Sand fly endosymbionts in Kenya: Rickettsia and Wolbachia associations with Leishmania and detection of Rickettsia africae

BACKGROUND: Sand flies are small hematophagous insects known as leishmaniasis vectors. Similar to most arthropods, they harbor nonobligate endosymbionts that may influence host adaptation and pathogen transmission, but these symbiont communities remain poorly characterized in Leishmania-endemic African sand flies. METHODS: We screened 1700 wild-caught phlebotomine sand flies (1266 females, 434 males) from Kenya's Baringo, Nakuru, and Kajiado counties, and 253 colony Phlebotomus duboscqi, for Rickettsia, Wolbachia, Spiroplasma, Cardinium, Arsenophonus, Microsporidia, and Leishmania by high-resolution melting analysis and sequencing of PCR products. RESULTS: In wild sand flies (Phlebotomus and Sergentomyia spp.), Wolbachia spp. were most common (8.5%, 145/1700), with particularly high prevalences in Ph. mireillae (92.3%, 12/13), Ph. guggisbergi (73.2%, 82/112), and Ph. saevus (48.6%, 18/37), followed by Spiroplasma (1.4%, 23/1700), Rickettsia (0.7%, 12/1700), Cardinium (0.4%, 6/1700), Tubulinosema sp. (0.1%, 1/1700), and various gut bacteria (1.8%, 30/1700). In addition, we detected Rickettsia africae, a tick-borne pathogen causing African tick-bite fever (ATBF), in Ph. martini (4.7%, 5/106), Ph. guggisbergi (1.8%, 2/112), S. schwetzi (0.4%, 1/263), S. clydei (0.5%, 2/440), and Sergentomyia sp. (0.3%, 1/371). Notably, R. africae DNA was found in one male Ph. martini and Rickettsia sp. DNA in one male S. clydei and one male S. schwetzi, consistent with infection rather than blood-meal contamination. Furthermore, Rickettsia endosymbionts were positively associated with Leishmania DNA (OR = 20.31; 95% CI [4.93, 77.03]; P < 0.0001), including within Phlebotomus (OR = 13.54; 95% CI [2.33, 78.88]; P = 0.0017). Wolbachia also correlated with Leishmania overall (OR = 2.46; 95% CI [1.17, 4.79]; P = 0.011), though not within individual fly genera. Colony Ph. duboscqi harbored only Serratia and other gut bacteria. CONCLUSIONS: Sand flies in Kenya harbored six endosymbionts, including the first detection of pathogenic R. africae in sand flies, and gut bacteria that may influence vector competence. The frequent co-occurrence of Rickettsia and Wolbachia endosymbionts with Leishmania indicates nonrandom associations between symbionts and parasite infection, without implying causality. These findings reveal previously undescribed sand-fly-microbe interactions, and highlight the need for experimental studies to test whether sand flies contribute to the ecology and potential transmission of ATBF.