The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.     

Enhancing our understanding of the molecular responses to hypoxia in mammals using Drosophila melanogaster.

Drosophila melanogaster has been used as a genetic model, especially in the past decade, to examine normative biological processes and disease conditions very effectively. These span a wide range of major issues such as aging, cancer, embryogenesis, neural development, apoptosis, and alcohol intoxication. Here, we detail how the Drosophila melanogaster can be used as a genetic model to study the molecular and genetic underpinnings of the response to hypoxia. In our study of the basis of anoxia tolerance, one of the potent approaches that we use is a mutagenesis screen to identify loss-of-function mutants that are anoxia sensitive. The major advantage of this approach is that it is not biased for any particular gene or gene product. Although our screen is in progress, we already have evidence that this approach is useful.     

Variation in response to CO2 in Drosophila melanogaster and D. simulans

A type of slow-recovery from high temperature CO2 exposure is a wild-type characteristic of Drosophila simulans. Species hybrids of wild-type D. melanogaster and D. simulans have intermediate recovery times. The response to nitrogen exposure at both high and low temperatures is the same for the two species suggesting that the observed CO2 response is more than just anoxia. Since there is no female bias in hybrids, and since injections of D. simulans extract into D. melanogaster produced no increase in CO2 sensitivity in the recipients, we conclude that the prolonged recovery time results from genomic differences between D. simulans and D. melanogaster, and is not due to a sigma-like infectious agent.     

The process of cardiorespiratory autoresuscitation in intact newborn rats

To examine the process of spontaneous autoresuscitation and the recovery of the hypoxic ventilatory response (HVR) after prolonged anoxia, we monitored respiratory frequency (f, by body plethysmography) and heart rate (HR, by ECG) in intact newborn rats (n = 12, day 2-4) before, during, and after 100% N2 exposure. The rat before anoxia showed signs of HVR: f changes at acute hypoxia (10% O2) and hyperoxia (100% O2). During anoxia, the spontaneous respiratory movement "gasping" appeared for 21 min (mean). At O2 restoration (with 100% O2), gasping stopped and no respiratory flow was detected for 1 min. One rat failed to autoresuscitate and had heart arrhythmia during the transient apnea, but 11 rats recovered respiration after the HR acceleration. Despite the successful autoresuscitation, the rats did not show HVR at 10 min into the recovery period and the recovery of HVR required more than 30 min. The results indicate that O2 inhalation is useful to trigger autoresuscitation even when the rat has already been in a state of profound hypoxic depression, but the rat becomes transiently insensitive to HVR after autoresuscitation. We estimate that reform of the respiratory control system in newborn rats is not yet firmly established to track HVR early in the recovery phase after prolonged anoxia.     

The dynamics of the induction and regression of puffs 87A, 87C and 93D in the polytene chromosomes of Drosophila melanogaster under the action of anoxia and high temperature]

A study was made of the heat shock puff activity in salivary glands of Drosophila melanogaster larvae after 5 and 20 min treatments with anoxia (dipping into physiological solution), heat shock (37 degrees C), and simultaneously with both the agents. The simultaneous treatment with heat shock and anoxia, as well as treatment with anoxia only blocked the induction of heat shock puffs. They appeared 10-15 min after the treatment during recovery under aerobic conditions. There was a super-additive effect of the simultaneous treatment on the heat shock puffing duration. A specific regulation of the 93D locus was observed. The 93D puff was induced by a 5 min simultaneous treatment with anoxia and heat shock and, as a rule, was not induced by the analogous 20 min treatment. The role of anoxia in blocking heat shock puff induction under simultaneous effects of heat shock and anoxia is discussed.     

Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster.

Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury. Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress. Here we report on a new role of trehalose in anoxia resistance in Drosophila. We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia. Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia. Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the P-element rescues totally the phenotype. We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation.     

A Drosophila CDK5alpha-like molecule and its possible role in response to O(2) deprivation.

