Early-life hypoxia alters adult physiology and reduces stress resistance and lifespan in Drosophila

Abstract

In many animals, short-term fluctuations in environmental conditions in early life often exert long-term effects on adult physiology. In Drosophila, one ecologically relevant environmental variable is hypoxia. Drosophila larvae live on rotting, fermenting food rich in microorganisms, an environment characterized by low ambient oxygen. They have therefore evolved to tolerate hypoxia. Although the acute effects of hypoxia in larvae have been well studied, whether early-life hypoxia affects adult physiology and fitness is less clear. Here, we show that Drosophila exposed to hypoxia during their larval period subsequently show reduced starvation stress resistance and shorter lifespan as adults, with these effects being stronger in males. We find that these effects are associated with reduced whole-body insulin signaling but elevated TOR kinase activity, a manipulation known to reduce lifespan. We also identify a sexually dimorphic effect of larval hypoxia on adult nutrient storage and mobilization. Thus, we find that males, but not females, show elevated levels of lipids and glycogen. Moreover, we see that both males and females exposed to hypoxia as larvae show defective lipid mobilization upon starvation stress as adults. These data demonstrate how early-life hypoxia can exert persistent, sexually dimorphic, long-term effects on Drosophila adult physiology and lifespan.

Keywords: Akt; Glucose; Glycogen; Insulin; Lifespan; Lipids; Metabolism; Sexual dimorphism; Starvation stress; TOR.

Fig. 1.

Effects of larval hypoxia on adult body mass and survival in Drosophila melanogaster. (A) An outline of the experimental protocol for examining the effects of larval hypoxia on adult physiology in D. melanogaster. For all experiments, w¹¹¹⁸ embryos were raised in normoxia. Upon hatching, they were transferred to food vials and kept in either normoxia or hypoxia (5% oxygen) for the duration of their larval period. The animals were then kept in normoxia throughout pupal development until they emerged as adults. Mated, 1-week-old adults were assayed for changes in their starvation stress survival, lifespan, gene expression and metabolite levels. (B) Male and female adult body masses from the normoxic and hypoxic groups. (C) Survival to adulthood of animals from the normoxic and hypoxic groups. Data were calculated as the percentage of eclosed adults from each group. Data are presented as box plots (25%, median and 75% values), with error bars indicating the minimum and maximum values, and individual data points shown as dots. *P<0.05; n.s., not significant; Student’s t-test.

Fig. 2.

Larval hypoxia has no effect on adult tolerance to hypoxia in D. melanogaster. (A,B) Hypoxia (1% oxygen) survival graphs for male (A) and female (B) adult Drosophila that had been exposed to either normoxia (blue lines) or hypoxia (brown lines) as larvae. Survival was measured after exposing adult animals to either a 16 h or 20 h hypoxia exposure. Data are presented as box plots (25%, median and 75% values), with error bars indicating the minimum and maximum values, and individual data points shown as dots. n.s., not significant; Student’s t-test.

Fig. 3.

Larval hypoxia leads to reduced adult tolerance to starvation stress and reduced adult lifespan in D. melanogaster. (A–D) Starvation survival curves (A,B) and survival curves (C,D) for male (A,C) and female (B,D) adult Drosophila that had been exposed to either normoxia (blue lines) or hypoxia (brown lines) as larvae. Data were analyzed using the Log-rank test.

Fig. 4.

Larval hypoxia leads to altered adult insulin and TOR signaling in D. melanogaster. (A,B) Levels of dILP mRNAs were measured by qRT-PCR in mated adult males (A) and females (B) after they had been exposed to either normoxia (blue plots) or hypoxia (brown plots) as larvae. Data are presented as box plots (25%, median and 75% values), with error bars indicating the minimum and maximum values, and individual data points shown as dots. *P<0.05; Student’s t-test. (C) Western blot analysis of phosphorylated Akt (pAkt; threonine 342 and serine 505), total Akt, phosphorylated S6K (pS6K) and total Actin (loading control), in lysates from mated adult flies after they had been exposed to either normoxia (N) or hypoxia (H) as larvae.

Fig. 5.

Larval hypoxia alters adult male, but not female, nutrient storage in D. melanogaster. (A) Levels of triacylglyceride (TAG), glycogen and glucose in wandering third-instar larvae that had been exposed to either normoxia (blue plots) or hypoxia (brown plots) for the duration of their larval development. (B,C) Levels of TAG, glycogen and glucose in mated adult male (B) and female (C) animals after they had been exposed to either normoxia (blue plots) or hypoxia (brown plots) as larvae. Data are presented as box plots (25%, median and 75% values), with error bars indicating the minimum and maximum values, and individual data points shown as dots. *P<0.05; n.s., not significant; Student's t-test.

Fig. 6.

Larval hypoxia leads to a sexually dimorphic effect on lipid mobilization during starvation stress in D. melanogaster. Changes in TAG levels upon 16 h of complete nutrient starvation in adult male and female animals after they had been exposed to either normoxia (blue plots) or hypoxia (brown plots) as larvae. Data were calculated as percentage change in TAG levels in starved animals compared with fed animals, and are presented as box plots (25%, median and 75% values), with error bars indicating the minimum and maximum values, and individual data points shown as dots. *P<0.05; two-way ANOVA followed by post hoc Student's t-test.