Holometabolous insects undergo dramatic morphological and physiological changes during ontogeny. In particular, the larvae of many holometabolous insects are specialized to feed in soil, water or dung, inside plant structures, or inside other organisms as parasites where they may commonly experience hypoxia or anoxia. In contrast, holometabolous adults usually are winged and live with access to air. Here, we show that larval Drosophila melanogaster experience severe hypoxia in their normal laboratory environments; third instar larvae feed by tunneling into a medium without usable oxygen. Larvae move strongly in anoxia for many minutes, while adults (like most other adult insects) are quickly paralyzed. Adults survive anoxia nearly an order of magnitude longer than larvae (LT50: 8.3 versus 1 h). Plausibly, the paralysis of adults is a programmed response to reduce ATP need and enhance survival. In support of that hypothesis, larvae produce lactate at 3× greater rates than adults in anoxia. However, when immobile in anoxia, larvae and adults are similarly able to decrease their metabolic rate, to about 3% of normoxic conditions. These data suggest that Drosophila larvae and adults have been differentially selected for behavioral and metabolic responses to anoxia, with larvae exhibiting vigorous escape behavior likely enabling release from viscous anoxic media to predictably normoxic air, while the paralysis behavior of adults maximizes their chances of surviving flooding events of unpredictable duration. Developmental remodeling of behavioral and metabolic strategies to hypoxia/anoxia is a previously unrecognized major attribute of holometabolism.

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Environmental changes during development have long-term effects on adult phenotypes in diverse organisms. Some of the effects play important roles in helping organisms adapt to different environments, such as insect polymorphism. Others, especially those resulting from an adverse developmental environment, have a negative effect on adult health and fitness. However, recent studies have shown that those phenotypes influenced by early environmental adversity have adaptive value under certain (anticipatory) conditions that are similar to the developmental environment, though evidence is mostly from morphological and behavioral observations and it is still rare at physiological and molecular levels. In the companion study, we applied a short-term starvation treatment to fifth instar honey bee larvae and measured changes in adult morphology, starvation resistance, hormonal and metabolic physiology and gene expression. Our results suggest that honey bees can adaptively respond to the predicted nutritional stress. In the present study, we further hypothesized that developmental starvation specifically improves the metabolic response of adult bees to starvation instead of globally affecting metabolism under well-fed conditions. Here, we produced adult honey bees that had experienced a short-term larval starvation, then we starved them for 12 h and monitored metabolic rate, blood sugar concentrations and metabolic reserves. We found that the bees that experienced larval starvation were able to shift to other fuels faster and better maintain stable blood sugar levels during starvation. However, developmental nutritional stress did not change metabolic rates or blood sugar levels in adult bees under normal conditions. Overall, our study provides further evidence that early larval starvation specifically improves the metabolic responses to adult starvation in honey bees.

Oxygen limitation plays a key role in many pathologies; yet, we still lack a fundamental understanding of the mechanisms responsible for variation in anoxia tolerance. Most vertebrate studies suggest that anoxia tolerance involves the ability to maintain cellular ATP despite the loss of aerobic metabolism. However, insects such as adult are able to survive long periods of anoxia (LT: ∼8 h) in a hypo-energetic state characterized by low [ATP]. In this study, we tested for possible mechanisms that allow adults to survive long periods of anoxia. Adults are paralyzed within 30 s, and after 2 h of anoxia, ATP was 3% of normal, extracellular potassium concentration ([K]) increased threefold, pH dropped 1 unit, yet survival was 100%. With 0.5-6 h of anoxia, adults maintained low but constant ATP levels while [K] and pH continued to change. When returned to normoxia, adults restored [K] and activity. With longer durations of anoxia, ATP levels decreased and [K] rose further, and both correlated tightly with decreased survival. This response contrasts with the anoxia-sensitive larval stage (LT: ∼1 h). During anoxia, larvae attempted escape for up to 30 min and after 2 h of anoxia, ATP was <1% of resting, [K] increased by 50%, hemolymph pH fell by 1 unit, and survival was zero. The superior anoxia tolerance of adult appears to be due to the capacity to maintain a paralytic hypometabolic state with low but non-zero ATP levels, and to be able to tolerate extreme extracellular ionic variability.

Animals vary tremendously in their capacities to survive anoxia, and the mechanisms responsible are poorly understood. Adult Drosophila melanogaster are rapidly paralyzed and survive up to 12 h of anoxia, whereas larvae vigorously attempt escape but then die if anoxia exceeds 2 h. Here we use nuclear magnetic resonance methods to compare the metabolome of larvae and adult D. melanogaster under normoxic conditions and after various anoxic durations up to 1 h. Glucose increased during anoxia in both larvae and adults, so anoxic death by carbohydrate limitation is unlikely for either stage. Lactate and alanine were the primary anaerobic end products in both adults and larvae. During the first 30 min of anoxia, larvae accumulated anaerobic end products (predominately lactate) at a higher rate, suggesting that larvae may experience greater initial acid-base disruption during anoxic exposures. Adult Drosophila did not possess higher levels of putative protective metabolites; however, these increased during anoxia in adults and decreased in larvae. Metabolites that decreased during anoxia in larvae included mannitol, xylitol, glycerol, betaine, serine, and tyrosine, perhaps due to use as fuels, antioxidants, or binding to denatured proteins. Adults showed significant increases in glycine, taurine, and the polyols glycerol, mannitol, and xylitol, suggesting that adults upregulate protective metabolites to prevent damage. Our results suggest that lower initial metabolic demand due to paralytic hypometabolism and capacities to upregulate protective metabolites may assist the better anoxia tolerance of adult Drosophila.