Cyclin-dependent kinase 5 activator (Cdk5alpha) is an activator of Cdk5 kinase activity and its expression is restricted to neurons. The complex of Ckd5/Cdk5alpha is essential for neurite outgrowth during neuronal differentiation and possibly also for neuronal degeneration. Here we report the isolation and characterization of a Drosophila Cdk5alpha-like molecule (dCdk5alpha). The gene encoding this molecule is localized in the Drosophila chromosome region of 31D1-31D2. The expression of this gene is differentially regulated with a very low level at earlier developmental stages and reaches the highest level in the adult. The C-terminal of this molecule shares high homology with the mammalian Cdk5alpha molecule. Constitutive over-expression of dCdk5alpha in transgenic flies significantly prolongs their recovery time from 5 min to O(2) deprivation or anoxia in older flies (15 days) but not in young ones (4 days). In addition, anoxia up-regulated the expression of this gene. Taken together, the results in this report and others provide a framework for genetically dissecting the functions of Cdk5alpha/Cdk5 complex in the CNS.     

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

By oxymetry and electron paramagnetic resonance (EPR), we investigated the effects of repeated anoxia/re-oxygenation (A/R) periods on the respiration and production of free radicals by synoviocytes (rabbit HIG-82 cell line and primary equine synoviocytes) and equine articular chondrocytes. Three periods of 20 min anoxia followed by re-oxygenation were applied to 10(7)cells; O(2) consumption was measured before anoxia and after each re-oxygenation. After the last A/R, cellular free radical formation was investigated by EPR spectroscopy with spin trapping technique (n=3 for each cell line). Both types of synoviocytes showed a high O(2) consumption, which was slowered after anoxia. By EPR with the spin trap POBN, we proved a free radical formation. Results were similar for equine and rabbit synoviocytes. For chondrocytes, we observed a low O(2) consumption, unchanged by anoxia, and no free radical production. These observations suggest an oxidant activity of synoviocytes, potentially important for the onset of osteoarthritis.     

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose.     

Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens. II. Chronic exposures.

Sex-linked recessive lethal mutations were induced in Drosophila melanogaster males by gaseous 1,2-dibromoethane at concentrations ranging from 0.2 to 2 parts per million. Significant numbers of mutations could be induced at all these concentrations. Pronounced germ-cell sensitivity differences were observed. For low exposures, spermatids and spermatocytes were about 10--20 times more sensitive than spermatozoa. The dose-effect relation was linear below 60 ppm . h for the 3 cell types. At higher exposures, sterility prevented mutation detection in spermatocytes and in spermatogonia. The lowest effective exposure for spermatozoa was 18 ppm . h (0.25 ppm for 72 h). In spermatids, the lowest exposure tested, 2.3 ppm . h (0.2 ppm for 11 h) induced 4 times the spontaneous mutation rate. Therefore, using prolonged exposure periods one may be able to detect concentrations in the range of parts per billion. Thus, Drosophila appears suitable as a system for detecting very low concentrations of gaseous mutagens in industrial, agricultural and environmental atmospheres.     

Effects of Oxygen Depletion on Norepinephrine- and Carbachol-Stimulated Phosphoinositide Turnover in Rat Brain Slices

We examined the effects of in vitro anoxia and in vivo hypoxia (8% O2/92% N2) on norepinephrine (NE)- and carbachol-stimulated phosphoinositide (PI) turnover in rat brain slices. The formation of 3H-labeled polyPI in cortical slices was impaired by in vitro anoxia and fully restored by reoxygenation. Accumulation of 3H-labeled myo-inositol phosphates (3H-IPs) stimulated by 10(-5) M NE was significantly reduced by anoxia (control at 60 min, 1,217 +/- 86 cpm/mg of protein; anoxia for 60 min, 651 +/- 82 cpm/mg; mean +/- SEM; n = 5; p less than 0.01), and reoxygenation following anoxia resulted in overshooting of the accumulation (control at 120 min, 1,302 +/- 70 cpm/mg; anoxia for 50 min plus oxygenation for 70 min, 1,790 +/- 126 cpm/mg; n = 5; p less than 0.01). The underlying mechanisms for the two phenomena--the decrease caused by anoxia and the overshooting caused by reoxygenation following anoxia--seemed to be completely different because of the following observations. (a) Although the suppression of NE-stimulated accumulation at low O2 tensions was also observed in Ca2+-free medium, the overshooting in response to reoxygenation was not. (b) Carbachol-stimulated accumulation was significantly reduced by anoxia and was restored by reoxygenation only to control levels. Thus, the postanoxic overshooting in accumulation of 3H-IPs seems to be a specific response to NE. (c) The decrease observed at low O2 tensions was due to a decrease in Emax value, whereas the postanoxic overshooting was due to a decrease in EC50 value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos

We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3-13 were subjected to O2 deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ~20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2 deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.     

