Molecular screening for Wolbachia endosymbionts in phlebotomine sand flies within the North Central Province, Sri Lanka.

Cutaneous leishmaniasis is a growing public health threat in Sri Lanka, transmitted primarily by phlebotomine sand flies. While biological control using Wolbachia has proven effective against other vector-borne diseases, its potential application in phlebotomine sand flies remains largely unexplored. Therefore, investigating the natural prevalence of these endosymbionts in Sri Lankan sand fly populations is well justified. To address this knowledge gap and evaluate the feasibility of symbiont-mediated control, we conducted a six-month entomological survey across four endemic sampling areas in the North Central Province. Captured sand flies were morphologically identified at genus level, and 90 females belonging to the genera Phlebotomus and Sergentomyia were screened for Wolbachia using wsp-targeted polymerase chain reaction (PCR). To provide complementary evidence and characterize the broader sand fly-associated bacterial community, high-throughput 16S rRNA gene amplicon sequencing was performed on a pooled sample of 25 females. Wolbachia was not detected in any of the individually screened specimens (0/90; observed prevalence, 0.0%; approximate rule-of-three upper 95% prevalence limit, 3.3%) or in the pooled 16S dataset. Sequencing generated 220,698 non-chimeric reads, and Serratia was the most abundant identifiable genus, accounting for 41.71% of bacterial reads. These findings indicate an absence of detectable Wolbachia under the methods applied, rather than its definitive absence from the wider regional sand fly population. The study provides baseline evidence relevant to evaluating the feasibility and limitations of symbiont-mediated strategies for leishmaniasis vector control in Sri Lanka.