adults, poststorage survival was unaffected by FTR, as was the case for female fecundity and male mating capacity. On the other hand, when cold storage occurred at pupal stage, poststorage survival and male mating capacity were altered under CLT, but not under FTR. After storage of pupae, female fecundity was lower under FTR compared to CLT, suggesting an energy trade-off between repair of chill damages and egg production. This study provides detailed information on the application and optimization of an FTR-based protocol for cold storage of D. suzukii that could be useful for the biocontrol of this pest.

Fluctuating thermal regime preserves physiological homeostasis and reproductive capacity in Drosophila suzukii.

Drosophila suzukii, an invasive species recently introduced in Europe, lays eggs in thin-skinned fruits and causes huge financial losses to fruit growers. One potential way to control this pest is the sterile insect technique (SIT) which demands a large stock of reproductive females to produce millions of sterile males to be released on demand. Unfortunately, Drosophila stocks age quickly, show declining fecundity when maintained at warm temperatures and conversely, they die from chill injury if they are maintained at constant low temperature. Here we investigate the potential of fluctuating thermal regime (FTR) as a storage method that harness the benefits of both warm and cold storage. Using a FTR with a daily warm period (1 h 20 at 25 °C) and cold period (20 h at 3 °C), interspaced by gradual heating and cooling, we compared longevity, fecundity and physiological condition between FTR females and females exposed to constant 25 °C and 3 °C. As hypothesised, FTR flies experienced much slower senescence (>3-fold increase in lifespan) and they preserved fecundity to a much higher age than flies from constant 25 °C. Flies maintained at constant 3 °C quickly died from chill injuries caused by a gradual loss of ion and water balance. In contrast, FTR flies were able to maintain ion and water balance (similar to 25 °C flies) as they were allowed to recover homeostasis during the short warm periods. Together these results demonstrate that FTR represents a useful protocol for storage of Drosophila stocks, and more broadly, this shows that the benefits of FTR are tightly linked with the insect ability to recover physiological homeostasis during the short warm periods.

Cold tolerance of third-instar Drosophila suzukii larvae.

Drosophila suzukii is an emerging global pest of soft fruit; although it likely overwinters as an adult, larval cold tolerance is important both for determining performance during spring and autumn, and for the development of temperature-based control methods aimed at larvae. We examined the low temperature biology of third instar feeding and wandering larvae in and out of food. We induced phenotypic plasticity of thermal biology by rearing under short days and fluctuating temperatures (5.5-19°C). Rearing under fluctuating temperatures led to much slower development (42.1days egg-adult) compared to control conditions (constant 21.5°C; 15.7days), and yielded larger adults of both sexes. D. suzukii larvae were chill-susceptible, being killed by low temperatures not associated with freezing, and freezing survival was not improved when ice formation was inoculated externally via food or silver iodide. Feeding larvae were more cold tolerant than wandering larvae, especially after rearing under fluctuating temperatures, and rearing under fluctuating temperatures improved survival of prolonged cold (0°C) to beyond 72h in both larval stages. There was no evidence that acute cold tolerance could be improved by rapid cold-hardening. We conclude that D. suzukii has the capacity to develop at low temperatures under fluctuating temperatures, but that they have limited cold tolerance. However, phenotypic plasticity of prolonged cold tolerance must be taken into account when developing low temperature treatments for sanitation of this species.