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. 2015 Jun 2;10(6):e0128976.
doi: 10.1371/journal.pone.0128976. eCollection 2015.

The Role of Inducible Hsp70, and Other Heat Shock Proteins, in Adaptive Complex of Cold Tolerance of the Fruit Fly (Drosophila melanogaster)

Affiliations

The Role of Inducible Hsp70, and Other Heat Shock Proteins, in Adaptive Complex of Cold Tolerance of the Fruit Fly (Drosophila melanogaster)

Tomáš Štětina et al. PLoS One. .

Abstract

Background: The ubiquitous occurrence of inducible Heat Shock Proteins (Hsps) up-regulation in response to cold-acclimation and/or to cold shock, including massive increase of Hsp70 mRNA levels, often led to hasty interpretations of its role in the repair of cold injury expressed as protein denaturation or misfolding. So far, direct functional analyses in Drosophila melanogaster and other insects brought either limited or no support for such interpretations. In this paper, we analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D. melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp70 gene.

Principal findings: We found that larvae of both strains show similar patterns of Hsps complex gene expression in response to long-term cold-acclimation and during recovery from chronic cold exposures or acute cold shocks. No transcriptional compensation for missing Hsp70 gene was seen in Hsp70- strain. The cold-induced Hsps gene expression is most probably regulated by alternative splice variants C and D of the Heat Shock Factor. The cold tolerance in Hsp70- null mutants was clearly impaired only when the larvae were exposed to severe acute cold shock. No differences in mortality were found between two strains when the larvae were exposed to relatively mild doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C.

Conclusions: The up-regulated expression of a complex of inducible Hsps genes, and Hsp70 mRNA in particular, is tightly associated with cold-acclimation and cold exposure in D. melanogaster. Genetic elimination of Hsp70 up-regulation response has no effect on survival of chronic exposures to 0°C or mild acute cold shocks, while it negatively affects survival after severe acute cold shocks at temperatures below -8°C.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic depiction of acclimation protocols and cold treatments used in this study.
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).
Fig 2
Fig 2. Survival in Drosophila melanogaster larvae of two strains, Oregon and Hsp70-, when exposed to different long-term acclimations (A, B, C).
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 (N) of larvae in each acclimation/ strain treatment are shown in parentheses.
Fig 3
Fig 3. Survival in Drosophila melanogaster 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.
Numbers flanking each data point show numbers (N) of larvae in each experiment. Sigmoid survival curves were fitted to data (goodness of fit, R2: 0.9895, Oregon; 0.9983, Hsp70-).
Fig 4
Fig 4. Survival in Drosophila melanogaster larvae of two strains, Oregon (blue full circles) and Hsp70- (red empty circles) acclimated under protocol C (15°C followed by 6°C/2 d) and then exposed to (A) chronic cold exposures (0°C), or (B) acute (1 h) cold shocks of variable intensity ranging from (A) 0 d to 7 d, or (B) 0°C to -12°C.
Numbers flanking each data point show numbers (N) of larvae in each experiment. Sigmoid survival curves were fitted to data [goodness of fit, R2: (A) 0.9124, Oregon; 0.9743, Hsp70-; (B) 0.8881, Oregon; 0.9916, Hsp70-]. Green symbols show analogous data collected for larvae that were acclimated under protocol B (15°C constant).
Fig 5
Fig 5. Gene expression response to different long-term acclimations (A, B, C) analyzed by Principal Component Analysis (PCA) in Drosophila melanogaster larvae of two strains, Oregon (blue symbols) and Hsp70- (red symbols).
Log2-transformed values of the fold-differences in relative mRNA levels (shown in S4 Fig) were fitted into the PCA model and a plot of principal components PC1 and PC2 is presented. The ellipsoids in lower part of Fig 5 delimit the areas of clustering of three biological replications of each treatment. The eigenvectors in the upper part of Fig 5 represent individual mRNAs.
Fig 6
Fig 6. 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 Drosophila melanogaster larvae of two strains, Oregon (blue symbols) and Hsp70- (red symbols).
Log2-transformed values of the fold-differences in relative mRNA levels (shown in S4 Fig) 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.

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