Metabolomic profiling of rapid cold hardening and cold shock in Drosophila melanogaster

A short exposure to a mild cold stress is sufficient to increase cold tolerance in many insects. This phenomenon, termed rapid cold hardening (RCH) expands the thermal interval that can be exploited by the insect. To investigate the possible role of altered metabolite levels during RCH, the present study used untargeted (1)H NMR metabolomic profiling to examine the metabolomic response in Drosophila melanogaster during the 72 h following RCH and cold shock treatment. These findings are discussed in relation to the costs and benefits of RCH that are measured in terms of survival and reproductive output. Cold shock caused a persistent disturbance of the metabolite profile that correlated well with a delayed onset of cold shock mortality. The disruption of metabolite homeostasis was smaller following RCH, where control levels were fully recovered after 72 h. RCH improved both survival and reproductive output after a subsequent cold shock but the RCH treatment alone was associated with costs in terms of reduced survival and reproductive output. The most pronounced changes following the RCH treatment were elevated levels of glucose and trehalose. Although, it is difficult to discern if a change in a specific metabolite is linked to physiological processes of adaptive, neutral or detrimental nature we observed that the onset and magnitude of the increased sugar levels correlated tightly with the improved chill tolerance following RCH. These findings suggest a putative role of cryoprotectants during RCH which are discussed in the light of the existing literature on the mechanistic background of RCH.     

Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster

Naturally occurring diurnal variations in temperature are sufficient to induce a rapid cold hardening (RCH) response in insects. RCH can increase cold tolerance by 1-2 degrees C and extend the temperature interval at which insects can remain active. While the benefits of RCH are well established, the underlying physiological mechanisms remain unresolved. In this study we investigated the role of RCH on expression of heat shock proteins (Hsp70) after a cold shock, and the effect of RCH on the composition of phospholipid fatty acids (PLFAs) in membranes of Drosophila melanogaster. These experiments were performed on both "control" flies and flies selected for cold resistance in order to additionally examine a possible target for selection for cold tolerance. RCH improved survival following cold shock at -4, -6 and -8 degrees C. No induction of Hsp70 was found following cold shock irrespective of the pre-treatment. In contrast, a 5h RCH treatment was sufficient to induce small, but significant, changes in the composition of PLFAs. Here, the polyunsaturated linoleic acid, 18:2(n-6), increased while monounsaturated (18:1) and saturated (14:0) PLFAs decreased in abundance. These changes were observed in both selection groups and caused a significant increase in the overall degree of unsaturation. This response is consistent with the membrane response typically found during cold acclimation in ectothermic animals and it is likely adaptive to maintain membrane function during cold. Cold selection resulted in PLFA changes (decrease of 18:0 and 18:1 and increase of 14:0 and 16:1), which may improve the ability to harden during RCH.     

Identification of a genetic network for an ecologically relevant behavioural phenotype in Drosophila melanogaster

Pupation site choice of Drosophila third‐instar larvae is critical for the survival of individuals, as pupae are exposed to various biotic and abiotic dangers while immobilized during the 3–4 days of metamorphosis. This singular behavioural choice is sensitive to both environmental and genetic factors. Here, we developed a high‐throughput phenotyping approach to assay the variation in pupation height in Drosophila melanogaster, while controlling for possibly confounding factors. We find substantial variation of mean pupation height among sampled natural stocks and we show that the Drosophila Genetic Reference Panel (DGRP) reflects this variation. Using the DGRP stocks for genome‐wide association (GWA) mapping, 16 loci involved in determining pupation height could be resolved. The candidate genes in these loci are enriched for high expression in the larval central nervous system. A genetic network could be constructed from the candidate loci, which places scribble (scrib) at the centre, plus other genes known to be involved in nervous system development, such as Epidermal growth factor receptor (Egfr) and p53. Using gene disruption lines, we could functionally validate several of the initially identified loci, as well as additional loci predicted from network analysis. Our study shows that the combination of high‐throughput phenotyping with a genetic analysis of variation captured from the wild can be used to approach the genetic dissection of an environmentally relevant behavioural phenotype.     

