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doi: 10.1371/journal.pone.0004546. Epub 2009 Feb 20.

The 70 kDa heat shock protein assists during the repair of chilling injury in the insect, Pyrrhocoris apterus

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The 70 kDa heat shock protein assists during the repair of chilling injury in the insect, Pyrrhocoris apterus

Vladimír Kostál et al. PLoS One. 2009.

Abstract

Background: The Pyrrhocoris apterus (Insecta: Heteroptera) adults attain high levels of cold tolerance during their overwintering diapause. Non-diapause reproducing adults, however, lack the capacity to express a whole array of cold-tolerance adaptations and show relatively low survival when exposed to sub-zero temperatures. We assessed the competence of non-diapause males of P. apterus for responding to heat- and cold-stresses by up-regulation of 70 kDa heat shock proteins (Hsps) and the role of Hsps during repair of heat- and cold-induced injury.

Principal findings: The fragments of P. apterus homologues of Hsp70 inducible (PaHsp70) and cognate forms (PaHsc70) were cloned and sequenced. The abundance of mRNA transcripts for the inducible form (qPCR) and corresponding protein (Western blotting) were significantly up-regulated in response to high and low temperature stimuli. In the cognate form, mRNA was slightly up-regulated in response to both stressors but very low or no up-regulation of protein was apparent after heat- or cold-stress, respectively. Injection of 695 bp-long Pahsp70 dsRNA (RNAi) caused drastic suppression of the heat- and cold-stress-induced Pahsp70 mRNA response and the up-regulation of corresponding protein was practically eliminated. Our RNAi predictably prevented recovery from heat shock and, in addition, negatively influenced repair of chilling injuries caused by cold stress. Cold tolerance increased when the insects were first exposed to a mild heat shock, in order to trigger the up-regulation of PaHsp70, and subsequently exposed to cold stress.

Conclusion: Our results suggest that accumulation of PaHsp70 belongs to a complex cold tolerance adaptation in the insect Pyrrhocoris apterus.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Heat shock-induced up-regulation of PaHsp70 and PaHsc70 expressions.
Males of Pyrrhocoris apterus were exposed to +41°C for 1 h (shaded column) and then allowed to recover at 25°C. (A) Relative levels of Pahsp70 and Pahsc70 mRNAs in the fat body were measured by qPCR (quantitative real–time PCR with Rp49 serving as a reference gene). The fat bodies (5 per each sample) were dissected prior to heat shock (time −1 h), at the end of it (time 0 h) and during recovery (up to time 24 h). (B) Protein levels were based on the results of Western blot hybridization. Whole procedure of electrophoresing, blotting, hybridization and densitometry was replicated three times for each sample. Data points are means±S.E.M. (C) An example of the results of standard PCR amplification with 25 cycles (note that a different method, i.e. q PCR was used to quantify the abundance of mRNA transcripts). The products were separated on 2% agarose and stained by ethidium bromide. It documents the temporal pattern of up-regulation of Pahsp70 mRNA in contrast to relatively stable levels of Pahsc70 an Rp49 mRNAs. (D) An example of SDS-PAGE shows equal loading of proteins (20 μg). The up-regulation of PaHsp70 was only weakly detectable (arrowheads). (E) The dashed-line rectangle area of SDS-PAGE is shown after Western blotting. The mouse monoclonal anti-Hsp70 primary antibody (clone BRM-22, Sigma) recognized both PaHsp70 and PaHsc70 proteins. Note clear up-regulation of PaHsp70 at times 1 h and 3 h in contrast to relatively stable signal of PaHsc70. (F) An example of Western blot signal quantification using Quantiscan (Biosoft) densitometry.
Figure 2
Figure 2. RNAi suppression of heat shock-induced up-regulation of PaHsp70 expression and its effect on survival.
Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the heat shock (+41°C for 1 h). The insects were either untreated (control) or injected two days prior to heat shock with: 2 μL of the injection buffer alone (blank); 2 μL (10 μg) of ß-galactosidase (ß-gal) dsRNA; or 2 μL (2 μg) of Pahsp70 dsRNA. Each column is a mean±S.E.M. of 3–4 independent samples (5 fat bodies per sample). The differences in mRNA levels were assessed by ANOVA followed by Tukey's multiple comparison test at p = 0.05 (columns flanked by different letters differ significantly). (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 39) and Pahsp70 dsRNA-injected (F, n = 40) insects after a severe heat shock (+45°C for 3 h) were assessed during recovery at 25°C for 7 days. The fit insects were those showing normal, rapid and coordinated crawling; the injured insects displayed signs of heat injury, i.e. slow, uncoordinated crawling or movements of body appendages only; and the dead insect did not respond to stimulation with a fine paintbrush. See Fig. 1 for more information.
Figure 3
Figure 3. Cold exposure-induced up-regulation of PaHsp70 and PaHsc70 expressions.
Males of Pyrrhocoris apterus were exposed to −5°C for 5 d (shaded area) and then allowed to recover at 25°C. (A) Relative levels of Pahsp70 and Pahsc70 mRNAs in the fat body were measured by qPCR (with Rp49 serving as a reference gene). The fat bodies (5 per each sample) were dissected prior to cold exposure (not shown), during it (time −1 h), at the end of it (time 0 h) and during recovery (up to time 24 h). (B) Protein levels were based on the results of (C) Western blot hybridization. Data points are means±S.E.M. of three Western blot replications. See Fig. 1 for more information.
Figure 4
Figure 4. RNAi suppression of cold exposure-induced up-regulation of PaHsp70 expression and its effect on survival.
Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the cold exposure to −5°C for 5 d. The insects were either untreated (control) or injected two days prior to heat shock with: 2 μL of the injection buffer alone (blank); or 2 μL (2 μg) of Pahsp70 dsRNA. (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 49) and Pahsp70 dsRNA-injected (F, n = 48) insects after the cold exposure. (G) Cross-tolerance was assesed by observing the survival in insects (n = 48) that were pretreated with a mild heat shock (+41°C for 1 h) prior to the cold exposure to −5°C for 5 d. See Figs. 1 and 2 for more information.

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