Responses of Drosophila melanogaster to atypical oxygen atmospheres

https://doi.org/10.1016/j.jinsphys.2011.01.005Get rights and content

Abstract

We examined physiological phenotypes of Drosophila melanogaster in hypoxic to hyperoxic atmospheres. We performed measurements on life span or behavioural function in 5, 21, 40, 60, and 80% O2, and combined this with literature data for 2% and 100% O2. O2 incubation resulted in a concentration-dependent reduction of life span in both hypoxia and hyperoxia, though different measures of life span were affected differently. We also examined how behavioural and metabolic functions were affected by exposure to hyperoxia (up to 60% O2). Climbing behaviour was measured as a fast (4 s) and slow (55 s) response in a negative geotaxis assay. In normoxia, both measures of climbing response declined exponentially until disappearing completely. Interestingly, survivorship was very high until the loss of climbing ability, after which it dropped rapidly. This pattern appeared accelerated in 40% O2. However, while flies in 60% O2 also apparently lost their fast climbing ability immediately prior to the drop in survivorship, they maintained considerable climbing ability over the longer trial. Metabolism, measured by CO2 release, did not change with age in normoxic flies, but was significantly lower in flies exposed to hyperoxia, particularly as the flies aged. There was, however, a slight increase in water loss rate with age in normoxia, while in hyperoxia, water loss was reduced. Uniquely, the water loss rates of flies in 60% O2 doubled immediately prior to the end of their life span. Because ageing results in generally irreversible functional declines, we examined if functional declines in hyperoxia (60% O2) were also irreversible, or whether some functioning could recover after a return to normoxia. After 7 days of recovery, water loss rates decreased, CO2 exhalation slightly increased, and climbing ability was partially recovered. Therefore, the effect of O2 on D. melanogaster function is non-linear, may be reversible, and may include unique phenotypes that arise at some O2 concentrations, and not others.

Research highlights

► Life span decreases as logarithmic function of oxygen in hypoxia and hyperoxia. ► Different patterns of age-associated locomotor dysfunction in normoxia and hyperoxia. ► Different patterns of age-specific respiratory function in normoxia and hyperoxia. ► Locomotor and metabolic dysfunction in hyperoxia can be recovered by return to normoxia.

Introduction

During the course of normal ageing, DNA (Hamilton et al., 2001), proteins (Levine and Stadtman, 2011), and cellular structures (Miquel et al., 1975) can become damaged by reactive oxygen species. This damage may impair metabolism and neural function, and contribute to mortality in adults (e.g., Martin et al., 2009a, Wicks et al., 2009). Frequently, model organisms are exposed to hyperoxic atmospheres to simulate or accelerate pathologies associated with oxygen damage (e.g., Rebrin and Sohal, 2006, Walker and Benzer, 2004). In the modern literature, hyperoxia (normoxia = 21% O2) is usually synonymous with 100% O2, but a paucity of information on the effects of moderate hyperoxia impedes our understanding of the mode of oxygen toxicity (Harrison et al., 2006). It is generally assumed that the loss of function in hyperoxia is a linear, graded function of oxygen content. This does indeed suggest that a pure oxygen atmosphere will result in the most pronounced loss of function, but this dependence on linearity assumes that novel or exaggerated phenotypes do not emerge at different oxygen concentrations.

The literature supporting a linear impact of oxygen content on Drosophila melanogaster function is conflicting, and largely relies on measured life spans. Baret et al. (1994) concluded that life span of D. melanogaster was linearly related to O2 concentrations from 20% to 50% O2; however, in hyperbaric hyperoxic atmospheres, the effect of O2 concentration was distinctly non-linear (Fenn et al., 1967). There is generally little information on the effect of intermediate hyperoxia (in this text, 22–100% O2) on decay of function or tissue damage in fruit flies (Harrison et al., 2006). At 50% O2, the decay of locomotor function was accelerated compared to normoxia, which supported the view that O2 damage caused the behavioural deficits in ageing flies (Miquel et al., 1975). Besides this single exposure, intermediate normobaric O2 concentrations are poorly described, so it remains an open question to what degree variation in O2 reduces life span and alters behavioural performance. Moreover, a consideration of the graded effects of O2 would benefit from more data on long-term exposures to hypoxia (<21% O2).

The effect of hyperoxia on D. melanogaster physiology may indeed be simply to accelerate the onset of the normal ageing phenotype. Overexpression and deletion of antioxidant genes, which alter endogenous levels of oxidative stress, have provided results consistent with this interpretation. For example, expression of SOD1 (Sun and Tower, 1999) or SOD2 (Sun et al., 2002) transgenes extends D. melanogaster lifespan, while deletion or loss of function mutations of Sod1 or Sod2 shortens lifespan (e.g., Martin et al., 2009a, Martin et al., 2009b). Some phenotypes are strikingly similar between hyperoxia and senescence, such as the onset of skeletal muscle mitochondrial whorls (Sacktor and Shimada, 1972, Walker and Benzer, 2004). Nonetheless, the physiological bases for the shared phenotypes or functional declines may differ. For instance, Miquel et al. (1975) compared flies in normoxia and 50% O2, and noted that while many tissue-level degenerative phenotypes are shared, the phenotypes in hyperoxia are frequently more pronounced than in senescence. At the cellular level, the redox state (as measured in the glutathione system) of ageing flies gradually and consistently becomes more oxidised, but flies exposed to 100% O2 show no obvious trend towards oxidation (Rebrin and Sohal, 2006). On the other hand, protein carbonylation rates are far greater in hyperoxia – and in hypoxia – than in normoxia (Rascón and Harrison, 2010). In the transcriptome, aged and 100% O2-exposed flies exhibit a 40% overlap, with one key difference being a downregulation of metabolic enzymes during senescence (Landis et al., 2004). While exposure to hyperoxia leads to marked destruction in the neural system (Miquel et al., 1975), where apoptotic bodies and neurodegeneration are readily induced (Gruenewald et al., 2009), caspase activity, a marker of apoptosis, is absent from senescent flies’ heads (Zheng et al., 2005). Similarly, Kloek et al. (1978) observed that vacuolation was absent from senescent flies’ brains, but widespread in brains of flies exposed to some hyperoxic conditions. Interestingly, they observed a threshold of >33% O2 for brain vacuolation, and increasing amounts of vacuolation thereafter (up to 55% O2). Together, these data suggest that, although O2 contributes to normal senescence, there may be fundamental differences in the functional phenotypes which result from chronic exposure to low O2 doses (in this case, normoxia) and those which result from acute exposure to massive doses.

