However, some male-killers have been reported from species where eggs are laid singly [31], so sibling interactions are of low intensity. Again, this could be explained if these bacteria have other effects, such as increasing host resistance to pathogens. The high prevalence of symbionts within and across species [32] could TPCA-1 concentration therefore be result of such symbionts that ‘employ’ multiple strategies, and may help explain their apparent success in invading new host populations or host species. In this study we have tested whether D. bifasciata infected with a male-killing strain of Wolbachia have greater protection

from viral pathogens. This strain of Wolbachia naturally infects 5-7% of female D. bifasciata resulting in close to 100% female broods at 18°C [33]. At elevated temperatures, infected males can be produced, and then the bacteria cause weak

cytoplasmic incompatibility when Small molecule library crossed to uninfected females [33]. In this study we examine whether this bacterium has a third phenotype by testing whether it confers protection Sapanisertib price from two RNA viruses. The first virus we used was Drosophila C virus (DCV), a positive sense RNA virus in the family Dicistroviridae [34] that naturally infects D. melanogaster in the wild [35, 36]. DCV commonly infects laboratory stocks of other Drosophila species [37], and can replicate when injected into a wide range of insects [38]. Secondly we used Flock House virus (FHV), a positive sense RNA virus in the family Nodaviridae [39]. It is not a natural pathogen of Drosophila (having been isolates from a coleopteran [40]), but will replicate in a broad range of insects and other taxa [41–44]. Wolbachia has been reported to increase the survival of D. melanogaster infected with both of these viruses GNA12 [17, 18]. Methods The Wolbachia-infected line of Drosophila bifasciata was collected in Japan in 1998 [33]. Since then (>140 generations) they have since been maintained by backcrossing infected females to males from an isofemale uninfected line present in the lab for 20 years. The two lines therefore

have the same nuclear genetic background. Because infected flies were maintained using male flies from the uninfected stock, other aspects of the flies (such as any commensal flora) will also be similar. The Wolbachia infection rate was 100% (no males were observed in the infected line). The flies were reared on agar-malt medium at ~18°C. We used reverse transcription (rt) PCR to check that the fly stocks we were using were not infected with DCV or FHV before the experiment. Total RNA was extracted from 40 individuals per line using Trizol reagent (Invitrogen Corp, San Diego, CA, USA) as described previously [45]. RNA was then reverse-transcribed with Promega Goscript reverse transcriptase (Promega Corp, Madison, WI, USA) using random hexamer primers.