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EFFECTS OF OXYGEN CONCENTRATION AND PRESSURE ON Drosophila melanogaster: OXIDATIVE STRESS, MITOCHONDRIAL ACTIVITY, AND SURVIVORSHIP Gerardo Bosco Department of Biomedical Science, University of Padua, Padua, Italy and Hyperbaric Center Association Hyperbaric Technicians ATIP, Padua, Italy
Martina Clamer and Elisa Messulam Department of Biomedical Science, University of Padua, Padua, Italy
Cristina Dare Department of Biology, University of Padua, Padua, Italy
Zhongjin Yang Department of Anesthesiology, Upstate Medical University, Syracuse, New York, USA
Mauro Zordan Department of Biology, University of Padua, Padua, Italy
Carlo Reggiani Hyperbaric Center Association Hyperbaric Technicians ATIP, Padua, Italy
Qinggang Hu Department of Anesthesiology, Upstate Medical University, Syracuse, New York, USA
Aram Megighian Hyperbaric Center Association Hyperbaric Technicians ATIP, Padua, Italy
Correspondence to: Zhongjin Yang, Department of Anesthesiology, Upstate Medical University, Syracuse, New York, NY 13210. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 88, No. 4, 222–234 (2015) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21217
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Organisms are known to be equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. In this study, the effects of extreme changes in oxygen availability and atmospheric pressure on physiological phenotypes of Drosophila melanogaster were investigated to explore adaptation mechanisms. The changes in citrate synthase activity (CSA), lifespan, and behavioral function in different atmospheric conditions were evaluated. In the CAS test, hyperoxia significantly increased CSA; both hypoxia and hyperbaric conditions caused a significant decrease in CSA. In the survivorship test, all changed atmospheric conditions caused a significant reduction in lifespan. The lifespan reduced more after hypoxia exposure than after hyperbaria exposure. In behavioral function test, when mechanical agitation was conducted, bang-sensitive flies showed a stereotypical sequence of initial muscle spasm, paralysis, and recovery. The percentage of individuals that displayed paralysis or seizure was measured on the following day and after 2 weeks from each exposure. The majority of flies showed seizure behavior 15 days after exposure, especially after 3 h of exposure. The percentage of individuals that did not undergo paralysis or seizure and was able to move in the vial, was also tested. The number of flies that moved and raised the higher level of the vial decreased after exposure. Animal’s speed decreased significantly 15 days after exposure to extreme environmental conditions. In summary, the alteration of oxygen availability and atmospheric pressure may lead to significant changes in mitochondria mass, lifespan, and behavioral function in C 2014 Wiley Periodicals, Inc. D. melanogaster. Keywords: hypoxia; hyperoxia; hyperbaria; mitochondrial mass; lifespan; behavioral function; Drosophila melanogaster
INTRODUCTION The changes in environmental conditions play an essential role in an organism’s evolution on Earth. Organisms are equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. Adaptation to the environment is one of major factors involved in the evolution of the species, which makes organisms better suited to their habitat. Oxygen availability was crucial to the evolution of organisms throughout the history of the Earth (Harrison and Haddad, 2011). The relationship among atmospheric oxygen tension, oxidative stress, and lifespan across the range of oxygen levels has been previously investigated. Rascon and Harrison (2010) observed that the change of adult longevity was in a complex and nonlinear manner when juveniles were reared in varying oxygen concentration. Both extreme high and low atmospheric oxygen levels heightened oxidative stress and reduced longevity (Rascon and Harrison, 2010). Skandalis et al. (2011) also reported that decrease in lifespan of flies was an O2 concentration dependent in both hypoxia and hyperoxia. Hyperoxia led to a marked decrease in the rapid, and to a lesser extent prolonged, climbing behavior, though only flies in moderate hyperoxia showed a decrease that paralleled flies in normoxia (Skandalis et al., 2011). Hyperoxia leads to a marked decrease in the rapid, and to a lesser extent prolonged climbing behavior. Metabolism and water loss were both reduced Archives of Insect Biochemistry and Physiology
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in hyperoxia. Eleven-day-old flies in the highest oxygen concentration nearly doubled their water loss rates. These and other studies have examined the effects of altered oxygen availability on insects by focusing, although not exclusively, on prolonged hypoxia and hyperoxia (2, 5, 10, 40, 90, 100%) to identify the changes in phenotypic plasticity, respiratory physiology, behavior, and aerobic/anaerobic metabolism, as well as lifespan (Freeman, 2000; Miro et al., 2004; Pichaud et al., 2010; Charette et al., 2011; Ali et al., 2012). The effects of changed oxygen availability on animals have been examined in order to understand the evolutionary consequences and mechanisms of adaptation to these environments (Zhao and Haddad, 2011; Zhao et al., 2012). However, the mechanism underlying the adaptation to changed atmospheric pressure has not been investigated. The aim of this study was to investigate the effects of extreme changes in oxygen availability and atmospheric pressure (hypoxia, hyperoxia, hyperbaria, and hyperbaric hyperoxia) on survivorship, seizure susceptibility, and mitochondrial mass in Drosophila melanogaster to explore adaptation mechanisms.
MATERIALS AND METHODS Fly Lines Two commonly used wild Drosophila type lines of Canton-S and W1118 were used. The flies were reared in a fly incubator under standard conditions (25 ± 1°C, 12:12h light/dark cycle) and raised on a sugar/yeast/agar medium (Robert and Standen, 1998) prior to and during the experiments.
