Ghosts of symbionts past: The hidden history of the dynamic association between filarial nematodes and their Wolbachia endosymbionts

ABSTRACT Many, but not all, parasitic filarial nematodes (Onchocercidae) carry intracellular, maternally-transmitted Wolbachia symbionts, and these alphaproteobacteria are targets for anti-filarial chemotherapeutic interventions for human disease. The symbionts of Onchocercidae derive from four of the major supergroups (C, D, F and J) defined within the genus Wolbachia. Using twenty-two whole genome sequences of filarial nematodes and genome sequences of their Wolbachia partners, we have explored the evolutionary history of the nematode-Wolbachia symbiosis. We screened the nuclear genome sequences of all the nematodes for nuclear Wolbachia transfers (NUWTs), fragments of the Wolbachia genome that have been integrated into the nuclear genome. Six species have no current Wolbachia infection. Setaria labiatopapillosa had no validated NUWTs and we interpret this to mean that this species was never infected with Wolbachia. In the other five species (Acanthocheilonema viteae, Cercopithifilaria (Ce.) johnstoni, Elaeophora elaphi, Loa (Lo.) loa and Onchocerca flexuosa) we found NUWTs, implying they have previously had and have now lost Wolbachia infections. For each NUWT locus, we identified the supergroup membership of the Wolbachia from which it originated, and found that the five Wolbachia-free species carried NUWTs derived from multiple supergroups, including a high shared rate of sequences derived from supergroup C. In Dirofilaria repens we identified a sample that carried two Wolbachia symbionts, one from supergroup C and one from supergroup F. In Dirofilaria immitis where live infection with a supergroup C Wolbachia is found, we identified NUWTs derived from an F Wolbachia, confirming that the F association predated the divergence of these Dirofilaria species. The supergroup D lineage of Wolbachia, as present in the human parasites Wuchereria bancrofti and Brugia malayi, derives from a replacement event. Madathamugadia (Md.) hiepei shows signs of multiple recent repeated endosymbiont replacement. From these data we infer that the history of Wolbachia in onchocercid nematodes includes not only cospeciation (as present in the Onchocerca-Dirofilaria group in association with supergroup C Wolbachia) and loss (in the five Wolbachia-free species), but also frequent symbiont replacement and dual infection. This dynamic pattern is challenging to models that assume host-symbiont mutualism.