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

Transcriptional analysis of selected stress proteins in larvae of the fruit fly, \kur{Drosophila melanogaster} (Diptera: Drosophilidae

We assessed influence of three acclimation regimes and influence of recovery after cold shock (exposure to 0°C for a period of time corresponding to Lt25) on the relative mRNA levels of selected stress proteins using qRT-PCR method. Larvae acclimated at 25°C showed relatively weak upregulation responses to cold shock. Much stronger responses were observed in the larvae that were cold-acclimated at 15°C or 15°C ? 6°C prior to cold shock. Two different general trends were distinguished in the response to cold acclimation and cold shock: (a) proteins from families SP70 and SP90 and splice variants c and d of the transcription factor HSF were upregulated in response to cold acclimation and the levels of their mRNA transcripts further increased after cold shock (for instance, the abundance of hsp70Aa mRNA increased up to 300-fold after cold shock (acclimation variant 15°C ? 6°C)); (b) four members of the small Hsp family (22, 23, 26 and 27 kDa) and splice variants a and b of the transcription factor HSF were down-regulated during cold acclimation (for instance, 10-fold in the case of hsp22) and the levels of their mRNA transcripts were either unchanged or increased only moderately after the cold shock. A third group of proteins, namely Hsc70, Hsp40 showed no or relatively small changes

Cold hardiness of larvae of the fruit fly, \kur{Drosophila melanogaster} (Diptera: Drosophilidae)

We assessed survival of larvae of the fruit fly, Drosophila melanogaster at low temperatures (0°C and 5°C) after rearing them under seven different acclimation regimes. Larvae that have developed in a standard diet at 25°C showed 50% mortality after 12,6 min of the exposure to 0°C (Lt50 = 0.21 h). In contrast, larvae that have developed in a diet enriched with glycerol at 15°C, and were cold acclimated at 5°C during last two days of their development, had Lt50 = 38.6 h. It means that it was possible to increase the Lt50 at 0°C more than 180-fold using simple manipulations with rearing temperatures and diet composition. The physiological differences in duration of larval development, fresh mass, dry mass, hydration and total contents of proteins, lipids and glycogen between the larvae belonging to different acclimation variants are described. The samples for future detailed metabolomic analysis were prepared

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

We assessed survival of larvae of the fruit fly, Drosophila melanogaster at 0°C after rearing them under three different acclimation regimes. 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 showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt50) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Detailed metabolomic analyses showed that long-term cold acclimation modified the metabolomic profiles of the larvae. The most prominent responses were accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM). In addition, restructuring of the glycerophospholipid composition of biological membranes was observed

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Gene expression response to recovery from chronic cold exposure (CE, solid lines) or cold shock (CS, dashed lines) analyzed by Principal Component Analysis (PCA) in <i>Drosophila melanogaster</i> larvae of two strains, Oregon (blue symbols) and Hsp70^- (red symbols).

<p>Log2-transformed values of the fold-differences in relative mRNA levels (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128976#pone.0128976.s004" target="_blank">S4 Fig</a>) were fitted into the PCA model and a plot of principal components PC1 and PC2 is presented. The ellipsoids in lower part of Fig 6 delimit the areas of clustering of three biological replications of each sample. Samples were taken at three different times (1h, 3h, 24h) of recovery at constant 18°C. The eigenvectors in the upper part of Fig 6 represent individual mRNAs.</p

Survival in <i>Drosophila melanogaster</i> larvae of two strains, Oregon (blue full circles) and Hsp70^- (red empty circles) acclimated at constant 22°C and then exposed to 1 h heat shocks of variable intensity ranging from +32°C to +38°C.

<p>Numbers flanking each data point show numbers (<i>N</i>) of larvae in each experiment. Sigmoid survival curves were fitted to data (goodness of fit, R^<b>2</b>: 0.9895, Oregon; 0.9983, Hsp70^-).</p

Survival in <i>Drosophila melanogaster</i> larvae of two strains, Oregon and Hsp70^-, when exposed to different long-term acclimations (A, B, C).

<p>Black columns show proportion of individuals that were able to form puparium and grey columns show proportion of individuals that finally emerged as fit adults. Numbers (<i>N</i>) of larvae in each acclimation/ strain treatment are shown in parentheses.</p

Schematic depiction of acclimation protocols and cold treatments used in this study.

<p>The experiments are represented horizontally and the main temperature and timing conditions are indicated. Vertical lines divide the experiments to four major parts: developmental acclimation, pre-treatment, cold treatment, and recovery. Larvae were acclimated under three different long-term acclimation (LTA) temperature protocols (A, B, C) and, when reaching the stage of fully grown 3rd instar, were exposed to pre-treatment [none, chronic (0°C/1.25 d), or acute (-4°C/1 h)], followed by various chronic or acute cold treatments, and recovery at constant 18°C. The pupariation and emergence of fit adults from puparia, as two criterions of survival, were checked in all experiments for 14 d of recovery. The samples for gene expression analysis were taken at time points indicated by arrows: at the end of each acclimation protocol, and on times 1, 3 and 24 h of recovery after the CE (0°C/1.25 d), or CS (-4°C/1 h).</p