Hypoxic/Hyperbaric Exposure Experiments Male and female adult flies (n = 200; 100 males and 100 females) from each of the two lines were collected and pooled into fly bottles. Fertilized eggs from the two lines were also collected on standard yeast–glucose–agar medium in Petri dishes (60 × 15 mm). Fly bottles or Petri dishes were placed inside Plexiglas hyperbaric chamber (diameter 17 cm, height 23 cm, volume 5.2 l, temperature 26°C). Five different experimental conditions were utilized: normobaricnormoxia, hypoxia (2% oxygen), hyperoxia (100% oxygen), hyperbaria (2.5 ATA), and hyperbaric hyperoxia (2.5 ATA with 100% oxygen). Each group of individuals from the two utilized strains (CS and W1118) were submitted to one of the above-mentioned experimental conditions for a short period (90 min) or for a long period (180 min). Fly bottles were placed inside hyperbaric chamber. A ROXY-8 oxygen regulation system (Sable Systems International, Las Vegas, NV) was used to monitor PO2 in the chamber approximately every 30 min by regulating the ratio of O2 to N2 . At the end of the experiment, the bottles with the live flies were removed from the hyperbaric chamber and quickly (within 30 min) transferred to the laboratory, where they were immediately anesthetized with CO2 and frozen at −70°C in liquid nitrogen for citrate synthase’s assay, or re-placed in the incubator for the subsequent behavioral or lifespan measurements. Petri dishes with fertilized eggs were immediately transferred to the fly incubator for lifespan measurements. Archives of Insect Biochemistry and Physiology
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Isolation of Mitochondria Mitochondria were extracted in duplicate from each sample containing approximately 200 whole flies. The samples were homogenized using a Dounce glass–glass mortar and lose-fitting pestle. A mannitol–sucrose buffer (pH 7.4) with 2% bull serum albumin (BSA) was added to bring the homogenate volume to 25 ml. The samples were then centrifuged at 1,500 × g (Beckman Avanti J–25 Centrifuge, Beckman 2550 rotor, Beckman Coulter Life Sciences, Indianapolis, IN ,USA) at 4°C for 6 min. The pellet was discarded by filtering the sample through fine mesh, and the supernatant was recentrifuged at 7,000 × g at 4°C for 6 min. This supernatant was discarded and the remaining pellet was resuspended in 20 ml of the mannitol–sucrose buffer without BSA and centrifuged at 7,000 × g under the same conditions listed above. The supernatant was once again discarded and the pellet was resuspended in 50 ml of buffer solution. Concentration of proteins was determined by the Biuret test before storage at –80°C. The extracted mitochondria samples were diluted with a hypotonic buffer to the desired concentration—normally a final volume of mitochondria homogenate between 10 and 50 μl. Prior to assay, the samples were subjected to three freeze–thaw cycles using liquid nitrogen in order to break the mitochondrial membranes. Mitochondrial Mass (Citrate Synthase Activity) Assay According to Reisch and Elpeleg (2007), the enzymatic activity of citrate synthase was measured at room temperature. In brief, citrate synthase activity (CSA) was measured by monitoring the reduction of dithio-bis-nitrobenzoic acid (DTNB, colorless) to thionitrobenzoic acid (yellow) with coenzyme A at 412 nm (=13,600 M−1 cm−1 ). The assay buffer consisted of 75 mM Tris-HCl (pH 8) in which 0.4 mM acetyl-CoA, 0.1 μM DTNB, 0.5 mM oxaloacetate, and 5 μg mitochondrial proteins were added. Lifespan Measurements After the experiments in the hyperbaric chamber, male adult flies (n = 40 for each group) were immediately (see above) transferred to fly bottles with fresh food, stored in the fly incubator, and monitored until they died. The times of death were recorded every 24 h; this test was completed when the last insect was observed dead. Every 2–3 days, animals were transferred to new fly bottles with fresh food for maintaining the best physiological conditions, and to avoid insect death due to other causes (sticking to the humid food, mold, or bacterial growth). A similar experimental protocol was used for lifespan measurements of adult male flies (n = 40 for each group) that had been developed from fertilized eggs exposed to the different pressure/oxygen conditions in the hyperbaric chamber. Behavioral Assays Seizure behavior (bang-sensitive paralysis) was analyzed as described by Kuebler and Tanouye (2000). One day and 15 days after hyperbaric chamber experiments, male flies (n = 80 for each group) were tested for bang-sensitive paralysis. Twelve hours before experiments, individual males were collected under CO2 anesthesia and placed in polystirene vials (six animals per vial), where they were fed with water alone through a piece of soaked paper. The test consisted of a mechanical shock delivered by vortexing the vial with flies at high speed (40 Hz) for 10 sec (Kuebler and Tanouye, 2000). Archives of Insect Biochemistry and Physiology
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Mechanical stimulation induces the appearance of “seizures” in susceptible animals. Seizures are characterized by a first phase of tonic–clonic behavior, followed by a paralysis phase and third slow-recovery phase, as compared to a human seizure and demonstrated by direct electrophysiological recordings of the giant fiber neuromotor pathway (Kuebler and Tanouye, 2000; Lee and Wu, 2002). The fly’s behavior was video-recorded for 30 sec using a standard webcam. Offline analyses of the recorded videos determined in each fly group (i) the percentage of flies that sustained an epileptic seizure and, between these, (ii) the percentage of flies that did not recover from seizure, and (iii) the percentage of flies that did recover from seizure (Kuebler and Tanouye, 2000). From the videos, the speed at which the flies recovered from seizure, including seizure-like symptoms, was also analyzed. Statistical Analyses To analyze lifespan data, and to compare survivorship between normal conditions and the other treatments groups, we used the log-rank (Mantel–Cox) test. Chi-square test was used to compare the number of individuals displaying distinct epileptic behaviors (see above) between different fly groups. ANOVA was used to compare speed, and all other results were analyzed using Tukey test. All data were graphed using GraphPad Prism 5.0. Quantitative differences were considered significant if P-values were less than 0.05.
RESULTS Mitochondrial Mass After 90-min treatments, mitochondrial mass was significantly altered in W1118 flies. Hyperoxia caused a significant increase in CSA, while hypoxia and hyperbaric conditions caused significant reduction in mitochondrial enzymatic activity as shown in Figure 1A. The similar changes were also observed after prolonged 3-h treatments, as shown in Figure 1B. As shown in Figure 1C, in Canton-S flies, 90-min hyperoxia treatments increased enzymatic activity, whereas treated with both hypoxia and hyperbaria caused a decrease in enzymatic activity. Hyperbaric hyperoxia had no significant effect on enzymatic activity. Three hours of treatment with hyperoxia, hyperbaria, and hypoxia led to a significant decrease in enzymatic activity. However, hyperbaric hyperoxia had no effect on enzymatic activity. Longevity Survivorship was significantly reduced after each treatment. There was a significant reduction in lifespan from 70–80 days to 20–30 days after exposure, especially after 10–15 days from exposure. The reduction in the survivorship was the greatest after short-term hypoxia, and was the least after exposure to hyperbaric conditions (Fig. 2 A and B). Prolonged treatments (3 h) also caused a significant decrease in longevity. The mortality mostly happened 20 days after the treatments. The prolonged exposure to hyperbaria caused mortality only 20–25 days after exposure, as shown in Figure 2C and D. The effects of different oxygen concentration and atmospheric pressure on larvae of these two fly lines were also evaluated. A significant reduction in longevity after both Archives of Insect Biochemistry and Physiology
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Figure 1. Variation in specific activity (SA) of citrate synthase (nmol min−1 mg−1 ) of fly lines W1118 and Canton-S (CS) among treatments of different duration ((A and C) 90 min, (B and D) 3 h). Results are expressed as mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA), four treatments all compared to the control, respectively (*P < 0.05, n = 200).
