Adaptation to Long-Term Hypoxia in Drosophila Figure 5. Suppression of genes encoding components of the respiratory chain complexes. A) Cluster map of changes in gene expression encoding components of respiratory chain complexes in NF and AF larvae. Each cluster contained 9 hybridizations of NF and 8 hybridizations of AF samples. B) Summary of alterations in each respiratory chain complex measured in larvae. C) Comparison of gene expression changes in each respiratory chain complex between larvae and adult. doi:10.1371/journal.pgen.1000221.g005 In summary, we have generated a Drosophila melanogaster strain that is very tolerant to severe hypoxic conditions through long- term experimental selection. Several adaptive changes have been identified in the hypoxia-selected AF flies that include up- regulation of multiple signal transduction pathways, modulation of cellular respiration enzymes, and polymorphic differences in metabolic enzymes such as TPI. While we believe that multiple pathways contribute to hypoxia-tolerant trait in this Drosophila strain, we demonstrate that hairy-mediated metabolic suppression is an important one. The adaptive mechanisms identified in this hypoxia-tolerant Drosophila model may also play a crucial role in protecting mammals from hypoxia injury. Materials and Methods Drosophila melanogaster Stocks, Hypoxia Selection, and Phenotypic Assays Twenty-seven isogenic Drosophila melanogaster lines (kindly provid- ed by Dr. Andrew Davis) were used as parental stocks for the long- term hypoxia-selection experiment as described previously [17]. Briefly, embryos collected from the parental population were divided into 6 groups, 3 groups of them were subjected to long-term hypoxia-selection, and 3 other groups were maintained under normoxia as controls. Both hypoxia-selection and control experi- ments were performed in specially designed population chambers (26 cm616 cm616 cm). These chambers were connected to either O2 at certain concentrations (balanced with N2, for the hypoxia- selection experiments) or to room air (21% O2, for the control experiments). The humidity in the chambers was maintained by passing the gas through water prior to going into the chambers. The flow speed was monitored by 565 Glass Tube Flowmeter (Concoa, Virginia Beach, VA), and the O2 level within the chamber was monitored with Diamond General 733 Clark Style Electrode (Diamond General Development Corp., Ann Arbor, MI). The selection was started at 8% O2 and this concentration was gradually decreased by 1% each 3 to 5 generations to keep the selection pressure. Embryos, 3rd instar larvae and adult flies were collected from each generation and stored at 280uC for analyses. The results presented in the current study were derived from expression arrays using larval and adult samples. The body weight of hypoxia-selected flies was determined at each generation. Male flies (n = 100) from hypoxia or control chambers were collected, weighed and used as the index of body weight. PLoS Genetics | www.plosgenetics.org 7 October 2008 | Volume 4 | Issue 10 | e1000221