Wolbachia, a maternally inherited intracellular bacterial species, can manipulate host insect reproduction by cytoplasmic incompatibility (CI), which results in embryo lethality in crosses between infected males and uninfected females. CI is encoded by two prophage genes, cifA and cifB. Wolbachia, coupled with the sterile insect technique, has been used in field trials to control populations of the dengue vector Aedes albopictus, but CI-inducing strains are not known to infect the malaria vector Anopheles gambiae. Here we show that cifA and cifB can induce conditional sterility in the malaria vector An. gambiae. We used transgenic expression of these Wolbachia-derived genes in the An. gambiae germline to show that cifB is sufficient to cause embryonic lethality and that cifB-induced sterility is rescued by cifA expression in females. When we co-expressed cifA and cifB in male mosquitoes, the CI phenotype was attenuated. In female mosquitoes, cifB impaired fertility, which was overcome by co-expression of cifA. Our findings pave the way towards using CI to control malaria mosquito vectors.

Wolbachia infections are a fascinating example of reproductive parasitism with strong potential to combat vector-borne diseases, due to their combined ability to spread in insect populations and block pathogen replication. Though the Wolbachia factors mediating the notable reproductive manipulation cytoplasmic incompatibility (CI) have now been identified as prophage WO genes cifA and cifB, the relative role of these genes is still intensely debated, with different models claiming that CI requires either both factors or cifB alone. Here we investigated whether cifA and cifB are sufficient to induce conditional sterility in the major malaria vector Anopheles gambiae, a species that appears to have limited susceptibility to invasion by Wolbachia. We report that CI can be fully recapitulated in these mosquitoes, and that cifB is sufficient to cause this reproductive manipulation. cifB-induced sterility is fully rescued by high levels of cifA expression in females. Surprisingly, however, when cifA is highly expressed in males alongside cifB, the CI phenotype is attenuated. cifB strongly impairs fertility also when expressed in the female germline, again mitigated by cifA. These data support a system whereby cifB and cifA must be fine-tuned to exercise CI and rescue, respectively, possibly explaining the limited success of Wolbachia at invading Anopheles. Our findings pave the way towards facilitating Wolbachia infections in anopheline vectors, for use in malaria control strategies.

Insecticide resistance is under strong selective pressure in Anopheles mosquitoes due to widespread usage of insecticides in vector control strategies. Resistance mechanisms likely cause changes that profoundly affect mosquito physiology, yet it remains poorly understood how selective pressures imposed by insecticides may alter the ability of the mosquito to host and transmit a Plasmodium infection. From pyrethroid-resistant field-derived Anopheles gambiae s.l. mosquitoes, we established resistant (RES) and susceptible (SUS) colonies by either selection for, or loss of insecticide resistance. We show increased oocyst intensity and growth rate as well as increased sporozoite prevalence and intensity in RES compared to SUS females infected with Plasmodium falciparum. The increase in infection intensity in RES females was not associated with the presence of the kdrL1014F mutation and was not impacted by inhibition of Cytochrome P450s. The lipid transporter lipophorin (Lp), which was upregulated in RES compared to SUS, was at least partly implicated in the increased intensity of P. falciparum but not directly involved in the insecticide resistance phenotype. Interestingly, we observed that although P. falciparum infections were not affected when RES females were exposed to permethrin, these females had decreased lipid abundance in the fat body following exposure, pointing to a possible role for lipid mobilization in response to damage caused by insecticide challenge. The finding that selection for insecticide resistance can increase P. falciparum infection intensities and growth rate reinforces the need to assess the overall impact on malaria transmission dynamics caused by selective pressures mosquitoes experience during repeated insecticide challenge.

No abstract