Author Summary Hypoxia-induced injury has been related to multiple pathological conditions. In order to render mammalian cells and tissues resistant to low O2 environment, we wished to first understand the mechanisms underlying hypoxia-tolerance in resistant animals. Therefore, we generated a D. melanogaster strain that is tolerant to severe hypoxic conditions through long-term experimen- tal selection. Several adaptive changes were identified in the hypoxia-selected flies that included up-regulation of multiple signal transduction pathways (such as Notch pathway, Insulin pathway, EGF receptor pathway, and Toll/ Imd pathway), modulation of cellular respiration enzymes, and polymorphic differences in metabolic enzymes (such as TPI). While we believe that multiple pathways contrib- ute to the hypoxia-tolerant trait in this Drosophila strain, we demonstrate that hairy-mediated metabolic suppres- sion is a critical mechanism for reducing the mismatch between supply and demand of O2. of development. Furthermore, we used a combination of bioinformatic, molecular and genetic strategies to investigate the role of specific genes in hypoxia tolerance. Results Experimental Selection of Hypoxia-Tolerant Drosophila melanogaster Twenty-seven wild-type isogenic lines constituted the parental population that we used for long-term experimental selection of a Drosophila melanogaster strain. At baseline, there was significant variability in hypoxia tolerance among these 27 lines, as determined by eclosion rate under 5% O2 and recovery time from anoxic stupor [17]. In order to determine the level of O2 at which to initiate the long-term selection experiment, we performed a pilot study by culturing F1 embryos of the parental flies under different levels of hypoxia (e.g., 8%, 6% or 4% O2) (Figure 1A). We found that their survival rate was reduced at 6% O2, and no adult flies were actually obtained at 4% O2. At 8% O2, however, the majority of embryos (.80%) completed their development and reached the adult stage. Therefore, hypoxia selection was initiated at 8% O2, an O2 level in which flies can develop throughout their life cycle. The O2 concentration was then gradually decreased by ,1% every 3 to 5 generations to maintain the selection pressure (except for the transition between 5% and 4% which necessitated a much longer time). By the 13th generation, flies were able to complete development and perpetually live in 5% O2; and by the 32nd generation, the AF flies could even live perpetually under a severer level (4% of O2), a lethal condition for NF. We hypothesized that this is due to, at least partially, newly occurring mutations or recombination and selection of favorable alleles in the AF population. To test this hypothesis, a subset of embryos obtained from selected flies were collected and cultured under normoxia for several consecutive generations. After 8 generations in normoxia, AF were re-introduced into the lethal hypoxic environment (4% O2), and again, the majority (.80%) of the flies completed their development and could be maintained in this extreme condition perpetually. This result strongly suggested that the hypoxia-tolerance in the selected flies is a heritable trait. Several remarkable phenotypic changes were observed in the hypoxia-selected flies (AF). As described previously [17], the AF flies have a shortened recovery time from anoxia-induced stupor [6], consume more oxygen in hypoxia, and show a significant Adaptation to Long-Term Hypoxia in Drosophila reduction in body weight and size. We also demonstrated that this reduction in size is due to the reduction in both cell number and cell size [17]. In the hypoxia chambers, at 5% O2 levels, adult flies had significantly decreased body weight: the decrease in male body weight was about 25% and was further reduced to about 40% under 4% O2 (Figure 1C and 1D). Interestingly, the reduction in body weight and size were reversed to normal when the AF embryos were grown under normoxia. Life span was also studied in our selected and na¨ıve flies. Hypoxia-selection did not affect lifespan (Figure 1B). Whole Genome Expression Profiles of Hypoxia-Selected Drosophila melanogaster Global gene expression profiles were examined in hypoxia- selected Drosophila melanogaster in the 3rd instar larval stage and in adults using cDNA microarrays that contained 13,061 known or predicted genes of the Drosophila melanogaster genome [16,18]. After analyzing the data sets with a significant cutoff of .1.5 fold difference and a false discovery rate (FDR) of ,0.05 [19], 2749 genes (1534 up- and 1215 down-regulated) were significantly altered in the larval stage, but only 138 genes (,20 times less than those in larvae) met this criteria (95 up- and 43 down-regulated) in the adult. The complete list of differentially regulated genes is detailed in Tables S1 and S2. Among them, 51 genes were found to be altered in both larval and adult stages with 23 up-regulated and 7 down-regulated genes (Figure 2A). Interestingly, most of the commonly up-regulated genes encode proteins that are related to immunity, and the majority of the commonly down-regulated genes encode proteins that are related to metabolism. The significantly altered genes were further analyzed, based upon the Gene Ontological categorizations (GO) [20], by GenMAPP and MAPPFinder [20,21]. Less than half of the differentially expressed genes (,40%) encode for proteins whose functions have not been characterized; the remaining encode proteins that are involved in numerous biological functions, such as development, metabolism, defense mechanisms and signal transduction (Tables S3 and S4). More than 30 biological processes were found to be altered in both larvae and adult and these were mostly related to either defense (especially immune responses, p,0.05) or metabolism (especially carbohydrate and peptide metabolism, p,0.05) (Figure 2B). The most affected processes in larvae were related to metabolism (1044 genes, especially carbohydrate metabolism, 135 genes, p,0.01). In addition, multiple components of signal transduction pathways were identified to be significantly altered in larvae and these included EGF, insulin, Notch, and Toll/Imd signaling pathways (Figure 3, p,0.05). To confirm the changes obtained from microarrays, 10 differentially expressed genes were randomly chosen and their expression levels were determined by real-time qRT-PCR using specific primers in larval samples (Table S5). The microarray results and the real-time PCR gave similar trends (r = 0.85, Figure S1). Changes in Genes Encoding Cellular Respiratory Enzymes Significant gene changes were identified in the family of genes regulating cellular respiration in AF, especially in larvae. The majority of these changes consisted of a down- rather than an up- regulation of gene expression (Table S3). Besides one pyruvate kinase isoform (CG12229), most of the genes encoding glycolytic enzymes were dramatically down-regulated (Figures 4A and 4B). Similarly, suppression was also found in the TCA cycle (Figures 4A and 4C), lipid b-oxidation, and respiratory chain complex genes in larvae (Figure 5). As shown in Figures 5B and 5C, among the 50 measured genes encoding components of the respiratory chain, 33 PLoS Genetics | www.plosgenetics.org 2 October 2008 | Volume 4 | Issue 10 | e1000221