Balanced stocks were used first to create homozygous females and hemizygous males of the two parental backgrounds, in order to include in the analysis any maternal effects of the mutation. Note that tko is an X-chromosomal gene. The initial outbreeding to create the balanced stocks restores a wild-type genetic background, but does not completely eliminate any potentially compensatory recessive alleles already in the wild-type backgrounds. To minimize the effects of any such alleles, the crossing scheme illustrated is both maximally wild-type and heterozygous, under which conditions we saw the most substantial accentuation of the mutant phenotype, compared with inbred tko^25t lines [115].

(a) The number of eggs laid per mated female was counted daily for individual mated females from the crosses indicated. Asterisks indicate significant differences (p<0.01, t-test). (b) Single mating pairs of the genotypes shown were observed for time to copulation.

(a, b) Proposed metabolic effects, based on differences in gene expression affecting nutrition and metabolism between (a) wild-type and (b) tko^25t flies. In wild-type flies glucose is metabolized via PEP to pyruvate, which is then fed to the TCA cycle mainly via the pyruvate dehydrogenase complex generating acetyl-CoA, with a small amount converted to oxaloacetate to replenish the TCA cycle intermediates as needed, maintaining a supply of carbon skeletons for biosynthesis. Surplus NADH is reoxidized via the ETC (complexes I, III and IV), generating potentially most of the cell's ATP needs at complex V. In tko^25t flies, the maximal activity of the ETC complexes is only 10–20% that of wild-type flies [18]. For simplicity, its greatly decreased contribution to NADH oxidation and ATP generation is omitted altogether in panel (b). Instead, the bulk of ATP must be supplied by glycolysis, with NADH reoxidation dependent on lactate dehydrogenase and similar shunts. Because pyruvate is, under such conditions, mainly shunted to lactate, the TCA cycle must be supplied from other sources, via the mobilization of dietary lipids, generating acetyl-CoA, PEP carboxykinase (I) diverting a small amount of PEP to oxaloacetate, and the mobilization of dietary protein and amino acid catabolism supplying these and other TCA cycle intermediates, as well as biosynthetic reactions directly. The modifications to metabolism in tko^25t flies are accompanied (c) by altered expression of genes connected with nutrient breakdown, absorption and transport, plus xenobiotic handling, affecting mainly the gut, Malpighian tubule and fat body. In addition, there is downregulation or delayed expression of genes connected with gametogenesis and skeletogenesis, and, notably in males, altered expression of genes controlling circadian and courtship behaviour, interpretable as a biological response to poor nutritional conditions.

PCR reactions analysed on agarose gels, using Wolbachia-specific 16S rDNA and Drosophila mitochondrial 12S rDNA primers. The 897 bp Wolbachia-specific product (arrowed) is detected only in the Wolbachia-infected strain obtained from the Bloomington Stock Center (wol), and not in wild-type (+) or tko^25t flies in either the Oregon R (OR) or Canton S (CS) backgrounds, nor in inbred laboratory stocks of the sesB¹ or to¹ mutants. The 180 bp mitochondrial DNA product is evident in all strains tested. M, 1 kb marker ladder.

RNA measurements were made and normalized as described in Materials and Methods. Means±SD of three sample runs of each of three biological replicates are shown. Significance at the p value shown was computed using a t-test. See also Table 3.