Adaptation to Long-Term Hypoxia in Drosophila Figure 8. Differences in enzymic activities of triose phosphate isomerase and pyruvate kinase and allelic frequency between AF and NF. A) Enzymic activity and hypoxia response of TPI in AF and NF. TPI activity was measured in 3rd instar larvae of AF and NF cultured under either normoxia or hypoxia. AF had a significantly reduced enzymic activity than NF under both hypoxic and normoxic conditions. In contrast to the effect of hypoxia in NF, hypoxia did not induce a significant reduction on enzyme activity in AF. Data were presented as means6SD (* p,0.05, #p.0.05). B) Enzymic activity and hypoxia response of PK in AF and NF. The enzymic activity of PK was measured in 3rd instar larvae of NF and AF cultured under either normoxia or hypoxia. There was no significant difference in PK activity between AF and NF cultured under normoxia. Hypoxia induced a significant reduction of PK activity in NF but not in AF. Data were presented as means6SD (* p,0.05, #p.0.05). C) Comparison of allelic frequency in the genomic region of TPI gene between AF and NF. The genomic locus encoding TPI gene was amplified by high fidelity PCR using specific primers. The PCR products were purified and cloned into pCR4-TOPO plasmid to create a plasmid library for the TPI alleles of AF and NF populations. Ten clones from the AF or NF TPI allele library were sequenced and compared by using ClustalW2 software and DnaSP 4.0. The statistical power for the comparison was calculated by GPower software 3.0.3. Seven 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 (p,0.001, CHI-Test). doi:10.1371/journal.pgen.1000221.g008 isopropanol. The DNA pellet was washed with 70% of ethanol and dissolved in TE buffer. The genomic locus encoding TPI gene was amplified by PCR using specific primers (forward primer: GTTTAAGGTCCGCA- GAGGTG, and reverse primer: ATTTTGGCAAGCCTGTT- GAT). All the coding exons and intronic flanking regions were amplified by polymerase chain reaction (PCR) using the high fidelity proofreading DNA polymerase, Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, CA). The PCR products were purified and cloned into pCR4-TOPO plasmid to create the plasmid library for TPI alleles of the AF and NF population. Cycle sequencing was performed on an ABI automated sequencer (Applied Biosystems, Foster City, CA) by Eton Biosciences, Inc. (San Diego, CA). Ten clones from AF or NF TPI allele library PLoS Genetics | www.plosgenetics.org 10 October 2008 | Volume 4 | Issue 10 | e1000221