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

Highly contiguous assemblies of 101 drosophilid genomes

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species

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 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

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

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 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