Prevalence of Wolbachia in natural sand fly (diptera: psychodidae) populations from Türkiye and its potential role in mitochondrial divergence

Abstract Background Phlebotomine sand flies are vectors of various pathogens, most notably Leishmania spp. Symbiotic bacteria have recently gained considerable attention owing to their effects on hosts and on other organisms co-infecting the same host. In this study, we investigated the natural Wolbachia infection status of sand fly taxa distributed in Türkiye and examined its potential role in driving the deep mitochondrial divergence observed within certain taxa. Methods We analysed 858 sand fly specimens, mostly collected between 2005 and 2016, with additional samples obtained in 2023. Specimens were morphologically identified, and the mitochondrial cox1 gene was sequenced for DNA barcoding. For selected taxa showing marked mitochondrial divergence, species delimitation methods were applied, and genetic diversity indices and neutrality tests were calculated. Wolbachia infection was detected via PCR amplification of the wsp gene, and strain diversity was characterised using multilocus sequence typing (MLST) of five housekeeping genes. Logistic regression was used to evaluate associations between infection status and mitochondrial lineage, sex or collection period. Results Wolbachia infection was detected in 16.67% of specimens, occurring exclusively in Phlebotomus papatasi, Ph. major s.l., Ph. tobbi, Ph. economidesi and Sergentomyia minuta. Analyses of wsp and MLST data identified all sequences as belonging to Supergroup A, with multiple strains present within and across host taxa. Infection among the five Ph. major s.l. lineages delineated by species delimitation was significantly associated with lineage, with lineages 3–5 showing a higher probability of infection. The reduced haplotype and nucleotide diversity, along with a significant negative deviation from neutrality observed in lineage 5, suggest a selective sweep likely driven by Wolbachia infection. Conclusions This study represents the first comprehensive screening of Wolbachia infection in sand fly taxa distributed across Türkiye, during which several novel Wolbachia strains were identified. Our findings suggest a potential role of Wolbachia infection in driving lineage differentiation within certain sand fly taxa. However, further detailed investigations are required to elucidate the mechanisms by which Wolbachia influences sand fly diversification and to assess the broader epidemiological implications related to sand fly-borne diseases (SFBDs). Graphical Abstract