short- and long-term exposure was observed. The mortality was mainly observed 25–30 days after each treatment. The short-term hyperbaric hyperoxia had the most negative impact on larvae’s survivorship, as shown in Figure 3. Seizure Susceptibility In response to mechanical agitation, flies demonstrated a stereotypical sequence of initial muscle spasm (uncoordinated movements), paralysis (lack of motion), and recovery (normal condition). The percentage of flies that displayed paralysis or seizure was measured on the day following treatment, and then 2 weeks after. The majority of flies showed seizure behavior 15 days after exposure, especially with the long-term treatments as shown in Figure 4 A and B. The percentage of flies that did not undergo paralysis or seizure, and was able to move in the vial (score), was also evaluated. The number of flies that moved and were able to reach at the higher level of the vial decreased after long-term exposure, and as shown in Figure 4C and D few flies were able to move 15 days after exposure. Recording the movements of the flies after exposure, we also calculated the time that they needed to move from one point to another in the vial. We measured adult walking speed by placing a single adult into a test tube and knocking the fly to the bottom of the tube according to Gilchrist (1996). The fly would then walk quickly up the tube, and we recorded the time for it to walk from bottom to the top (distance 8 cm). We immediately reran each fly and analyzed its average speed. The flies’ speed decreased significantly 15 Archives of Insect Biochemistry and Physiology
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Figure 2. Survivorship curves of W1118 and Canton-S: variation in longevity among treatments of different O2 concentration, pressure, and duration (90 min and 3 h). Log-rank (Mantel–Cox) test was used to compare lifespan between groups.
days after the exposure to extreme environmental conditions, especially after long-term treatments, as shown in Figure 5.
DISCUSSION Adaptive plasticity is an essential ability for organisms to respond to environmental challenges. In the present study, the impact of altered oxygen availability and atmospheric pressure on mitochondrial mass, lifespan, and behavioral function in D. melanogaster was evaluated. The main findings are as follows: (1) increased oxygen availability caused an increase in mitochondrial mass (higher CSA); (2) decreased oxygen availability, or increased atmospheric pressure, caused a decrease in mitochondrial mass (lower CSA); and (3) the acutely changed oxygen availability and atmospheric pressure decreased lifespan. The effect of changed oxygen availability, either hypoxia or hyperoxia on lifespan of D. melanogaster has been controversial. Rascon and Harrison examined (1) the effects of oxygen on adult longevity and (2) the effects of the oxygen concentration experienced by larvae on adult lifespan by rearing D. melanogaster in three oxygen atmospheres throughout larval development (10, 21, and 40 kPa), then measuring the lifespan of adults in five oxygen tensions (2, 10, 21, 40, 100 kPa; Rascon and Harrison, 2010). The rearing of juveniles in varying oxygen concentration affected lifespan in a complex manner, and the effect of different oxygen tensions on adult lifespan was nonlinear, with reduced Archives of Insect Biochemistry and Physiology
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Figure 3. Survivorship curves of flies born of eggs that experimented extreme environmental conditions: variation in longevity among treatments of different O2 concentration, pressure, and duration (90 min and 3 h). Log-rank (Mantel–Cox) test was used to compare lifespan between groups.
longevity and heightened oxidative stress at extreme high and low atmospheric oxygen levels. Moderate hypoxia (10 kPa) extended maximum, but not mean, lifespan (Miro et al., 2004). Skandalis et al. (2011) examined physiological phenotypes of D. melanogaster in hypoxic to hyperoxic atmospheres. They performed measurements on lifespan in 5, 21, 40, 60, and 80% O2 and observed that O2 incubation resulted in a concentrationdependent reduction in lifespan in both hypoxia and hyperoxia (Skandalis et al., 2011). The results in the present study are in agreement with Skandalis et al.’s (2011) study that changed oxygen availability; either hypoxia or hyperoxia considerably reduced the lifespan of D. melanogaster. Our study further suggests that acute changes in atmospheric pressure also reduce lifespan. We also observed that the reduced lifespan was associated with the reduced mitochondrial activity (less energy production). Both genetic and environmental factors determine the lifespan. Exactly how the interaction between the genetic and environmental factors affect lifespan is still not fully understood. However, environmental oxygen concentration is known to affect body size, growth rates, developmental rates, and cell cycle duration in Drosophila (Harrison and Harrison, 2006). In the history of the life evolution on Earth, the structural, behavioral, and physiological alterations are important strategies for organisms to adapt to a changed environment. All these alteration may be based on the equilibrium between energy production and consumption. The enzyme citrate synthase exists in nearly all living cells in the mitochondrial matrix and acts as a pace-making enzyme in the first step of the citric acid cycle, which is a key component of the metabolic pathway by which all aerobic organisms generate energy. The enzyme citrate synthase is essential for the survival of Archives of Insect Biochemistry and Physiology
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Figure 4. Percentage of the total number of flies that experimented extreme environmental conditions and displayed seizure behavior after mechanical stimulation. Data are referred to short-term (90 min) and long-term treatments (3 h); analyses were carried out after 24 h (1° Bang test) and 15 days from the exposure (2° Bang test). Statistical analyses were performed using Chi-square test (*P < 0.05).
Figure 5. Variation in animal’s speed after mechanical stimulation among treatments of different duration. Data are referred to short- and long-term exposure (90 min and 3 h) and analyses were carried out after 24 h and 15 days from the exposure. Statistical analyses were performed using two-way ANOVA (*P < 0.05).