Reorganization of membrane lipids during fast and slow cold hardening in Drosophila melanogaster

Abstract Rapid cold hardening is a naturally occurring phenomenon in insects that is thought to be responsible for increased cold tolerance during diurnal variations in temperature. The underlying physiological mechanisms are still not fully resolved but, in Drosophila melanogaster (Meigen 1830), rapid cold hardening is accompanied by specific changes in the membrane lipid composition. To further understand the link between rapid cold hardening and adjustments in the membrane lipid composition, the present study investigates how different rates of cooling affect thermotolerance and the composition of phospholipid fatty acids. Female Drosophila are cooled gradually from 25 to 0 °C at 0.01, 0.05, 0.1 or 0.5 °C min^?1, respectively, and, subsequently, phospholipid fatty acid composition and survival after a 1‐h cold shock at ?5 °C is measured. The rapid cold hardening treatments all influence cold tolerance differently so that short and intermediate rapid cold hardening treatments (0.05, 0.1 or 0.5 °C min^?1 cooling rates) increase cold shock survival, whereas the slow cooling treatment (0.01 °C min^?1) decreases survival relative to an untreated control. The intermediate rapid cold hardening treatments (0.05 or 0.1 °C min^?1) induce a similar type of response characterized by an increase in the molar percentage of linoleic acid, 18:2(n‐6), at the expense of 16:0 and 18:1(n‐9), which leads to an increase in the degree of unsaturation. The slowest cooling treatment (0.01 °C min^?1) results in a large increase in cis‐16:1(n‐7) and significant reductions in the saturated phospholipid fatty acids 16:0, 18:0 and the unsaturated 16:1(n‐9) and 18:2(n‐6) fatty acids. These changes cause a slight decrease in the average length of the phospholipid fatty acids and an increase in the overall ratio of unsaturated vs. saturated fatty acids. These findings demonstrate that the rate of cooling is important for both the reorganization of membrane lipids, and for the degree of acquired cold tolerance during rapid cold hardening, and they suggest an important role for rapid cold hardening during diurnal rather than seasonal temperature changes.     

Short-term hardening effects on survival of acute and chronic cold exposure by Drosophila melanogaster larvae

We quantified the variation and plasticity in cold tolerance among four larval stages of four laboratory strains of Drosophila melanogaster in response to both acute (<2 h of cold exposure) and chronic (7 h of cold exposure) cold exposure. We observed significant differences in basal cold tolerance between the strains and among larval stages. Early larval instars were generally more tolerant of acute cold exposures than third-instar larvae. However, wandering larvae were more tolerant of chronic cold exposures than the other stages. Early stages also displayed a more pronounced rapid cold-hardening response than the later stages. Heat pre-treatment did not confer a significant increase in cold tolerance to any of the strains at any stage, pointing to different mechanisms being involved in resolving heat- and cold-elicited damage. However, when heat pre-treatment was combined with rapid cold-hardening as sequential pre-treatments, both positive (heat first) and negative (heat second) effects on cold tolerance were observed. We discuss possible mechanisms underlying cold-hardening and the effects of acute and chronic cold exposures.     

Brief carbon dioxide exposure blocks heat hardening but not cold acclimation in Drosophila melanogaster

Carbon dioxide is a commonly used anaesthetic in Drosophila research. While any detrimental effects of CO2 exposure on behaviour or traits are largely unknown, a recent study observed significant effects of CO2 exposure on rapid cold hardening and chill-coma recovery in Drosophila melanogaster. In this study we investigated the effect of a brief CO2 exposure on heat hardening and cold acclimation in D. melanogaster, measuring heat knockdown and chill-coma recovery times of flies exposed to CO2 for 1 min after hardening or acclimation. CO2 anaesthesia had a significant negative effect on heat hardening, with heat knockdown rates in hardened flies completely reduced to those of controls after CO2 exposure. Chill-coma recovery rates also significantly increased in acclimated flies that were exposed to CO2, although not to the same extent seen in the heat populations. CO2 exposure had no impact on heat knockdown rates of control flies, while there was a significant negative effect of the anaesthetic on chill-coma recovery rates of control flies. In light of these results, we suggest that CO2 should not be used after hardening in heat resistance assays due to the complete reversal of the heat hardening process upon exposure to CO2.     

Induction of male recombination in Drosophila melanogaster by chemical treatment

Acridine orange (AO), ethidium bromide (EB), ethyl methanesulfonate (EMS) and 8- ethoxycaffeine ( EOC ) were fed to larvae of Drosophila melanogaster in order to test their capacity for the induction of meiotic recombination in males. Our results show that AO and EB increase significantly the male recombination frequencies. No relationship between chromosome breakage ability and male recombination induction was found since EMS and EOC , two effective chromosome-breaking agents, were unable to increase the male recombination.     

