Long-Term Cold Acclimation Extends Survival Time at 0°C and Modifies the Metabolomic Profiles of the Larvae of the Fruit Fly Drosophila melanogaster

Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately -5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum.We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt(50)) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt(50) (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines.Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring

How consistent are the transcriptome changes associated with cold acclimation in two species of the Drosophila virilis group?

This work was financially support by a Marie Curie Initial Training Network grant, “Understanding the evolutionary origin of biological diversity” (ITN-2008–213780 SPECIATION), grants from the Academy of Finland to A.H. (project 132619) and M.K. (projects 268214 and 272927), a grant from NERC, UK to M.G.R. (grant NE/J020818/1), and NERC, UK PhD studentship to D.J.P. (NE/I528634/1).For many organisms the ability to cold acclimate with the onset of seasonal cold has major implications for their fitness. In insects, where this ability is widespread, the physiological changes associated with increased cold tolerance have been well studied. Despite this, little work has been done to trace changes in gene expression during cold acclimation that lead to an increase in cold tolerance. We used an RNA-Seq approach to investigate this in two species of the Drosophila virilis group. We found that the majority of genes that are differentially expressed during cold acclimation differ between the two species. Despite this, the biological processes associated with the differentially expressed genes were broadly similar in the two species. These included: metabolism, cell membrane composition, and circadian rhythms, which are largely consistent with previous work on cold acclimation/cold tolerance. In addition, we also found evidence of the involvement of the rhodopsin pathway in cold acclimation, a pathway that has been recently linked to thermotaxis. Interestingly, we found no evidence of differential expression of stress genes implying that long-term cold acclimation and short-term stress response may have a different physiological basis.PostprintPeer reviewe

Functional Characterization of the Frost Gene in Drosophila melanogaster: Importance for Recovery from Chill Coma

BACKGROUND: Almost all animals, including insects, need to adapt to temperature fluctuations. The molecular basis of thermal adaptation is not well understood, although a number of candidate genes have been proposed. However, a functional link between candidate genes and thermal tolerance has rarely been established. The gene Frost (Fst) was first discovered when Drosophila flies were exposed to cold stress, but the biological function(s) of Fst has so far not been characterized. Because Fst is up-regulated after a cold stress, we tested whether it was essential for chill-coma recovery. METHODOLOGY/PRINCIPAL FINDINGS: A marked increase in Fst expression was detected (by RT-PCR) during recovery from cold stress, peaking at 42-fold after 2 h. The GAL4/UAS system was used to knock down expression of Fst and recovery ability was assessed in transgenic adults following 12 h of chill coma at 0 degrees C. The ability to recover from cold stress (short-, medium- and long-term) was significantly altered in the transgenic adults that had Fst silenced. These findings show that Fst plays an essential role in the recovery from chill coma in both males and females. CONCLUSIONS/SIGNIFICANCE: The Frost gene is essential for cold tolerance in Drosophila melanogaster and may play an important role in thermal adaptation

Metabolomics of aging assessed in individual parasitoid wasps

Metabolomics studies of low-biomass organisms, such as small insects, have previously relied on the pooling of biological samples to overcome detection limits, particularly using NMR. We show that the differentiation of metabolite profiles of individual 1 mg parasitoid wasps of different ages is possible when using a modified sample preparation and a combination of untargeted NMR and LC-MS based metabolomics. Changes were observed between newly emerged and older wasps in glycerolipids, amino acids and circulatory sugars. This advance in chemical profiling has important implications for the study of the behaviour and ecology of parasitoids and many other species of small organisms because predictions and observations are typically made at the level of the individual. Thus, the metabolomic state of low-biomass individuals can now be related to their behaviour and ecological performance. We discuss specifically the utility of age-related metabolomic profiling but our new approach can be applied to a wide range of biological research

Rapid Growth Reduces Cold Resistance: Evidence from Latitudinal Variation in Growth Rate, Cold Resistance and Stress Proteins

Background: Physiological costs of rapid growth may contribute to the observation that organisms typically grow at submaximal rates. Although, it has been hypothesized that faster growing individuals would do worse in dealing with suboptimal temperatures, this type of cost has never been explored empirically. Furthermore, the mechanistic basis of the physiological costs of rapid growth is largely unexplored. Methodology/Principal Finding: Larvae of the damselfly Ischnura elegans from two univoltine northern and two multivoltine southern populations were reared at three temperatures and after emergence given a cold shock. Cold resistance, measured by chill coma recovery times in the adult stage, was lower in the southern populations. The faster larval growth rates in the southern populations contributed to this latitudinal pattern in cold resistance. In accordance with their assumed role in cold resistance, Hsp70 levels were lower in the southern populations, and faster growing larvae had lower Hsp70 levels. Yet, individual variation in Hsp70 levels did not explain variation in cold resistance. Conclusions/Significance: We provide evidence for a novel cost of rapid growth: reduced cold resistance. Our results indicate that the reduced cold resistance in southern populations of animals that change voltinism along the latitudinal gradient may not entirely be explained by thermal selection per se but also by the costs of time constraint-induced higher growth rates. This also illustrates that stressors imposed in the larval stage may carry over and shape fitness in the adul

Meat Feeding Restricts Rapid Cold Hardening Response and Increases Thermal Activity Thresholds of Adult Blow Flies, Calliphora vicina (Diptera: Calliphoridae)

Virtually all temperate insects survive the winter by entering a physiological state of reduced metabolic activity termed diapause. However, there is increasing evidence that climate change is disrupting the diapause response resulting in non-diapause life stages encountering periods of winter cold. This is a significant problem for adult life stages in particular, as they must remain mobile, periodically feed, and potentially initiate reproductive development at a time when resources should be diverted to enhance stress tolerance. Here we present the first evidence of protein/meat feeding restricting rapid cold hardening (RCH) ability and increasing low temperature activity thresholds. No RCH response was noted in adult female blow flies (Calliphora vicina Robineau-Desvoidy) fed a sugar, water and liver (SWL) diet, while a strong RCH response was seen in females fed a diet of sugar and water (SW) only. The RCH response in SW flies was induced at temperatures as high as 10°C, but was strongest following 3h at 0°C. The CTmin (loss of coordinated movement) and chill coma (final appendage twitch) temperature of SWL females (-0.3 ± 0.5°C and -4.9 ± 0.5°C, respectively) was significantly higher than for SW females (-3.2 ± 0.8°C and -8.5 ± 0.6°C). We confirmed this was not directly the result of altered extracellular K+, as activity thresholds of alanine-fed adults were not significantly different from SW flies. Instead we suggest the loss of cold tolerance is more likely the result of diverting resource allocation to egg development. Between 2009 and 2013 winter air temperatures in Birmingham, UK, fell below the CTmin of SW and SWL flies on 63 and 195 days, respectively, suggesting differential exposure to chill injury depending on whether adults had access to meat or not. We conclude that disruption of diapause could significantly impact on winter survival through loss of synchrony in the timing of active feeding and reproductive development with favourable temperature conditions