Hypoxia induces major effects on cell cycle kinetics and protein expression in Drosophila melanogaster embryos

Hypoxia induces a stereotypic response in Drosophila melanogaster embryos: depending on the time of hypoxia, embryos arrest cell cycle activity either at metaphase or just before S phase. To understand the mechanisms underlying hypoxia-induced arrest, two kinds of experiments were conducted. First, embryos carrying a kinesin-green fluorescent protein construct, which permits in vivo confocal microscopic visualization of the cell cycle, showed a dose-response relation between O2 level and cell cycle length. For example, mild hypoxia (Po2 approximately 55 Torr) had no apparent effect on cell cycle length, whereas severe hypoxia (Po2 approximately 25-35 Torr) or anoxia (Po2 = 0 Torr) arrested the cell cycle. Second, we utilized Drosophila embryos carrying a heat shock promoter driving the string (cdc25) gene (HS-STG3), which permits synchronization of embryos before the start of mitosis. Under conditions of anoxia, we induced a stabilization or an increase in the expression of several G1/S (e.g., dE2F1, RBF2) and G2/M (e.g., cyclin A, cyclin B, dWee1) proteins. This study suggests that, in fruit fly embryos, 1) there is a dose-dependent relationship between cell cycle length and O2 levels in fruit fly embryos, and 2) stabilized cyclin A and E2F1 are likely to be the mediators of hypoxia-induced arrest at metaphase and pre-S phase.     

Recovery of the ventilatory response to hypoxia in normal adults

Recovery of the initial ventilatory response to hypoxia was examined after the ventilatory response had declined during sustained hypoxia. Normal young adults were exposed to two consecutive 25-min periods of sustained isocapnic hypoxia (80% O2 saturation in arterial blood), separated by varying interludes of room air breathing or an increased inspired O2 fraction (FIO2). The decline in the hypoxic ventilatory response during the 1st 25 min of hypoxia was not restored after a 7-min interlude of room air breathing; inspired ventilation (VI) at the end of the first hypoxic period was not different from VI at the beginning and end of the second hypoxic period. After a 15-min interlude of room air breathing, the hypoxic ventilatory response had begun to recover. With a 60-min interlude of room air breathing, recovery was complete; VI during the second hypoxic exposure matched VI during the first hypoxic period. Ventilatory recovery was accelerated by breathing supplemental O2. With a 15-min interlude of 0.3 FIO2 or 7 min of 1.0 FIO2, VI of the first and second hypoxic periods were equivalent. Both the decline and recovery of the hypoxic ventilatory response were related to alterations in tidal volume and mean inspiratory flow (VT/TI), with little alteration in respiratory timing. We conclude that the mechanism of the decline in the ventilatory response with sustained hypoxia may require up to 1 h for complete reversal and that the restoration is O2 sensitive.     

Oxygen reperfusion damage in an insect

The deleterious effects of anoxia followed by reperfusion with oxygen in higher animals including mammals are well known. A convenient and genetically well characterized small-animal model that exhibits reproducible, quantifiable oxygen reperfusion damage is currently lacking. Here we describe the dynamics of whole-organism metabolic recovery from anoxia in an insect, Drosophila melanogaster, and report that damage caused by oxygen reperfusion can be quantified in a novel but straightforward way. We monitored CO(2) emission (an index of mitochondrial activity) and water vapor output (an index of neuromuscular control of the spiracles, which are valves between the outside air and the insect's tracheal system) during entry into, and recovery from, rapid-onset anoxia exposure with durations ranging from 7.5 to 120 minutes. Anoxia caused a brief peak of CO(2) output followed by knock-out. Mitochondrial respiration ceased and the spiracle constrictor muscles relaxed, but then re-contracted, presumably powered by anaerobic processes. Reperfusion to sustained normoxia caused a bimodal re-activation of mitochondrial respiration, and in the case of the spiracle constrictor muscles, slow inactivation followed by re-activation. After long anoxia durations, both the bimodality of mitochondrial reactivation and the recovery of spiracular control were impaired. Repeated reperfusion followed by episodes of anoxia depressed mitochondrial respiratory flux rates and damaged the integrity of the spiracular control system in a dose-dependent fashion. This is the first time that physiological evidence of oxygen reperfusion damage has been described in an insect or any invertebrate. We suggest that some of the traditional approaches of insect respiratory biology, such as quantifying respiratory water loss, may facilitate using D. melanogaster as a convenient, well-characterized experimental model for studying the underlying biology and mechanisms of ischemia and reperfusion damage and its possible mitigation.     