Induction of somatic and male crossing-over by bleomycin in Drosophila melanogaster

Bleomycin (BLM) is well known as an antibiotic as well as for its antineoplastic activity. A clinical preparation of BLM was tested for its recombinogenicity in a higher eukaryotic organism, Drosophila melanogaster. Feeding of the F1 larvae on a medium with BLM increased somatic crossing-over spots on female tergites and induced recombination in male germ cells. However, nonlinear dose-response curves were obtained. Malformed tergites were also observed in females treated with BLM.     

Estimation of microsatellite mutation rates in Drosophila melanogaster

Microsatellite mutations were studied in a set of 175 mutation accumulation lines, all of them independently derived from a completely homozygous population of Drosophila melanogaster and maintained under strong inbreeding during 80 generations. We assayed 28 microsatellites and detected two mutations. One mutation consisted of a single addition of a dinucleotide repeat and the other was a deletion of five trinucleotide repeats. The average mutation rate was 5.1 x 10(-6), in full agreement with previous estimates from two different sets of mutation accumulation lines.     

Complexity of the cold acclimation response in Drosophila melanogaster

Insects can increase their resistance to cold stress when they are exposed to non-lethal conditions prior to the stress; these plastic responses are normally described only in terms of immediate effects on mortality. Here we examine in Drosophila melanogaster the short- and longer-term effects of different conditions on several measures of cold resistance, but particularly chill coma recovery. Short-term exposure to sublethal temperature (cold hardening) did not decrease chill coma recovery times even though it decreased mortality. Exposure to 12 degrees C for 2 days (acclimation) decreased chill coma recovery times for a range of stressful temperatures when flies were cultured at 25 degrees C, but did not usually affect recovery times when flies were cultured at 19 degrees C. In contrast, 2-day exposure to 12 degrees C decreased mortality regardless of rearing temperature. Rearing at 19 degrees C decreased mortality and chill coma recovery time relative to rearing at 25 degrees C. Acclimation increased the eclosion rate of eggs from stressed females, but did not affect development time or size of the offspring. These results indicate that plastic responses to cold in D. melanogaster are complex when resistance is scored in different ways, and that effects can extend across generations.     

Dietary sugars affect cold tolerance of Drosophila melanogaster

In spite of the extensive knowledge of the biology and the genetics of Drosophila melanogaster, the mechanisms by which this fly builds up cold tolerance remain poorly understood. Recent studies have reported that acclimation-mediated acquisition of cold tolerance is associated with moderate accumulation of sugars in drosophilids. However, it is not known whether there is a genuine causative link between cold tolerance and body sugar accumulation in Drosophila flies. We thus tested whether increasing body sugars levels, via dietary enrichment, will promote the cold tolerance of D. melanogaster adults. We gradually augmented the concentration of four different sugars (sucrose, fructose, glucose and trehalose) in rearing diets and tested the basal cold tolerance (acute and chronic). Using SIM-GC/MS approach, we verified whether feeding of larvae and adults on sugar-enriched diets was associated with increasing body sugars. We also tested whether development, body mass, fat stores, metabolites composition and metabolic pathways were altered by these dietary manipulations. The data confirm an effective incorporation of all sugars. Contrary to the expectation, cold tolerance was negatively affected by exogenous sugars, especially when supplemented at high concentrations. Rearing on high-sugar doses induced system-wide metabolic alteration associated with carbohydrate metabolism imbalance, a developmental delay and a fresh mass reduction. Our data show that high dietary sugars create a metabolic imbalance and negatively affect cold tolerance. This study provides an intriguing connection between nutritional conditions and thermal trait. It also underlines that careful attention should be given to dietary factors when studying thermal traits.     

昆虫快速冷耐受的研究进展

快速冷耐受作为一种非常有效的耐寒策略,可在短时间内显著增强昆虫的耐寒性,使得昆虫能够在全球气候变暖的大背景下应对更加频繁的气候波动,具有重要的生态意义.快速冷耐受现象在昆虫中首次发现以来,得到了广泛的研究,尤其近年来,在其内在机制研究方面取得了很大进展.本文综述了近年来快速冷耐受现象的研究进展,对快速冷耐受的特征、生理生化和分子机制,以及生态学意义等进行了总结、归纳与分析,为昆虫快速冷耐受的研究提供参考.     

Increase in cold-shock tolerance by selection of cold resistant lines in Drosophila melanogaster

Abstract.

