A host-parasite-endosymbiont relationship: twisted-wing insects provide a new twist to Wolbachia infections in Australian tephritid fruit fly species

Wolbachia are maternally inherited endosymbionts in 50% of insects and can affect host reproduction and fitness. Occasional horizontal transfer of Wolbachia between species occurs. In some insects, field surveys consistently detect Wolbachia at low prevalence and titres without clear host effects. This enigmatic detection could be due to recurring horizontal Wolbachia acquisition or presence of concealed Wolbachia-infected hosts such as endoparasites. Previously, two Wolbachia strains have been reported at low prevalence and titre in seven Australian tephritid fruit fly species. Here, we performed whole-genome sequencing analyses of field-caught Wolbachia-positive flies. Besides the fruit fly mitochondrial genomes, we unexpectedly found complete mitogenomes of a twisted-wing insect, Dipterophagus daci (Strepsiptera), suggesting that the Wolbachia in the flies is actually linked to concealed parasitisation. We performed the first genetic characterisation of this strepsipteran and its relationship with Wolbachia and tephritids. Using a new PCR-diagnostic tool we detected D. daci in Wolbachia-positive flies that were not visibly parasitised (stylopised). In contrast, most Wolbachia-negative flies were negative for D. daci. Then, we dissected D. daci specimens from stylopised flies and confirmed by qPCR that Wolbachia infects D. daci, but also found potential somatic Wolbachia infections in stylopised flies. Our study highlights that early stages of strepsipteran parasitisation that are not externally visible have led to the incorrect previous assignment of Wolbachia infection to the tephritids. Wolbachia detection in some host taxa may be due to the presence of undetected strepsipterans or other parasites, obscuring ecological studies of this common endosymbiont and its horizontal transmission