Disrupting the timing of Wolbachia-induced male-killing

Several lineages of maternally inherited symbionts have evolved the ability to kill infected females' sons, a phenomenon known as male-killing. Male-killing varies in its timing, from early (death during embryogenesis) to late (mortality of late larval instars). Following the observation that treatment of male-killer infected adult females Hypolimnas bolina with tetracycline, a bacteriostatic antibiotic, produces a delay in the timing of male death, we hypothesized that early male-killers possess the ability to kill males through bacterial activity outside of embryogenesis. We verified this hypothesis by showing that treatment of surviving larvae with the bacteriocidal antibiotic rifampicin rescues males. This discounted the hypothesis that delayed death occurred due to postponed effects of toxins produced at earlier stages, and thus supported the importance of bacterial activity in the larval phase in delayed male-killing. These results argue against the view that early male-killing is achieved by specifically targeting an early developmental process within the sex determination pathway.