• 1 In Drosophila melanogaster, the cold-shock tolerance of adult flies at -7°C increased 22% after a prior 2h exposure to 4°C as measured by LD₅₀, the dose (degree minutes of exposure to subzero temperature) which resulted in 50% mortality.
• 2 Cold-shock tolerance was further significantly increased by selecting cold resistant lines by exposure of adults (1) to 4°C for 2 h (short-term chilling), or (2) to -7°C for 80–120 min (cold shock), or (3) to short-term chilling followed by cold-shock.
• 3 After ten generations of selection, the greatest increase in cold-shock tolerance was found in flies selected using the combined exposure of short-term chilling and cold shock. LD₅₀s increased 33% in comparison with the unselected control strain when no chilling pre-treatment was given prior to cold shock at -7°C.
• 4 The rapid cold-hardening response increased 82% in the line selected by the short-term chilling and cold-shock regime.
• 5 The enhanced cold-shock tolerance was relatively stable since no decrease was observed after four generations without selection.
• 6 This report shows the role of short-term adaptation as well as selection in the capacity to survive low temperatures in non-diapausing stages of insects.

     

Effects of cold- and heat hardening on thermal resistance in Drosophila melanogaster

The effects of cold- and heat hardening on resistance to both low and high temperature stress was examined in Drosophila melanogaster lines selected for resistance to either cold or heat. The hardening effect was positive when the hardening was of the same type as the stress in all selection regimes. The effect of cold hardening on survival after heat stress was further examined in the lines selected for cold resistance and corresponding controls. A cross-protection effect (increased heat resistance after cold hardening) was present and this effect was lower in the lines selected for resistance to cold than in the controls. The level of Hsp70 expression induced by a non-lethal cold hardening was examined, showing that cold hardening induced Hsp70 expression. The results suggest that the cross-protection effect is at least partly due to Hsp70 expression induced by cold exposure.     

Induction of chromosome aberrations by chemical mutagens in neural ganglia of Drosophila melanogaster

Chromatid aberrations induced by various concentrations of bleomycin, cyclophosphamide and mitomycin C were analyzed in neural ganglia of third-instar larvae of Drosophila melanogaster. A clear dose response was observed with increasing dose after treatment with bleomycin and mitomycin C, whereas no effect was observed after treatment with cyclophosphamide. A comparison with published data for the induction of sex-linked recessive lethals showed that, at least for the 3 drugs tested, the use of both tests eliminates false negatives and might comprise a useful procedure for testing mutagenicity in Drosophila.     

Enhancement of supercooling capacity and survival by cold acclimation, rapid cold and heat hardening in Spodoptera exigua

Insects can increase their resistance to cold stress by prior exposure to non-lethal cold temperatures. Here, we investigated the supercooling capacity and survival of eggs, 3rd and 5th instar larvae, and pupae of Spodoptera exigua (Lepidoptera: Noctuidae) during CA, and responses to various pre-treatment protocols, including constant temperatures, thermoperiods, and RCH, RHH, RCH + RHH and RHH + RCH combined with thermoperiods. Only acclimated eggs demonstrated a significant decrease in SCP, from −20.7 ± 0.3 to −22.9 ± 0.3 °C, among all experimental groups compared to non-acclimated stages. Survival increased by 17.5% for eggs, 40.0% and 13.3% for 3rd and 5th instar larvae, and by 20.0% for pupae after CA. Compared to controls, survival of eggs under the conditions of thermoperiod (5:15 °C), thermoperiod (5:15 °C) + RHH, and thermoperiod (5:15, 10:20, and 15:25 °C) + RCH significantly increased. In addition, survival of 3rd and 5th instar larvae and pupae increased under the conditions of thermoperiod (5:15 °C) and thermoperiod (5:15 °C) + RCH, possibly due to the induction of heat shock proteins or cryoprotectants. However, the pre-treatments of thermoperiod + RCH + RHH and thermoperiod + RHH + RCH did not significantly enhance survival of any developmental stage. These adaptive responses may allow S. exigua to enhance supercooling capacity and survival in response to seasonal or unexpected diurnal decreases in environmental temperatures.     

Induction by 5-bromodeoxyuridine of sex-linked lethal mutations in spermatogenous cells of Drosophila melanogaster

Injection of adult males of Drosophila melanogaster with a solution of 5-bromodeoxyuridine (BUdR) induced sex-linked lethal mutations but no chromosomal rerrangements. Application of the brood method suggests that the vast majority of the detected sex-linked lethals were induced in spermatozoa or spermatids. The findings suggest that secondary effects rather than an immediate direct involvement of DNA account for the mutagenic action of BUdR in D. melanogaster.