Most cited article - PubMed ID 15037645
On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus
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.
- Keywords
- freeze tolerance, insects, mitochondrial swelling, oxidative phosphorylation, permeability transition, respiration,
- Publication type
- Journal Article MeSH
Physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored. In contrast, recovery from cold stress received much less attention. Here we report on recovery of drosophilid fly larvae (Chymomyza costata) from three different levels of cold stress: supercooling to -10 °C, freezing at -30 °C, and cryopreservation at -196 °C. Analysis of larval CO2 production suggested that recovery from all three cold stresses requires access to additional energy reserves to support cold-injury repair processes. Metabolomic profiling (targeting 41 metabolites using mass spectrometry) and custom microarray analysis (targeting 1,124 candidate mRNA sequences) indicated that additional energy was needed to: clear by-products of anaerobic metabolism, deal with oxidative stress, re-fold partially denatured proteins, and remove damaged proteins, complexes and/or organelles. Metabolomic and transcriptomic recovery profiles were closely similar in supercooled and frozen larvae, most of which successfully repaired the cold injury and metamorphosed into adults. In contrast, the majority of cryopreseved larvae failed to proceed in ontogenesis, showed specific metabolic perturbations suggesting impaired mitochondrial function, and failed to up-regulate a set of 116 specific genes potentially linked to repair of cold injury.
- MeSH
- Drosophilidae * genetics metabolism MeSH
- Stress, Physiological * MeSH
- Cryopreservation * methods MeSH
- Larva MeSH
- Metabolomics methods MeSH
- Preservation, Biological MeSH
- Cold-Shock Response MeSH
- Gene Expression Profiling MeSH
- Freezing * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The cryopreservation techniques proposed for embryos of the fruit fly Drosophila melanogaster are not yet ready for practical use. Alternative methods for long-term storage of D. melanogaster strains, although urgently needed, do not exist. Herein, we describe a narrow interval of low temperatures under which the larvae of D. melanogaster can be stored in quiescence for up to two months. The development of larvae was arrested at the pre-wandering stage under fluctuating thermal regime (FTR), which simultaneously resulted in diminishing the accumulation of indirect chill injuries. Our physiological, metabolomic, and transcriptomic analyses revealed that compared to larvae stored at constant low temperatures, the larvae stored under FTR conditions were able to decrease the rates of depletion of energy substrates, exploited brief warm episodes of FTR for homeostatic control of metabolite levels, and more efficiently exerted protection against oxidative damage.
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.
- MeSH
- Acclimatization MeSH
- Drosophila melanogaster physiology MeSH
- Stress, Physiological * MeSH
- RNA, Messenger metabolism MeSH
- Cold Temperature * MeSH
- Drosophila Proteins genetics metabolism physiology MeSH
- HSP70 Heat-Shock Proteins genetics metabolism physiology MeSH
- Cold-Shock Response genetics MeSH
- Gene Expression Regulation MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- RNA, Messenger MeSH
- Drosophila Proteins MeSH
- HSP70 Heat-Shock Proteins MeSH
BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSION: 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.
- MeSH
- Acclimatization * MeSH
- Amino Acids metabolism MeSH
- Survival Analysis MeSH
- Time Factors MeSH
- Drosophila melanogaster metabolism physiology MeSH
- Larva metabolism physiology MeSH
- Fatty Acids metabolism MeSH
- Carbohydrate Metabolism MeSH
- Metabolome * MeSH
- Cold Temperature * adverse effects MeSH
- Polymers metabolism MeSH
- Freezing adverse effects MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Amino Acids MeSH
- Fatty Acids MeSH
- Polymers MeSH
- polyol MeSH Browser
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.
- MeSH
- Acclimatization genetics MeSH
- Insecta physiology MeSH
- Insect Proteins physiology MeSH
- Wound Healing genetics MeSH
- RNA, Messenger analysis MeSH
- HSP70 Heat-Shock Proteins genetics physiology MeSH
- Heat-Shock Response MeSH
- Cold Climate MeSH
- Temperature MeSH
- Up-Regulation genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Insect Proteins MeSH
- RNA, Messenger MeSH
- HSP70 Heat-Shock Proteins MeSH
