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

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SUMMARY Coping with seasonal changes in temperature is an important factor underlying the ability of insects to survive over the harsh winter conditions in the northern temperate zone, and only a few drosophilids have been able to colonize sub-polar habitats. Information on their winter physiology is needed as it may shed light on the adaptive mechanisms of overwintering when compared with abundant data on the thermal physiology of more southern species, such as Drosophila melanogaster. Here we report the first seasonal metabolite analysis in a Drosophila species. We traced changes in the cold tolerance and metabolomic profiles in adult Drosophila montana flies that were exposed to thermoperiods and photoperiods similar to changes in environmental conditions of their natural habitat in northern Finland. The cold tolerance of diapausing flies increased noticeably towards the onset of winter; their chill coma recovery times showed a seasonal minimum between late autumn and early spring, whereas their survival after cold exposure remained high until late spring. The flies had already moderately accumulated glucose, trehalose and proline in autumn, but the single largest change occurred in myo-inositol concentrations. This increased up to 400-fold during the winter and peaked at 147nmolmg -1 fresh mass, which is among the largest reported accumulations of this compound in insects. Supplementary material available online a

Winter survival in caterpillars of <i>Cydia pomonella</i> exposed to semi-natural conditions.

a<p>Laboratory-reared caterpillars were used for this experiment. They were gradually cold-acclimated prior to transfer outdoors on 3 October 2010. Groups (i) and (ii) remained outdoors for the whole winter. Groups (iii–v) were moved back to the laboratory on 5 January 2011 and exposed to simulated winter conditions in incubators (under constant darkness). All groups were moved to constant 25°C on 10 March 2011 to check their survival (pupation). See text for more details.</p

Glycogen.

<p>Seasonal whole-body and tissues changes of glycogen contents in field-sampled caterpillars of <i>Cydia pomonella</i> during 2010/2011. Each point is the mean ± S.D. (whole body, <i>n</i> = 5 individuals; tissues, <i>n</i> = 3 replicates, 3 individuals each). Influence of sampling date on glycogen content was tested by ANOVA followed by Bonferroni's post hoc test (means flanked with different letters are significantly different).</p

Glutamine.

<p>Seasonal whole-body and tissues changes of glutamine concentrations in field-sampled caterpillars of <i>Cydia pomonella</i> during 2010/2011. Each point is the mean ± S.D. (<i>n</i> = 3 replicates, 3 individuals each). Influence of sampling date on glutamine concentration was tested by ANOVA followed by Bonferroni's post hoc test (means flanked with different letters are significantly different).</p

Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (<i>Cydia pomonella</i>)

<div><p>Background</p><p>The codling moth (<i>Cydia pomonella</i>) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations.</p><p>Principal Findings</p><p>We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately −15.3°C during summer to −26.3°C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to −15°C, even in partially frozen state.</p><p>Conclusion</p><p>Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).</p></div

Fresh mass, dry mass, total lipid mass.

<p>Gradual losses of FM, DM and total lipid mass (all masses are in mg) in caterpillars of <i>Cydia pomonella</i> during their overwintering in the field in 2011/2012. Each point is the mean ± S.D. (<i>n</i> = 10 individuals). Black symbols are for larvae that were analyzed at the beginning of November, while red symbols are for larvae, in which gradual loss of FM was measured in approximately 14 d-intervals throughout the cold season and their DM and total lipids were analyzed in April (see text for details). The larvae were located on tree trunks (see text for details) and the course of ambient temperatures was recorded in 2 h-intervals.</p

Osmolality and SCP.

<p>Seasonal changes of hemolymph osmolality and whole-body supercooling point (SCP) of field-sampled caterpillars of <i>Cydia pomonella</i> during 2010/2011. Each point is the mean ± S.D. (osmolality, <i>n</i> = 10 individuals; SCP, <i>n</i> = 8 individuals). Influence of sampling date on parameter was tested by ANOVA followed by Bonferroni's post hoc test (means flanked with different letters are significantly different). Inset shows that Pearson's correlation between osmolality and SCP was relatively tight (close to statistical significance).</p

Cold tolerance.

<p>Survival at subzero temperatures in supercooled and partially frozen states in the field-sampled caterpillars of <i>Cydia pomonella</i> during 2010/2011. Each point is the percentage of survivors in a sample of <i>n</i> larvae (<i>n</i> = flanking number). Supercooled larvae were exposed either to −5°C for 14 d or to −15°C for 7 d. Partially frozen larvae were exposed to −5°C for 1 h.</p