Adaptation to Long-Term Hypoxia in Drosophila Figure 4. Suppression of genes encoding glycolytic and TCA cycle enzymes. A) Schematic illustration of the alterations in genes encoding glycolytic and TCA cycle enzymes (green: down-regulation, black: not significantly changed, red: up-regulation). B) Cluster map of changes in genes encoding glycolytic enzymes in NF and AF larvae. C) Cluster map of changes in genes encoding TCA cycle enzymes in NF and AF larvae. Each cluster contained 9 hybridizations of NF and 8 hybridizations of AF samples. doi:10.1371/journal.pgen.1000221.g004 necessarily behave differently in hypoxia [6], and b) we chose two different mutants of hairy and they have similar effects. Another interesting observation in this work is related to the enzymatic activities of triose-phosphate isomerase (TPI) and pyruvate kinase (PK) in AF. We hypothesized that, if there is a genetic basis for the change in enzyme activity, we should be able to detect the alterations in AF flies that were cultured in both hypoxia and normoxia, and the enzyme activity should maintain different from those in NF, whether in hypoxia or normoxia. Indeed, as expected, we found that the expression level and enzymatic activity of TPI were significantly lower in AF than those in NF in both hypoxia and normoxia. In addition, we identified 7 polymorphic differences in the genomic locus of the TPI gene in AF which demonstrated a genetic modification of this locus in AF strain. In contrast, the activity of PK in AF did not differ from that of NF in normoxia, although its activity was significantly higher in AF under hypoxia (Figure 8B). This result demonstrated that there was an inhibition of this enzyme in NF under hypoxia, and such hypoxia-induced inhibition was abolished in AF. On the surface, this result might weaken our argument, a potential explanation for such a difference between PK and TPI is that the regulation of PK is much more complicated. Indeed, there are 7 genes encoding PK in D. melanogaster. In AF, we found that 1 gene (i.e., Pyk) was down- regulated and another gene (i.e., CG12229) was up-regulated. Therefore, in addition to possible genetic rearrangements, other factors may play a role to keep higher activity of PK in AF under hypoxia and to minimize the difference between AF and NF in normoxia. If the changes that led to enhanced survival in extremely low O2 levels in the AF population are genetic in nature, as we demonstrated in this work, it is important to note that the breakthroughs in survival to lower and lower O2 levels took place after a relatively small number of generations in the Drosophila. This relatively small number of generations was also found when previous investigators were studying other phenomena such as geotaxis [15]. If translated into human years, the 32 generations that were needed to alter the phenotype and genotype in Drosophila can be approximated to about a thousand years. Although changes in the DNA code with selection pressure could take a much longer period of time (potentially thousands and millions of years in ‘‘Darwinian time’’), this work demonstrated that such changes in DNA and phenotype could proceed at a much faster rate, presumably because of the trait itself or the selection pressure applied. PLoS Genetics | www.plosgenetics.org 6 October 2008 | Volume 4 | Issue 10 | e1000221