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organisms. Compromised mitochondrial function has been linked to numerous diseases. Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. Among many theories of aging, one of the most promising is the free radical theory. This theory contends that senescence is the result of the accumulation of cellular and tissue damage caused by the reactive oxygen species (ROS). Mitochondria are one of the enzymatic sources of ROS and could also be a major target for ROSmediated damage (Wallace, 2005). Citrate synthase is commonly used as a quantitative enzyme marker for the presence of intact mitochondria. Maximal CSA is routinely used as a marker of aerobic capacity and mitochondrial density in cells. Citrate synthase has also been used to assess oxidative capacity (Reynolds et al., 2004; Gleixner et al., 2008), respiratory capacity (Freeman, 2000; Gleixner et al., 2008), and mitochondrial volume density (Gleixner et al., 2008). Changes in mitochondrial function may affect protein metabolism since protein synthesis and ubiquitin-dependent protein degradation are ATP-dependent. The present study shows that mitochondrial enzymatic activity depends on oxygen availability, which increases when oxygen is higher (hyperoxia) and decreases when oxygen is lower (hypoxia). The altered mitochondrial function (either increased or decreased CSA) may affect protein metabolism, and therefore cause the changes in behavioral function and survivorship. The links between aging and oxidative stress has been suggested and was later refined into the mitochondrial free radical theory of aging (Harman, 1972; Turrens, 2003). Overly produced ROS can damage cellular function and influence the rate of senescence in organisms. Nonetheless, the complex dynamics between oxygen concentration, oxidative damage, and senescence remain largely unclear. Either extreme hyperoxia or extreme hypoxia can induce increased oxidative stress. Rearing in pure oxygen (100% O2 ) has been shown to increase the rate of ROS production (Chance et al., 1979 ; Beckman and Ames, 1998) and oxidative injury of mitochondrial enzymes (Walker and Benzer, 2004) in Drosophila. It has also been observed that extreme hypoxia, followed by reoxygenation (reperfusion,) increases ROS production and causes oxidative injury (Chandel et al., 1998; Duranteau et al., 1998; Dirmeier et al., 2002). Oxidative stress may occur in both 100% oxygen and extreme hypoxia depends on a functional mitochondrial respiratory chain (Dirmeier et al., 2002), suggesting that the relationship between oxygen partial pressure and oxidative damage is a nonlinear relationship and perhaps, parabolic in shape. The nature of the relationship between atmospheric oxygen level and organism oxidative stress needs further investigation. It is now clear that much of the ROS production in mammalian cells is a toxic byproduct of the mitochondrial energy production pathway, an oxidative phosphorylation process (Forster and Estabrook, 1993; Ali et al., 2012). Overly produced ROS leads to the oxidation of mitochondrial lipids, proteins, and DNA, and the damage of their function (Holloszy et al., 1970; Hoppeler, 1986; Spina et al., 1996). The free radical theory of aging, understood as the decline of biological function on time, is complemented with the concept that lifespan is a consequence of oxygen toxicity (Klok et al., 2010; Perkins et al., 2012). When the free radical theory of aging (Perkins et al., 2012) is focused on mitochondria, it becomes more attractive as the mitochondrial hypothesis of aging (Harman, 1972). This hypothesis considers mitochondria as the pacemaker of tissue aging due to the continuous production of ROS and nitrogen species, which are kept in regulated steady-state concentration (Fergestad et al., 2008; Forster and Estabrook, 1993). The increased steady-state concentrations of ROS constitute the chemical basis of the biological situation of oxidative stress and imply an increased rate of intramitochondrial free radical reactions (Parker et al., 2011). Previous studies demonstrated that exposure Archives of Insect Biochemistry and Physiology
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to altered atmospheric partial pressure of oxygen increases ROS levels and oxidative stress; this increase in oxidative stress depends on mitochondrial activity and it can be the result of increased generation or decreased elimination of ROS (Miro et al., 2004; Pichaud et al., 2010; Charette et al., 2011; Ali et al., 2012). ROS could damage cells, tissues, and the entire organism. Prolonged exposure to high levels of oxygen generates excessive ROS, induces oxidative stress responses, affects immune response and DNA integrity, and causes cell death (Webster et al., 1987; Mantell and Lee, 2000; Lee and Choi, 2003; Tandara and Mustoe, 2004; Barker et al., 2006; Bhandari et al., 2007; Masalunga et al., 2007; Ogawa et al., 2007). The production of ROS has not been measured in the present study, however, abruptly changed oxygen availability may increase ROS production, as reflected by changes in mitochondrial function, and therefore, affects lifespan. The results from the present study demonstrate that organisms respond to short- and long-term exposure to hypoxia, hyperoxia, hyperbaria, and hyperoxic hyperbaria, along with the changes in mitochondrial function (altered CSA). The magnitude of changes in mitochondrial function is also in relation to the duration of exposure. We found that the adult’s survivorship was mostly affected by exposure to hypoxia and hyperbaria, and the larvae’s survivorship was mainly affected by exposure to hyperbaric hyperoxia. It has been observed that the newborn mammals were more resistant to hyperoxia challenge than adults (Zhao et al., 2010). Although this study focused on insect, the results were similar to that in mammal studies; D. melanogaster has similar O2 response pathways to those of mammals and research on flies has enhanced our understanding of oxidant stress (Chen et al., 1997; Haddad et al., 1997; Khurana, 2008; Morrow and Tanguay, 2008), possibly reflecting different O2 defense mechanisms between young and adult (Zhao et al , 2010). In summary, the results from the present study provide evidence that the alteration of environmental conditions, in terms of oxygen concentration and atmospheric pressure, can alter mitochondria mass and enzymatic activity. These altered mitochondrial functions may be responsible for the impaired behavioral function and the reduced lifespan. Further experiments are required to better understand the relationship between mitochondrial alterations and survivorship of flies coping with the environmental challenges. Our study may provide a basis for future studies to evaluate the effects of atmospheric oxygen and pressure on a variety of traits, including aerobic metabolism, behavioral, physiological, phenotypic, and genetic aspects.
ACKNOWLEDGMENTS We wish to thank Marcelyn Cook and Kimberly Hare for their English editorial efforts.
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Masalunga C, Rozycki HJ, Mainali ES. 2007. The impact of hyperoxia on the neonatal and adult developing dendritic cell. Pediatr Res 62(1):78–82. Miro O, Alonso JR, Lopez S, Beato A, Casademont J, Cardellach F. 2004. [Ex vivo analysis of mitochondrial function in patients attended in an emergency department due to carbon monoxide poisoning]. Med Clin (Barc) 122(11):401–406. Morrow G, Tanguay RM. 2008. Mitochondria and ageing in Drosophila. Biotechnol J 3(6):728–739. Ogawa Y, Tasaka S, Yamada W, Saito F, Hasegawa N, Miyasho T, Ishizaka A. 2007. Role of Toll-like receptor 4 in hyperoxia-induced lung inflammation in mice. Inflamm Res 56(8):334–338. Parker L, Padilla M, Du Y, Dong K, Tanouye MA. 2011. Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures. Genetics 187(2):523–534. Perkins G, Hsiao YH, Yin S, Tjong J, Tran MT, Lau J, Xue J, Liu S, Ellisman MH, Zhou D. 2012. Ultrastructural modifications in the mitochondria of hypoxia-adapted Drosophila melanogaster. PLoS One 7(9):e45344. Pichaud N, Chatelain EH, Ballard JW, Tanguay R, Morrow G, Blier PU. 2010. Thermal sensitivity of mitochondrial metabolism in two distinct mitotypes of Drosophila simulans: evaluation of mitochondrial plasticity. J Exp Biol 213(Pt 10):1665–1675. Rascon B, Harrison JF. 2010. Lifespan and oxidative stress show a non-linear response to atmospheric oxygen in Drosophila. J Exp Biol 213(Pt 20):3441–3448. Reisch AS, Elpeleg O. 2007. Biochemical assays for mitochondrial activity: assays of TCA cycle enzymes and PDHc. Methods Cell Biol 80:199–222. Reynolds ER, Stauffer EA, Feeney L, Rojahn E, Jacobs B, McKeever C. 2004. Treatment with the antiepileptic drugs phenytoin and gabapentin ameliorates seizure and paralysis of Drosophila bang-sensitive mutants. J Neurobiol 58(4):503–513. Roberts DB, Standen GN. 1998. The elements of Drosophila biology and genetics, pp. 1–54 in Drosophila: A Practical Approach, edited by D. B. ROBERTS. IRL Press, Oxford. Skandalis DA, Stuart JA, Tattersall GJ. 2011. Responses of Drosophila melanogaster to atypical oxygen atmospheres. J Insect Physiol 57(4):444–451. Spina RJ, Chi MM, Hopkins MG, Nemeth PM, Lowry OH, Holloszy JO. 1996. Mitochondrial enzymes increase in muscle in response to 7–10 days of cycle exercise. J Appl Physiol 80(6):2250–2254. Tandara AA, Mustoe TA. 2004. Oxygen in wound healing—more than a nutrient. World J Surg 28(3):294–300. Turrens JF. 2003. Mitochondrial formation of reactive oxygen species. J Physiol 552(Pt 2):335–344. Walker DW, Benzer S. 2004. Mitochondrial "swirls" induced by oxygen stress and in the Drosophila mutant hyperswirl. Proc Natl Acad Sci USA 101(28):10290–10295. Wallace DC. 2005. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407. Webster NR, Toothill C, Cowen PN. 1987. Tissue responses to hyperoxia. Biochemistry and pathology. Br J Anaesth 59(6):760–771. Zhao HW, Haddad GG. 2011. Review: hypoxic and oxidative stress resistance in Drosophila melanogaster. Placenta 32(Suppl 2):S104–S108. Zhao HW, Zhou D, Nizet V, Haddad GG. 2010. Experimental selection for Drosophila survival in extremely high O2 environments. PLoS One 5(7):e11701. Zhao HW, Ali SS, Haddad GG. 2012. Does hyperoxia selection cause adaptive alterations of mitochondrial electron transport chain activity leading to a reduction of superoxide production? Antioxid Redox Signal 16(10):1071–1076.
