Figure 2. Gene expression profiles in hypoxia-selected Dro- sophila melanogaster in larvae and adult flies. A) Comparison of gene expression alterations in hypoxia-selected Drosophila melanoga- ster in the larva and adult fly. Expression levels of 2749 genes (1534 up- and 1215 down-regulated) in larvae and 138 genes (95 up- and 43 down-regulated) in the adult were significantly altered in AF (fold change.1.5 and q,0.05). Red: up-regulation; Green: down-regulation. B) Summary of significantly altered biological processes in larvae and adult hypoxia-selected Drosophila melanogaster (p,0.05). doi:10.1371/journal.pgen.1000221.g002 tolerance, we determined the survival rate of these two hairy loss- of-function mutants at 6% of O2. This mild level of O2 was used since it is sufficient to show differences between the mutants and controls. As shown in Figure 7B, both hairy loss-of-function mutants exhibited much lower survival rate (p,0.0001, CHI-Test) as compared to controls, proving the contribution of hairy to hypoxia tolerance in flies. Differences in DNA Sequence between AF and NF Since some of the current experiments suggested that hypoxia tolerance was a heritable trait in the AF population, we studied these flies further to determine the genetic basis of this heritability. Two types of experiments were performed. First, we examined the activity levels of 2 key glycolytic enzymes, triose phosphate isomerase (TPI) and pyruvate kinase (PK) in NF and AF. We argued that, if there was a genetic basis for the change in enzyme activity, such activity level would be altered not only in the hypoxia-cultured flies but also in those cultured in normoxia, i.e., the enzyme activity would be different from that in NF, whether in hypoxia or normoxia. As shown in Figures 8A and 8B, the activity of TPI in AF was indeed reduced when these flies were cultured in Adaptation to Long-Term Hypoxia in Drosophila either normoxia or hypoxia. When PK activity was assessed, however, AF had a higher level activity under hypoxia and stayed at the same level in normoxia without a significant increase as compared to NF. Second, the genomic locus encoding TPI was sequenced to determine whether there were any differences between AF and NF. We chose to sequence the TPI locus, because, unlike PK, TPI is encoded by a single gene. As expected, a number of significant polymorphic differences were identified in the AF population that included 3 SNPs (277T/C, 253A/G, 251A/T) and 1 indel (274 to 266) in the cis-regulatory region, 1 synonymous SNP (1051A/G) in the coding region, 1 SNP (1480A/G) in the 39-untranslated region, and 1 SNP (1662C/T) in the downstream region (Figure 8C, Table S7), demonstrating significant genetic differences in the AF population (p,0.001, CHI-Test). Discussion The current laboratory selection experiments have shown that the hypoxia-selected flies have garnered, with ongoing genera- tions, a spectacular ability to survive extremely low O2 conditions. To put this ability in perspective, these flies live perpetually now at an oxygen level that is equivalent to that above Mount Everest by ,4,000 meters. This phenotypic breakthrough occurred after 32 generations of selection. One of the most profound phenotypic abnormalities is a reduction in body size in the AF flies. This substantial decrease in body size was presumably of physiologic relevance, which is likely related to shorten the O2 diffusion distances for improved survival. It is very interesting to note that, though we show evidence of heritability of the hypoxia survival trait in the selected flies, the change of body size and weight did not seem to be heritable, as body size was reverted to normal, even after a single generation in room air. The goal of the current study was to determine the genetic and molecular basis of adaptation to long-term (i.e., over generations) hypoxic environments. Since survival under long term hypoxia is a complex trait, we expected this adaptation to be controlled by a number of pathways and genes. Indeed, our microarray and genetic analyses have shown that the survival of both larvae and adult flies was accompanied by alterations in a number of major signaling pathways, such as EGF, Insulin, Notch and Toll/Imd pathways. In this regard, there are a number of interesting observations made from our studies. First, the number of significantly altered genes was much larger in larvae than in the adults. More than 20% of all measured genes were significantly altered in the larval samples whereas only ,1% changed in the adults, using the same statistical criteria. This major difference between larvae and adults may have a number of explanations, including the fact that larval cells undergo rapid growth, proliferation and differentiation as compared to adults. Second, although adaptive changes were found in both larvae and adult flies, it is possible that these changes were induced through both genetic and/or epigenetic physiologic regulation. Since we found that a) the hypoxia-selected flies could live for a number of generations in normoxia and survive again when introduced back in 4% hypoxia, b) some glycolytic enzymes maintained their activity in the AF population at a lower (or higher) level not only during hypoxia but also in normoxia (e.g., TPI and PK), and c) a number of polymorphic changes existed in AF as compared to NF, our data provided direct evidence for actual genetic alterations that were responsible for, at least in part, tolerance to severe hypoxia. Third, several signal transduction pathways were altered in the selected flies, i.e., EGF, Insulin, Notch and Toll/Imd pathway, which have also been found to be activated by hypoxic stimuli in PLoS Genetics | www.plosgenetics.org 4 October 2008 | Volume 4 | Issue 10 | e1000221