Impact of Asaia bacteria on Leishmania major development in sand flies: implications for vector control strategies

Background: Asaia spp., bacteria originally isolated from tropical plants, have also been identified in various insect species, including blood-feeding ones. Their ability to colonize different host tissues and transmit vertically between generations makes these bacteria good candidates for paratransgenesis. However, most existing data derived from studies on mosquitoes and other important vectors, such as phlebotomine sand flies (Diptera: Psychodidae), remain understudied. In this study, we investigated the ability of wild-type Asaia siamensis, Asaia krungthepensis, and a genetically modified strain of Asaia expressing the Wolbachia surface protein (AsaiaWSP) to colonize Phlebotomus duboscqi. In addition, we studied their vertical transmission and their interactions with Leishmania major during superinfection. Methods: Phlebotomus duboscqi females were provided with Asaia via a sugar meal. Bacterial presence and vertical transmission were assessed using both cultivation and polymerase chain reaction (PCR). In superinfection experiments, females were first offered sugar containing Asaia, followed by a blood meal infected with Le. major. The outcomes of superinfection were assessed by cultivation, PCR, and microscopically. Statistical analyses were performed using Fisher’s exact or Chi-squared tests. Results: All tested Asaia strains colonized the gut of Ph. duboscqi. Both A. siamensis and A. krungthepensis were vertically transmitted to the progeny via egg smearing. These bacteria did not affect the infection rate and intensity of Le. major infection on days 2 and 5 post blood meal (pbm). However, by day 8 pbm, both species significantly reduced Le. major infection intensity. Moreover, A. krungthepensis significantly increased the proportion of metacyclic forms. Interestingly AsaiaWSP did not have a significant effect on Le. major development in Ph. duboscqi. Conclusions: We demonstrated for the first time that A. siamensis and A. krungthepensis can infect Ph. duboscqi and be vertically transmitted to the next generation via egg smearing. These bacteria affect the late phase of Le. major infection, which could have important epidemiological consequences.