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EFFECTS OF OXYGEN CONCENTRATION AND PRESSURE ON Drosophila melanogaster: OXIDATIVE STRESS, MITOCHONDRIAL ACTIVITY, AND SURVIVORSHIP Gerardo Bosco Department of Biomedical Science, University of Padua, Padua, Italy and Hyperbaric Center Association Hyperbaric Technicians ATIP, Padua, Italy
Martina Clamer and Elisa Messulam Department of Biomedical Science, University of Padua, Padua, Italy
Cristina Dare Department of Biology, University of Padua, Padua, Italy
Zhongjin Yang Department of Anesthesiology, Upstate Medical University, Syracuse, New York, USA
Mauro Zordan Department of Biology, University of Padua, Padua, Italy
Carlo Reggiani Hyperbaric Center Association Hyperbaric Technicians ATIP, Padua, Italy
Qinggang Hu Department of Anesthesiology, Upstate Medical University, Syracuse, New York, USA
Aram Megighian Hyperbaric Center Association Hyperbaric Technicians ATIP, Padua, Italy
Correspondence to: Zhongjin Yang, Department of Anesthesiology, Upstate Medical University, Syracuse, New York, NY 13210. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 88, No. 4, 222–234 (2015) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21217
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Organisms are known to be equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. In this study, the effects of extreme changes in oxygen availability and atmospheric pressure on physiological phenotypes of Drosophila melanogaster were investigated to explore adaptation mechanisms. The changes in citrate synthase activity (CSA), lifespan, and behavioral function in different atmospheric conditions were evaluated. In the CAS test, hyperoxia significantly increased CSA; both hypoxia and hyperbaric conditions caused a significant decrease in CSA. In the survivorship test, all changed atmospheric conditions caused a significant reduction in lifespan. The lifespan reduced more after hypoxia exposure than after hyperbaria exposure. In behavioral function test, when mechanical agitation was conducted, bang-sensitive flies showed a stereotypical sequence of initial muscle spasm, paralysis, and recovery. The percentage of individuals that displayed paralysis or seizure was measured on the following day and after 2 weeks from each exposure. The majority of flies showed seizure behavior 15 days after exposure, especially after 3 h of exposure. The percentage of individuals that did not undergo paralysis or seizure and was able to move in the vial, was also tested. The number of flies that moved and raised the higher level of the vial decreased after exposure. Animal’s speed decreased significantly 15 days after exposure to extreme environmental conditions. In summary, the alteration of oxygen availability and atmospheric pressure may lead to significant changes in mitochondria mass, lifespan, and behavioral function in C 2014 Wiley Periodicals, Inc. D. melanogaster. Keywords: hypoxia; hyperoxia; hyperbaria; mitochondrial mass; lifespan; behavioral function; Drosophila melanogaster
INTRODUCTION The changes in environmental conditions play an essential role in an organism’s evolution on Earth. Organisms are equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. Adaptation to the environment is one of major factors involved in the evolution of the species, which makes organisms better suited to their habitat. Oxygen availability was crucial to the evolution of organisms throughout the history of the Earth (Harrison and Haddad, 2011). The relationship among atmospheric oxygen tension, oxidative stress, and lifespan across the range of oxygen levels has been previously investigated. Rascon and Harrison (2010) observed that the change of adult longevity was in a complex and nonlinear manner when juveniles were reared in varying oxygen concentration. Both extreme high and low atmospheric oxygen levels heightened oxidative stress and reduced longevity (Rascon and Harrison, 2010). Skandalis et al. (2011) also reported that decrease in lifespan of flies was an O2 concentration dependent in both hypoxia and hyperoxia. Hyperoxia led to a marked decrease in the rapid, and to a lesser extent prolonged, climbing behavior, though only flies in moderate hyperoxia showed a decrease that paralleled flies in normoxia (Skandalis et al., 2011). Hyperoxia leads to a marked decrease in the rapid, and to a lesser extent prolonged climbing behavior. Metabolism and water loss were both reduced Archives of Insect Biochemistry and Physiology
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in hyperoxia. Eleven-day-old flies in the highest oxygen concentration nearly doubled their water loss rates. These and other studies have examined the effects of altered oxygen availability on insects by focusing, although not exclusively, on prolonged hypoxia and hyperoxia (2, 5, 10, 40, 90, 100%) to identify the changes in phenotypic plasticity, respiratory physiology, behavior, and aerobic/anaerobic metabolism, as well as lifespan (Freeman, 2000; Miro et al., 2004; Pichaud et al., 2010; Charette et al., 2011; Ali et al., 2012). The effects of changed oxygen availability on animals have been examined in order to understand the evolutionary consequences and mechanisms of adaptation to these environments (Zhao and Haddad, 2011; Zhao et al., 2012). However, the mechanism underlying the adaptation to changed atmospheric pressure has not been investigated. The aim of this study was to investigate the effects of extreme changes in oxygen availability and atmospheric pressure (hypoxia, hyperoxia, hyperbaria, and hyperbaric hyperoxia) on survivorship, seizure susceptibility, and mitochondrial mass in Drosophila melanogaster to explore adaptation mechanisms.
