Molecular identification and characterization of Wolbachia and Cardinium with co-occurrence of Leishmania spp. in Culicoides biting midges (Diptera: Ceratopogonidae) from leishmaniasis-affected areas of Thailand

Biting midges of the genus Culicoides have been identified as potential vectors for the transmission of species of the Leishmania subgenus Mundinia , the causative agents of autochthonous leishmaniasis in Thailand. Vector competence may be potentially affected by bacterial endosymbionts; however, there is no known correlation between these endosymbionts and Leishmania parasites in Culicoides biting midges. In this study, we aimed to explore the prevalence and association of bacterial endosymbionts and the detection of Leishmania DNA in Culicoides spp. Female midges were captured at five sampling sites in areas with autochthonous leishmaniasis in northern and southern Thailand. Culicoides species were identified using both morphological characteristics and cox 1 sequencing. The presence of Wolbachia , Cardinium , and Leishmania DNA in individual midges was molecularly screened targeting the wsp and 16S rRNA genes, and the ITS1 region, respectively. All amplification products were sequenced and subjected to phylogenetic analysis. A total of 593 female midges were collected, comprising 21 species of Culicoides and one species of Culicoides ( Trithecoides ). The Wolbachia isolates from infected Culicoides spp. were phylogenetically classified into supergroups A, B, and F. Six Wolbachia putative strains belonged to clade Wol-b (Wol-b1 to Wol-b6), two to clade Wol-a (Wol-a1 and Wol-a2), and one to clade Wol-f (Wol-f1). Three Wolbachia strains were identified as w Kerlac, w Beva_B, and w CauA. Moreover, both Cardinium groups A and C were identified. It is noteworthy that co-infections between bacterial endosymbionts and Leishmania spp. showed a significant association. To the best of our knowledge, this study provides the first evidence of Wolbachia and Cardinium in Culicoides spp. from leishmaniasis-affected areas in Thailand. