Caveats of this study

It should be noted that these experiments were performed with mosquitoes transiently infected in the somatic tissues with Wolbachia, rather than a stable maternally inherited infection. It remains to be seen whether a stable wAlbB infection will enhance WNV in a similar way. Wolbachia density in mosquito somatic tissues (as opposed to germline) was found to explain differences in virus infection in Aedes mosquitoes [41]. Thus, it seems likely that if stable infection in Cx. tarsalis has a similar somatic tissue distribution to a transient infection it may induce a similar virus enhancement phenotype. However, this must be tested empirically. It is also unknown whether virus enhancement is limited to WNV or occurs more broadly. Finally, we tested a single Wolbachia strain, and it is unknown whether virus enhancement is specific to wAlbB or occurs with diverse Wolbachia strains.

Previous studies have shown that pathogen suppression by Wolbachia has the potential to be a novel method for controlling vector-borne diseases [4], [42]–[44]. Not all mosquito species are naturally infected with Wolbachia, but non-infected species may support infection once introduced and these novel infections often effectively inhibit various pathogens [5], [45]. Our experiments indicate that following adult microinjection, Wolbachia is capable of establishing both somatic and germline infection in Cx. tarsalis but does not inhibit WNV infection, dissemination or transmission. In contrast with other studies showing pathogen inhibition by Wolbachia, our data suggest that Wolbachia may in fact increase WNV infection rates in Cx. tarsalis, particularly at early time points. Increased early infection has the potential to shorten the extrinsic incubation period of the pathogen, which can dramatically increase the reproductive rate of the virus [19]. It has become increasingly clear that Wolbachia does not always suppress pathogens in insects [46]. For example, the cereal crop pest Spodoptera exempta is more susceptible to nucleopolydrovirus mortality in the presence of Wolbachia [47]. In the mosquitoes An. gambiae An. stephensi, Ae. fluviatilis and Cx. pipiens, Wolbachia enhances Plasmodium berghei, P. yoelii, P. gallinaceum and P. relictum, respectively [17]–[20]. Enhancement may be dependent on the host-Wolbachia strain-pathogen system of interest, as Wolbachia strains that block one pathogen yet enhance another have been documented [9], [17]. Wolbachia-mediated pathogen enhancement may be a common yet often ignored phenomenon, which merits attention when designing Wolbachia-based strategies for disease control [46].

Intracellular infection with bacteria may alter the cellular environment in multiple ways, including bacterial manipulation to avoid host immune defenses [48]. Though the exact Wolbachia-mediated inhibition mechanism is unknown, studies have suggested that Wolbachia indirectly modulates mosquito immunity [40], [49]. Wolbachia can activate the Toll pathway, stimulating a cascade of events that have been correlated with inhibition of dengue and Plasmodium in mosquitoes [39], [50], [51]. In contrast, in Cx. tarsalis, wAlbB infection significantly downregulated REL1 (the activator of the Toll pathway), suggesting that in this system Wolbachia may down regulate antiviral Toll-based immunity leading to increased virus infection. However, while statistically significant, this decrease in REL1 expression was modest, and further study is required to determine the precise mechanism of Wolbachia-based WNV enhancement in this system.

To our knowledge this is first study showing Wolbachia can potentially enhance a vector-borne pathogen that causes human disease. Our results, combined with other Wolbachia enhancement studies [17]–[20], [46]–[47], suggest that field deployment of Wolbachia-infected mosquitoes should proceed with caution. Wolbachia effects on all potential pathogens in the study area should be determined. Additionally, several studies have shown that Wolbachia is capable of horizontal transfer to other insect species which could have unforeseen effects on non-target insects [52]–[54]. A lack of understanding of Wolbachia-pathogen-mosquito interactions could impact efficacy of disease control programs. Cx. tarsalis is a competent vector for many human pathogens, and further studies that assess alternative Wolbachia strains and viruses in Cx. tarsalis may elucidate the importance of host background on pathogen interference phenotypes in this medically important mosquito species.