MATERIALS AND METHODS Fly Lines Two commonly used wild Drosophila type lines of Canton-S and W1118 were used. The flies were reared in a fly incubator under standard conditions (25 ± 1°C, 12:12h light/dark cycle) and raised on a sugar/yeast/agar medium (Robert and Standen, 1998) prior to and during the experiments.
Hypoxic/Hyperbaric Exposure Experiments Male and female adult flies (n = 200; 100 males and 100 females) from each of the two lines were collected and pooled into fly bottles. Fertilized eggs from the two lines were also collected on standard yeast–glucose–agar medium in Petri dishes (60 × 15 mm). Fly bottles or Petri dishes were placed inside Plexiglas hyperbaric chamber (diameter 17 cm, height 23 cm, volume 5.2 l, temperature 26°C). Five different experimental conditions were utilized: normobaricnormoxia, hypoxia (2% oxygen), hyperoxia (100% oxygen), hyperbaria (2.5 ATA), and hyperbaric hyperoxia (2.5 ATA with 100% oxygen). Each group of individuals from the two utilized strains (CS and W1118) were submitted to one of the above-mentioned experimental conditions for a short period (90 min) or for a long period (180 min). Fly bottles were placed inside hyperbaric chamber. A ROXY-8 oxygen regulation system (Sable Systems International, Las Vegas, NV) was used to monitor PO2 in the chamber approximately every 30 min by regulating the ratio of O2 to N2 . At the end of the experiment, the bottles with the live flies were removed from the hyperbaric chamber and quickly (within 30 min) transferred to the laboratory, where they were immediately anesthetized with CO2 and frozen at −70°C in liquid nitrogen for citrate synthase’s assay, or re-placed in the incubator for the subsequent behavioral or lifespan measurements. Petri dishes with fertilized eggs were immediately transferred to the fly incubator for lifespan measurements. Archives of Insect Biochemistry and Physiology
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Isolation of Mitochondria Mitochondria were extracted in duplicate from each sample containing approximately 200 whole flies. The samples were homogenized using a Dounce glass–glass mortar and lose-fitting pestle. A mannitol–sucrose buffer (pH 7.4) with 2% bull serum albumin (BSA) was added to bring the homogenate volume to 25 ml. The samples were then centrifuged at 1,500 × g (Beckman Avanti J–25 Centrifuge, Beckman 2550 rotor, Beckman Coulter Life Sciences, Indianapolis, IN ,USA) at 4°C for 6 min. The pellet was discarded by filtering the sample through fine mesh, and the supernatant was recentrifuged at 7,000 × g at 4°C for 6 min. This supernatant was discarded and the remaining pellet was resuspended in 20 ml of the mannitol–sucrose buffer without BSA and centrifuged at 7,000 × g under the same conditions listed above. The supernatant was once again discarded and the pellet was resuspended in 50 ml of buffer solution. Concentration of proteins was determined by the Biuret test before storage at –80°C. The extracted mitochondria samples were diluted with a hypotonic buffer to the desired concentration—normally a final volume of mitochondria homogenate between 10 and 50 μl. Prior to assay, the samples were subjected to three freeze–thaw cycles using liquid nitrogen in order to break the mitochondrial membranes. Mitochondrial Mass (Citrate Synthase Activity) Assay According to Reisch and Elpeleg (2007), the enzymatic activity of citrate synthase was measured at room temperature. In brief, citrate synthase activity (CSA) was measured by monitoring the reduction of dithio-bis-nitrobenzoic acid (DTNB, colorless) to thionitrobenzoic acid (yellow) with coenzyme A at 412 nm (=13,600 M−1 cm−1 ). The assay buffer consisted of 75 mM Tris-HCl (pH 8) in which 0.4 mM acetyl-CoA, 0.1 μM DTNB, 0.5 mM oxaloacetate, and 5 μg mitochondrial proteins were added. Lifespan Measurements After the experiments in the hyperbaric chamber, male adult flies (n = 40 for each group) were immediately (see above) transferred to fly bottles with fresh food, stored in the fly incubator, and monitored until they died. The times of death were recorded every 24 h; this test was completed when the last insect was observed dead. Every 2–3 days, animals were transferred to new fly bottles with fresh food for maintaining the best physiological conditions, and to avoid insect death due to other causes (sticking to the humid food, mold, or bacterial growth). A similar experimental protocol was used for lifespan measurements of adult male flies (n = 40 for each group) that had been developed from fertilized eggs exposed to the different pressure/oxygen conditions in the hyperbaric chamber. Behavioral Assays Seizure behavior (bang-sensitive paralysis) was analyzed as described by Kuebler and Tanouye (2000). One day and 15 days after hyperbaric chamber experiments, male flies (n = 80 for each group) were tested for bang-sensitive paralysis. Twelve hours before experiments, individual males were collected under CO2 anesthesia and placed in polystirene vials (six animals per vial), where they were fed with water alone through a piece of soaked paper. The test consisted of a mechanical shock delivered by vortexing the vial with flies at high speed (40 Hz) for 10 sec (Kuebler and Tanouye, 2000). Archives of Insect Biochemistry and Physiology
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Mechanical stimulation induces the appearance of “seizures” in susceptible animals. Seizures are characterized by a first phase of tonic–clonic behavior, followed by a paralysis phase and third slow-recovery phase, as compared to a human seizure and demonstrated by direct electrophysiological recordings of the giant fiber neuromotor pathway (Kuebler and Tanouye, 2000; Lee and Wu, 2002). The fly’s behavior was video-recorded for 30 sec using a standard webcam. Offline analyses of the recorded videos determined in each fly group (i) the percentage of flies that sustained an epileptic seizure and, between these, (ii) the percentage of flies that did not recover from seizure, and (iii) the percentage of flies that did recover from seizure (Kuebler and Tanouye, 2000). From the videos, the speed at which the flies recovered from seizure, including seizure-like symptoms, was also analyzed. Statistical Analyses To analyze lifespan data, and to compare survivorship between normal conditions and the other treatments groups, we used the log-rank (Mantel–Cox) test. Chi-square test was used to compare the number of individuals displaying distinct epileptic behaviors (see above) between different fly groups. ANOVA was used to compare speed, and all other results were analyzed using Tukey test. All data were graphed using GraphPad Prism 5.0. Quantitative differences were considered significant if P-values were less than 0.05.
