Physiology, fitness and disease phenotypes are complex traits exhibiting continuous variation in natural populations. To understand complex trait gene functions transgenic lines of undefined genetic background are often combined to assess quantitative phenotypes ignoring the impact of genetic polymorphisms. Here, we used inbred wild-type strains of the Drosophila Genetics Reference Panel to assess the phenotypic variation of six physiological and fitness traits, namely, female fecundity, survival and intestinal mitosis upon oral infection, defecation rate and fecal pH upon oral infection, and terminal tracheal cell branching in hypoxia. We found continuous variation in the approximately 150 strains tested for each trait, with extreme values differing by more than four standard deviations for all traits. In addition, we assessed the effects of commonly used Drosophila UAS-RNAi transgenic strains and their backcrossed isogenized counterparts, in the same traits plus baseline intestinal mitosis and tracheal branching in normoxia, in heterozygous conditions, when only half of the genetic background was different among strains. We tested 20 non-isogenic strains (10 KK and 10 GD) from the Vienna Drosophila Resource Center and their isogenized counterparts without Gal4 induction. Survival upon infection and female fecundity exhibited differences in 50% and 40% of the tested isogenic vs. non-isogenic pairs, respectively, whereas all other traits were affected in only 10–25% of the cases. When 11 isogenic and their corresponding non-isogenic UAS-RNAi lines were expressed ubiquitously with Gal4, 4 isogenic vs. non-isogenic pairs exhibited differences in survival to infection. Furthermore, when a single UAS-RNAi line was crossed with the same Gal4 transgene inserted in different genetic backgrounds, the quantitative variations observed were unpredictable on the basis of pure line performance. Thus, irrespective of the trait of interest, the genetic background of commonly used transgenic strains needs to be considered carefully during experimentation.

Most physiology, fitness and disease phenotypes are considered complex traits since they exhibit continuous variation in natural populations. The use of homozygous inbred strains allows the assessment of the impact of genetic composition on complex phenotypes. Nevertheless, to understand gene function, we often combine transgenic lines to assess the phenotype of interest in a heterozygous state. Here, using inbred wild-type Drosophila strains, we assessed the phenotypic variation of six physiology and fitness traits: female fecundity, survival and intestinal mitosis upon oral infection, defecation rate and fecal pH upon oral infection, and terminal tracheal cell branching in hypoxia. Indeed, we found continuous variation in all traits tested. To estimate the extent to which small genetic background differences affected these traits, we assessed the effects of commonly used Drosophila UAS-RNAi transgenic strains and their backcrossed isogenized counterparts in the same assays. We tested 20 non-isogenic strains (10 KK and 10 GD) from the Vienna Drosophila Resource Center and their isogenic counterparts without Gal4 induction and found that the different phenotypes were variably sensitive to the genetic background. For example, survival upon infection and female fecundity exhibited differences in 50% and 40% of the tested isogenic vs. non-isogenic pairs, respectively, whereas all other phenotypes were affected in only 10-25% of the cases. We also observed that the genetic background affects Gal4 transgenes unpredictably. Thus, depending on the trait of interest, the genetic background of commonly used transgenic strains needs to be considered carefully during experimentation.