The effect of selection for desiccation resistance on cold tolerance of Drosophila melanogaster

Abstract.  Low temperature and desiccation stress are thought to be mechanistically similar in insects, and several studies indicate that there is a degree of cross-tolerance between them, such that increased cold tolerance results in greater desiccation tolerance and vice versa . This assertion is tested at an evolutionary scale by examining basal cold tolerance, rapid cold-hardening (RCH) and chill coma recovery in replicate populations of Drosophila melanogaster selected for desiccation resistance (with controls for both selection and concomitant starvation) for over 50 generations. All of the populations display a RCH response, and there is no effect of selection regime on RCH or basal cold tolerance, although there are differences in basal cold tolerance between sampling dates, apparently related to inter-individual variation in development time. Flies selected for desiccation tolerance recover from chill coma slightly, but significantly, faster than control and starvation-control flies. These findings provide little support for cross-tolerance between survival of near-lethal cold and desiccation stress in D. melanogaster .     

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.     

Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae)

High levels (about 4,000-fold) of resistance to dieldrin were isolated by screening field-collected populations of Drosophila melanogaster (Meigen). The resistance was made homozygous following 2-4 generations of selection. A single, major gene mapping to the left arm of chromosome III was solely responsible for resistance. The implications of the recovery of resistant mutants from field populations of D. melanogaster are discussed.     

High resolution mapping of candidate alleles for desiccation resistance in Drosophila melanogaster under selection

The ability to counter periods of low humidity is an important determinant of distribution range in Drosophila. Climate specialists with low physiological tolerance to desiccation stress are restricted to the tropics and may lack the ability to further increase resistance through evolution. Although the physiological adaptations to desiccation stress are well studied in Drosophila and other ectotherms, factors underlying evolutionary responses remain unknown because of a paucity of genetic data. We address this issue by mapping evolutionary shifts in D. melanogaster under selection for desiccation resistance. Genomic DNA from five independent replicate selected, and control lines were hybridized to high density Affymetrix Drosophila tiling arrays resulting in the detection of 691 single feature polymorphisms (SFPs) differing between the treatments. While randomly distributed throughout the genome, the SFPs formed specific clusters according to gene ontology. These included genes involved in ion transport and respiratory system development that provide candidates for evolutionary changes involving excretory and respiratory water balance. Changes to genes related to neuronal control of cell signaling, development, and gene regulation provide candidates to explore novel biological processes in stress resistance. Sequencing revealed the nucleotide shifts in a subset of the SFPs and highlighted larger regions of genomic diversity surrounding SFPs. The association between natural desiccation resistance and a 463-bp region of the 5' promoter region of the Dys gene undergoing allele frequency changes in response to selection in the experimental evolution lines was tested in an independent population from Coffs Harbour, Australia. The allele frequencies of 23 SNPs common to the two populations were inferred from the parents of the 10% most and 10% least resistant Coffs Harbour flies. The frequencies of the selected alleles were higher at all sites, with three sites significantly associated with the resistant Coffs Harbour flies. This study illustrates how rapid mapping can be used for discovering natural molecular variants associated with survival to low humidity and provides a wealth of candidate alleles to explore the genetic basis of physiological differences among resistant and susceptible Drosophila populations and species.     

Nitric oxide involvement in Drosophila immunity.

The augmented production of nitric oxide (NO) was observed during the hemocyte-mediated melanotic encapsulation responses of Drosophila melanogaster and D. teissieri. When introduced into the hemocoel of D. melanogaster larvae, NO activated the gene encoding the antimicrobial peptide Diptericin. These observations, together with previous studies documenting the production of superoxide anion (O(*-)(2)) and H(2)O(2) in immune-challenged Drosophila, provide evidence that reactive intermediates of both oxygen (ROI) and nitrogen (RNI) constitute a part of the cytotoxic arsenal employed by Drosophila in defense against both microbial pathogens and eukaryotic parasites. These ROI and RNI appear to represent an evolutionarily conserved innate immune response that is mediated by regulatory proteins that are homologous to those of mammalian species.