RESULTS Mitochondrial Mass After 90-min treatments, mitochondrial mass was significantly altered in W1118 flies. Hyperoxia caused a significant increase in CSA, while hypoxia and hyperbaric conditions caused significant reduction in mitochondrial enzymatic activity as shown in Figure 1A. The similar changes were also observed after prolonged 3-h treatments, as shown in Figure 1B. As shown in Figure 1C, in Canton-S flies, 90-min hyperoxia treatments increased enzymatic activity, whereas treated with both hypoxia and hyperbaria caused a decrease in enzymatic activity. Hyperbaric hyperoxia had no significant effect on enzymatic activity. Three hours of treatment with hyperoxia, hyperbaria, and hypoxia led to a significant decrease in enzymatic activity. However, hyperbaric hyperoxia had no effect on enzymatic activity. Longevity Survivorship was significantly reduced after each treatment. There was a significant reduction in lifespan from 70–80 days to 20–30 days after exposure, especially after 10–15 days from exposure. The reduction in the survivorship was the greatest after short-term hypoxia, and was the least after exposure to hyperbaric conditions (Fig. 2 A and B). Prolonged treatments (3 h) also caused a significant decrease in longevity. The mortality mostly happened 20 days after the treatments. The prolonged exposure to hyperbaria caused mortality only 20–25 days after exposure, as shown in Figure 2C and D. The effects of different oxygen concentration and atmospheric pressure on larvae of these two fly lines were also evaluated. A significant reduction in longevity after both Archives of Insect Biochemistry and Physiology
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Figure 1. Variation in specific activity (SA) of citrate synthase (nmol min−1 mg−1 ) of fly lines W1118 and Canton-S (CS) among treatments of different duration ((A and C) 90 min, (B and D) 3 h). Results are expressed as mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA), four treatments all compared to the control, respectively (*P < 0.05, n = 200).
short- and long-term exposure was observed. The mortality was mainly observed 25–30 days after each treatment. The short-term hyperbaric hyperoxia had the most negative impact on larvae’s survivorship, as shown in Figure 3. Seizure Susceptibility In response to mechanical agitation, flies demonstrated a stereotypical sequence of initial muscle spasm (uncoordinated movements), paralysis (lack of motion), and recovery (normal condition). The percentage of flies that displayed paralysis or seizure was measured on the day following treatment, and then 2 weeks after. The majority of flies showed seizure behavior 15 days after exposure, especially with the long-term treatments as shown in Figure 4 A and B. The percentage of flies that did not undergo paralysis or seizure, and was able to move in the vial (score), was also evaluated. The number of flies that moved and were able to reach at the higher level of the vial decreased after long-term exposure, and as shown in Figure 4C and D few flies were able to move 15 days after exposure. Recording the movements of the flies after exposure, we also calculated the time that they needed to move from one point to another in the vial. We measured adult walking speed by placing a single adult into a test tube and knocking the fly to the bottom of the tube according to Gilchrist (1996). The fly would then walk quickly up the tube, and we recorded the time for it to walk from bottom to the top (distance 8 cm). We immediately reran each fly and analyzed its average speed. The flies’ speed decreased significantly 15 Archives of Insect Biochemistry and Physiology
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Figure 2. Survivorship curves of W1118 and Canton-S: variation in longevity among treatments of different O2 concentration, pressure, and duration (90 min and 3 h). Log-rank (Mantel–Cox) test was used to compare lifespan between groups.
days after the exposure to extreme environmental conditions, especially after long-term treatments, as shown in Figure 5.
DISCUSSION Adaptive plasticity is an essential ability for organisms to respond to environmental challenges. In the present study, the impact of altered oxygen availability and atmospheric pressure on mitochondrial mass, lifespan, and behavioral function in D. melanogaster was evaluated. The main findings are as follows: (1) increased oxygen availability caused an increase in mitochondrial mass (higher CSA); (2) decreased oxygen availability, or increased atmospheric pressure, caused a decrease in mitochondrial mass (lower CSA); and (3) the acutely changed oxygen availability and atmospheric pressure decreased lifespan. The effect of changed oxygen availability, either hypoxia or hyperoxia on lifespan of D. melanogaster has been controversial. Rascon and Harrison examined (1) the effects of oxygen on adult longevity and (2) the effects of the oxygen concentration experienced by larvae on adult lifespan by rearing D. melanogaster in three oxygen atmospheres throughout larval development (10, 21, and 40 kPa), then measuring the lifespan of adults in five oxygen tensions (2, 10, 21, 40, 100 kPa; Rascon and Harrison, 2010). The rearing of juveniles in varying oxygen concentration affected lifespan in a complex manner, and the effect of different oxygen tensions on adult lifespan was nonlinear, with reduced Archives of Insect Biochemistry and Physiology
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Figure 3. Survivorship curves of flies born of eggs that experimented extreme environmental conditions: variation in longevity among treatments of different O2 concentration, pressure, and duration (90 min and 3 h). Log-rank (Mantel–Cox) test was used to compare lifespan between groups.
longevity and heightened oxidative stress at extreme high and low atmospheric oxygen levels. Moderate hypoxia (10 kPa) extended maximum, but not mean, lifespan (Miro et al., 2004). Skandalis et al. (2011) examined physiological phenotypes of D. melanogaster in hypoxic to hyperoxic atmospheres. They performed measurements on lifespan in 5, 21, 40, 60, and 80% O2 and observed that O2 incubation resulted in a concentrationdependent reduction in lifespan in both hypoxia and hyperoxia (Skandalis et al., 2011). The results in the present study are in agreement with Skandalis et al.’s (2011) study that changed oxygen availability; either hypoxia or hyperoxia considerably reduced the lifespan of D. melanogaster. Our study further suggests that acute changes in atmospheric pressure also reduce lifespan. We also observed that the reduced lifespan was associated with the reduced mitochondrial activity (less energy production). Both genetic and environmental factors determine the lifespan. Exactly how the interaction between the genetic and environmental factors affect lifespan is still not fully understood. However, environmental oxygen concentration is known to affect body size, growth rates, developmental rates, and cell cycle duration in Drosophila (Harrison and Harrison, 2006). In the history of the life evolution on Earth, the structural, behavioral, and physiological alterations are important strategies for organisms to adapt to a changed environment. All these alteration may be based on the equilibrium between energy production and consumption. The enzyme citrate synthase exists in nearly all living cells in the mitochondrial matrix and acts as a pace-making enzyme in the first step of the citric acid cycle, which is a key component of the metabolic pathway by which all aerobic organisms generate energy. The enzyme citrate synthase is essential for the survival of Archives of Insect Biochemistry and Physiology
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Figure 4. Percentage of the total number of flies that experimented extreme environmental conditions and displayed seizure behavior after mechanical stimulation. Data are referred to short-term (90 min) and long-term treatments (3 h); analyses were carried out after 24 h (1° Bang test) and 15 days from the exposure (2° Bang test). Statistical analyses were performed using Chi-square test (*P < 0.05).
Figure 5. Variation in animal’s speed after mechanical stimulation among treatments of different duration. Data are referred to short- and long-term exposure (90 min and 3 h) and analyses were carried out after 24 h and 15 days from the exposure. Statistical analyses were performed using two-way ANOVA (*P < 0.05).
