Perforated 50 ml centrifuge tube

The cytoplasmic-incompatibility level was assessed at Generation 19 by crossing infected, cured, and uninfected flies in all possible combinations except crosses between infected and cured flies (Table S1). Five one-day old virgin females were allowed to mate with five males of the same age from a different line in a mating cage, three cages per cross. Drosophila nigrosparsa, unlike D. melanogaster or D. simulans, females only lay eggs after seven days of mating and the larvae hatch two days after being laid. Thus, flies were allowed to mate for seven days. Males were removed on the eighth day, and each female was individualized into a perforated 50-ml centrifuge tube (Sarstedt, Germany) supplied with grape-juice agar, malt food, and live yeast. The number of eggs laid per female and the number of hatched larvae were counted on Day 9 and Day 14, respectively. Significance in hatching was analyzed using a generalized linear mixed model fit by maximum likelihood with a binomial error structure and logit link function implemented in the package lme4 (Bates et al., 2015) . The number of eggs laid and the infection status of females were used as fixed effects and lines as random effect.