Widespread and diverse Wolbachia infections in sand flies from visceral leishmaniasis resurgence areas in China: potential candidates for sand fly control

Sand fly-borne visceral leishmaniasis (VL) has re-emerged in parts of North-central China, while conventional insecticidal control is constrained by the predominantly exophilic ecology of sand flies. Wolbachia -based approaches have shown field utility in medical vector control, but baseline data on Wolbachia infection and strain diversity in sand flies from VL resurgence settings in China remain limited. Sand flies were collected during 2023–2024 at sentinel sites located in VL resurgence areas across four provincial-level administrative regions in China (Beijing Municipality, Shanxi Province, Henan Province, and Inner Mongolia Autonomous Region). Wolbachia was detected and typed by PCR and sequencing of the Wolbachia surface protein ( wsp ) gene, followed by estimation of pool positivity and bias-corrected maximum likelihood estimation (MLE)-based individual infection prevalence, haplotype inference, and phylogenetic and haplotype-network analyses. Three sand fly species— Phlebotomus chinensis , Ph. smirnovi , and Sergentomyia squamirostris —were identified across the surveyed sites. Overall, 268 pools (10 individuals per pool; 2680 specimens) were screened for Wolbachia , and 194 pools were PCR-positive (72.4%). Bias-corrected MLE estimates indicated variable but generally high individual-level Wolbachia prevalence across Phlebotomus sampling sites, ranging from 8.6% to 100.0% in Ph. chinensis and reaching 10.4% in Ph. smirnovi , whereas Se. squamirostris was negative. WSP -based typing resolved eight haplotypes in Wolbachia from the two Phlebotomus species examined, spanning supergroups A and B; notably, Hap4 clustered with cytoplasmic incompatibility-associated Wolbachia lineages (e.g., wPip) reported in mosquitoes. Wolbachia is highly prevalent in Phlebotomus sand flies from VL resurgence areas in North-central China and exhibits wsp -defined diversity across supergroups A and B. The detection of a wPip-related haplotype (Hap4), together with a locally prevalent Phlebotomus -associated clade, provides a baseline for prioritizing strain-level characterization and functional evaluation of Wolbachia for potential sand fly management in re-emergent VL foci.