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organisms. Compromised mitochondrial function has been linked to numerous diseases. Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. Among many theories of aging, one of the most promising is the free radical theory. This theory contends that senescence is the result of the accumulation of cellular and tissue damage caused by the reactive oxygen species (ROS). Mitochondria are one of the enzymatic sources of ROS and could also be a major target for ROSmediated damage (Wallace, 2005). Citrate synthase is commonly used as a quantitative enzyme marker for the presence of intact mitochondria. Maximal CSA is routinely used as a marker of aerobic capacity and mitochondrial density in cells. Citrate synthase has also been used to assess oxidative capacity (Reynolds et al., 2004; Gleixner et al., 2008), respiratory capacity (Freeman, 2000; Gleixner et al., 2008), and mitochondrial volume density (Gleixner et al., 2008). Changes in mitochondrial function may affect protein metabolism since protein synthesis and ubiquitin-dependent protein degradation are ATP-dependent. The present study shows that mitochondrial enzymatic activity depends on oxygen availability, which increases when oxygen is higher (hyperoxia) and decreases when oxygen is lower (hypoxia). The altered mitochondrial function (either increased or decreased CSA) may affect protein metabolism, and therefore cause the changes in behavioral function and survivorship. The links between aging and oxidative stress has been suggested and was later refined into the mitochondrial free radical theory of aging (Harman, 1972; Turrens, 2003). Overly produced ROS can damage cellular function and influence the rate of senescence in organisms. Nonetheless, the complex dynamics between oxygen concentration, oxidative damage, and senescence remain largely unclear. Either extreme hyperoxia or extreme hypoxia can induce increased oxidative stress. Rearing in pure oxygen (100% O2 ) has been shown to increase the rate of ROS production (Chance et al., 1979 ; Beckman and Ames, 1998) and oxidative injury of mitochondrial enzymes (Walker and Benzer, 2004) in Drosophila. It has also been observed that extreme hypoxia, followed by reoxygenation (reperfusion,) increases ROS production and causes oxidative injury (Chandel et al., 1998; Duranteau et al., 1998; Dirmeier et al., 2002). Oxidative stress may occur in both 100% oxygen and extreme hypoxia depends on a functional mitochondrial respiratory chain (Dirmeier et al., 2002), suggesting that the relationship between oxygen partial pressure and oxidative damage is a nonlinear relationship and perhaps, parabolic in shape. The nature of the relationship between atmospheric oxygen level and organism oxidative stress needs further investigation. It is now clear that much of the ROS production in mammalian cells is a toxic byproduct of the mitochondrial energy production pathway, an oxidative phosphorylation process (Forster and Estabrook, 1993; Ali et al., 2012). Overly produced ROS leads to the oxidation of mitochondrial lipids, proteins, and DNA, and the damage of their function (Holloszy et al., 1970; Hoppeler, 1986; Spina et al., 1996). The free radical theory of aging, understood as the decline of biological function on time, is complemented with the concept that lifespan is a consequence of oxygen toxicity (Klok et al., 2010; Perkins et al., 2012). When the free radical theory of aging (Perkins et al., 2012) is focused on mitochondria, it becomes more attractive as the mitochondrial hypothesis of aging (Harman, 1972). This hypothesis considers mitochondria as the pacemaker of tissue aging due to the continuous production of ROS and nitrogen species, which are kept in regulated steady-state concentration (Fergestad et al., 2008; Forster and Estabrook, 1993). The increased steady-state concentrations of ROS constitute the chemical basis of the biological situation of oxidative stress and imply an increased rate of intramitochondrial free radical reactions (Parker et al., 2011). Previous studies demonstrated that exposure Archives of Insect Biochemistry and Physiology
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to altered atmospheric partial pressure of oxygen increases ROS levels and oxidative stress; this increase in oxidative stress depends on mitochondrial activity and it can be the result of increased generation or decreased elimination of ROS (Miro et al., 2004; Pichaud et al., 2010; Charette et al., 2011; Ali et al., 2012). ROS could damage cells, tissues, and the entire organism. Prolonged exposure to high levels of oxygen generates excessive ROS, induces oxidative stress responses, affects immune response and DNA integrity, and causes cell death (Webster et al., 1987; Mantell and Lee, 2000; Lee and Choi, 2003; Tandara and Mustoe, 2004; Barker et al., 2006; Bhandari et al., 2007; Masalunga et al., 2007; Ogawa et al., 2007). The production of ROS has not been measured in the present study, however, abruptly changed oxygen availability may increase ROS production, as reflected by changes in mitochondrial function, and therefore, affects lifespan. The results from the present study demonstrate that organisms respond to short- and long-term exposure to hypoxia, hyperoxia, hyperbaria, and hyperoxic hyperbaria, along with the changes in mitochondrial function (altered CSA). The magnitude of changes in mitochondrial function is also in relation to the duration of exposure. We found that the adult’s survivorship was mostly affected by exposure to hypoxia and hyperbaria, and the larvae’s survivorship was mainly affected by exposure to hyperbaric hyperoxia. It has been observed that the newborn mammals were more resistant to hyperoxia challenge than adults (Zhao et al., 2010). Although this study focused on insect, the results were similar to that in mammal studies; D. melanogaster has similar O2 response pathways to those of mammals and research on flies has enhanced our understanding of oxidant stress (Chen et al., 1997; Haddad et al., 1997; Khurana, 2008; Morrow and Tanguay, 2008), possibly reflecting different O2 defense mechanisms between young and adult (Zhao et al , 2010). In summary, the results from the present study provide evidence that the alteration of environmental conditions, in terms of oxygen concentration and atmospheric pressure, can alter mitochondria mass and enzymatic activity. These altered mitochondrial functions may be responsible for the impaired behavioral function and the reduced lifespan. Further experiments are required to better understand the relationship between mitochondrial alterations and survivorship of flies coping with the environmental challenges. Our study may provide a basis for future studies to evaluate the effects of atmospheric oxygen and pressure on a variety of traits, including aerobic metabolism, behavioral, physiological, phenotypic, and genetic aspects.
ACKNOWLEDGMENTS We wish to thank Marcelyn Cook and Kimberly Hare for their English editorial efforts.
LITERATURE CITED Ali SS, Hsiao M, Zhao HW, Dugan LL, Haddad GG, Zhou D. 2012. Hypoxia-adaptation involves mitochondrial metabolic depression and decreased ROS leakage. PLoS One 7(5):e36801. Barker GF, Manzo ND, Cotich KL, Shone RK, Waxman AB. 2006. DNA damage induced by hyperoxia: quantitation and correlation with lung injury. Am J Respir Cell Mol Biol 35(3):277–288. Beckman KB, Ames BN. 1998. The free radical theory of aging matures. Physiol Rev 78(2):547–581. Bhandari V, Choo-Wing R, Homer RJ, Elias JA. 2007. Increased hyperoxia-induced mortality and acute lung injury in IL-13 null mice. J Immunol 178(8):4993–5000. Archives of Insect Biochemistry and Physiology
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