We raised flies in hypoxic and hyperoxic conditions to examine how life span, metabolism, and locomotor function are altered, compared to normoxia. We report two indices of life span, the ages at which 10% or 100% of the population has died. We monitored locomotor function using negative geotaxis, both as the number of flies that climbed to a threshold height within 4 s, and the number that climbed within 55 s. We then measured flies’ metabolic activities by their carbon dioxide release rate and the rate of water vapor loss. Water vapor loss is commonly used to indicate spiracular opening frequency (e.g., Lighton and Schilman, 2007). Finally, we examined whether the flies’ hyperoxic phenotypes were reversible if the flies were removed from hyperoxia and allowed to recover in normoxia.

Section snippets

Fly breeding

Breeding populations of flies (strain Oregon-R) were raised in ambient laboratory conditions, on a cornmeal/yeast/agar diet. Male flies <24 h old were collected and sorted on a chill table between −2 °C and −4 °C degrees (BioQuip Products, Inc); cold anaesthetisation was used to prevent confounding effects of anoxia (Rascón and Harrison, 2010). Flies were placed on a Kimwipe to prevent direct contact with the metal surface, and sorted within 15–20 min; in no case did flies die after cold exposure.

Life span reduction by both hypoxia and hyperoxia

In our experiments, flies’ LS0 were the same in hypoxia (5% O2) and normoxia, though LS90 was shorter in hypoxia; both LS0 and LS90 were decreased in hyperoxia (Fig. 1). We compared our data to published life spans in hypoxia and hyperoxia, and the results were similar (Fig. 2). LS90 was maximum in normoxia, and significantly reduced by incubation in hypoxia or hyperoxia (Fig. 2a). LS0 instead peaked at 10% O2, as per the observations of Rascón and Harrison (2010; Fig. 2b). The available data

The effect of O2 on life span

In general, our observations confirm the reduction of D. melanogaster life span in both hypoxic and hyperoxic oxygen atmospheres (Fig. 2; Rascón amd Harrison 2010). Our analysis of original and published data suggests that the effects of O2 atmospheres on life span, from extreme hypoxia (2% O2) to extreme hyperoxia (100% O2), are best modelled by non-linear equations:LS90=0.28ln O2+0.19, O221%0.58ln O2+2.76, O221%LS0=0.59 ln O20.04, O210%0.52 ln O2+2.52, O210%

The logarithmic

Acknowledgements

We gratefully acknowledge the emotional, intellectual, nutritional, and technical support provided by our colleagues Viviana Cadena, Marc Charette, Charles Darveau, Danielle Levesque, Melissa Page, Ellen Robb, Kurtis Salloway, and Brent Wiens. We also gratefully acknowledge two anonymous referees whose comments substantially improved and clarified the manuscript. This research was supported by Natural Sciences and Engineering Research Council of Canada grants to GJT and JAS.

References (40)

  • J.P. Phillips et al.

    Targeted neuronal gene expression and longevity in Drosophila

    Experimental Gerontology

    (2000)
  • I. Rebrin et al.

    Comparison between the effects of aging and hyperoxia on glutathione redox state and protein mixed disulfides in Drosophila melanogaster

    Mechanisms of Ageing and Development

    (2006)
  • P. Vigne et al.

    Diet dependent longevity and hypoxic tolerance of adult Drosophila melanogaster

    Mechanisms of Ageing and Development

    (2007)
  • S. Wicks et al.

    Hypoxia rescues early mortality conferred by superoxide dismutase deficiency

    Free Radical Biology & Medicine

    (2009)
  • W.O. Fenn et al.

    Oxygen poisoning in Drosophila

    Journal of General Physiology

    (1967)
  • M. Ferguson et al.

    Age-associated decline in mitochondrial respiration and electron transport in Drosophila melanogaster

    Biochemical Journal

    (2005)
  • M.L. Hamilton et al.

    Does oxidative damage to DNA increase with age?

    Proceedings of the National Academy of Sciences of the United States of America

    (2001)
  • S.K. Hetz et al.

    Insects breathe discontinuously to avoid oxygen toxicity

    Nature

    (2005)
  • G.P. Kloek et al.

    Survivorship and life expectancy of Drosophila melanogaster populations in abnormal oxygen-normal pressure regimes

    Aviation, Space, and Environmental Medicine

    (1976)
  • G.P. Kloek et al.

    Oxygen-induced brain vacuolation in Drosophila and a possible threshold for this response

    Aviation, Space, and Environmental Medicine

    (1978)
  • 1

    Currently at the Universität Ulm, Ulm, Germany.

    View full text