DataSheet1_Extracellular freezing induces a permeability transition in the inner membrane of muscle mitochondria of freeze-sensitive but not freeze-tolerant Chymomyza costata larvae.PDF

Background: Many insect species have evolved the ability to survive extracellular freezing. The search for the underlying principles of their natural freeze tolerance remains hampered by our poor understanding of the mechanistic nature of freezing damage itself.Objectives: Here, in search of potential primary cellular targets of freezing damage, we compared mitochondrial responses (changes in morphology and physical integrity, respiratory chain protein functionality, and mitochondrial inner membrane (IMM) permeability) in freeze-sensitive vs. freeze-tolerant phenotypes of the larvae of the drosophilid fly, Chymomyza costata.Methods: Larvae were exposed to freezing stress at −30°C for 1 h, which is invariably lethal for the freeze-sensitive phenotype but readily survived by the freeze-tolerant phenotype. Immediately after melting, the metabolic activity of muscle cells was assessed by the Alamar Blue assay, the morphology of muscle mitochondria was examined by transmission electron microscopy, and the functionality of the oxidative phosphorylation system was measured by Oxygraph-2K microrespirometry.Results: The muscle mitochondria of freeze-tolerant phenotype larvae remained morphologically and functionally intact after freezing stress. In contrast, most mitochondria of the freeze-sensitive phenotype were swollen, their matrix was diluted and enlarged in volume, and the structure of the IMM cristae was lost. Despite this morphological damage, the electron transfer chain proteins remained partially functional in lethally frozen larvae, still exhibiting strong responses to specific respiratory substrates and transferring electrons to oxygen. However, the coupling of electron transfer to ATP synthesis was severely impaired. Based on these results, we formulated a hypothesis linking the observed mitochondrial swelling to a sudden loss of barrier function of the IMM.</p

Heat shock-induced up-regulation of PaHsp70 and PaHsc70 expressions.

<p>Males of <i>Pyrrhocoris apterus</i> were exposed to +41°C for 1 h (shaded column) and then allowed to recover at 25°C. (A) Relative levels of <i>Pahsp70</i> and <i>Pahsc70</i> mRNAs in the fat body were measured by qPCR (quantitative real–time PCR with <i>Rp49</i> 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>i.e.</i> 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 <i>Pahsp70</i> mRNA in contrast to relatively stable levels of <i>Pahsc70</i> an <i>Rp49</i> 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.</p

RNAi suppression of cold exposure-induced up-regulation of PaHsp70 expression and its effect on survival.

<p>Relative levels of <i>Pahsp70</i> mRNA (A), PaHsp70 protein (B) and <i>Pahsc70</i> mRNA (C) were measured in the fat bodies of male <i>Pyrrhocoris apterus</i> 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 <i>Pahsp70</i> dsRNA. (D) An example of Western blotting. (E, F) Survival in blank-injected (E, <i>n</i> = 49) and <i>Pahsp70</i> dsRNA-injected (F, <i>n</i> = 48) insects after the cold exposure. (G) Cross-tolerance was assesed by observing the survival in insects (<i>n</i> = 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004546#pone-0004546-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004546#pone-0004546-g002" target="_blank">2</a> for more information.</p

RNAi suppression of heat shock-induced up-regulation of PaHsp70 expression and its effect on survival.

<p>Relative levels of <i>Pahsp70</i> mRNA (A), PaHsp70 protein (B) and <i>Pahsc70</i> mRNA (C) were measured in the fat bodies of male <i>Pyrrhocoris apterus</i> 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 <i>ß-galactosidase</i> (<i>ß-gal</i>) dsRNA; or 2 µL (2 µg) of <i>Pahsp70</i> 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 <i>p</i> = 0.05 (columns flanked by different letters differ significantly). (D) An example of Western blotting. (E, F) Survival in blank-injected (E, <i>n</i> = 39) and <i>Pahsp70</i> dsRNA-injected (F, <i>n</i> = 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>i.e.</i> slow, uncoordinated crawling or movements of body appendages only; and the dead insect did not respond to stimulation with a fine paintbrush. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004546#pone-0004546-g001" target="_blank">Fig. 1</a> for more information.</p

Cold exposure-induced up-regulation of PaHsp70 and PaHsc70 expressions.

<p>Males of <i>Pyrrhocoris apterus</i> were exposed to −5°C for 5 d (shaded area) and then allowed to recover at 25°C. (A) Relative levels of <i>Pahsp70</i> and <i>Pahsc70</i> mRNAs in the fat body were measured by qPCR (with <i>Rp49</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004546#pone-0004546-g001" target="_blank">Fig. 1</a> for more information.</p

The effect of cold pre-treatments on survival of <i>Drosophila melanogaster</i> larvae after the chronic cold exposure (CE) to 0°C for 5 days.

<p>¹ Acute pre-treatment, 1 h at -4°C; chronic pre-treatment, 1.25 d at 0°C. Both pre-treatments were followed by recovery at 18°C for 2 h.</p><p>² Statistical significance of the difference between control (no pre-treatment) and pre-treated larvae was tested using Fishers' exact test. Significant differences are shown in bold letters: *, <i>P</i> < 0.05; ns, not significant.</p><p>³ The relative risk of death expresses the chance of death in control larvae (not pre-treated) in comparison to pre-treated larvae.</p><p>The effect of cold pre-treatments on survival of <i>Drosophila melanogaster</i> larvae after the chronic cold exposure (CE) to 0°C for 5 days.</p

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

Gene expression response to different long-term acclimations (A, B, C) 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 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.</p

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

<div><p>Background</p><p>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 <i>Hsp70</i> 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 <i>Drosophila melanogaster</i> 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 <i>D</i>. <i>melanogaster</i>: the wild-type and the Hsp70^- null mutant lacking all six copies of <i>Hsp70</i> gene.</p><p>Principal Findings</p><p>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 <i>Hsp70</i> 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.</p><p>Conclusions</p><p>The up-regulated expression of a complex of inducible Hsps genes, and <i>Hsp70</i> mRNA in particular, is tightly associated with cold-acclimation and cold exposure in <i>D</i>. <i>melanogaster</i>. Genetic elimination of <i>Hsp70</i> 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 temperaures below -8°C.</p></div

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