Molecular co-occurrence of Leishmania (Mundinia) spp., Wolbachia, and Cardinium in cave-associated phlebotomine sand flies from Northern Thailand: genetic diversity and endosymbiont–parasite associations

Abstract Background Leishmaniasis caused by Leishmania ( Mundinia ) species is an emerging public health concern in Thailand, yet information on insect vectors and their associated bacterial endosymbionts remains limited in endemic areas of the country. This study investigated the occurrence of Leishmania , Wolbachia , and Cardinium in phlebotomine sand flies collected from cave- and livestock-associated environments in Northern Thailand and characterized the genetic diversity of detected endosymbionts. Methods Female sand flies were collected from two tourist caves and a livestock-associated site in Chiang Rai Province, Northern Thailand. Specimens were identified morphologically and screened for Leishmania DNA using qPCR assays targeting genus-level 18S rRNA and species-specific markers, together with ITS1-PCR coupled with nanopore sequencing. Endosymbionts were detected using PCR targeting the Wolbachia surface protein ( wsp ) gene and the Cardinium 16S rRNA gene. Associations between endosymbiont status and Leishmania DNA detection were evaluated using Firth’s penalized logistic regression. Genetic diversity was assessed using haplotype-based analyses and phylogenetic reconstruction, and a haplotype network was generated for Wolbachia . Representative Wolbachia WSP variants were further analyzed using amino acid sequence comparison and three-dimensional structural modeling. Results A total of 298 sand flies representing eight taxa across the genera Phlebotomus , Sergentomyia , and Idiophlebotomus were collected from three sites in Chiang Rai Province, Northern Thailand. Sand fly species composition differed among collection sites, suggesting habitat-associated structuring of sand fly communities. Leishmania DNA was detected in 78 specimens (26.2%), with both L. martiniquensis and L. orientalis identified, including four co-detections. Wolbachia was detected in 228 specimens (76.5%), comprising 124 single infections and 104 co-infections with Cardinium , while Cardinium -only infections were rare (1.3%). The overall association between endosymbiont status and Leishmania DNA detection was not statistically significant (penalized likelihood ratio test, P = 0.083). However, in category-specific comparison, the Wolbachia -only group showed lower odds of Leishmania DNA detection than endosymbiont-negative sand flies (OR = 0.49, 95% CI: 0.25–0.97; P = 0.041). Genetic analyses revealed 16 Wolbachia wsp haplotypes and 14 Cardinium 16S rRNA haplotypes, with Wolbachia clustering within Supergroups A and B and Cardinium grouping into Groups A and C, representing the first report of their genetic diversity in sand flies from Thailand. Structural analysis of WSP identified a 9-amino-acid insertion (KDSTKQVTD) within the HVR4 region of a Sergentomyia barraudi variant, corresponding to predicted localized conformational variation while preserving the conserved β-barrel core structure. This insertion occurred within a surface-exposed loop region potentially involved in host–symbiont interactions. Conclusions Sand fly populations in Northern Thailand harbor genetically diverse Wolbachia and Cardinium haplotypes spanning multiple phylogenetic groups alongside detection of multiple Leishmania spp. Despite the non-significant overall association across endosymbiont categories, the lower odds of Leishmania DNA detection observed in the Wolbachia -only group suggest a potential biological interaction influencing parasite occurrence. Structural variability in Wolbachia surface proteins further demonstrates molecular diversity within natural sand fly-associated Wolbachia populations and warrants further investigation of endosymbiont–parasite interactions in phlebotomine sand flies.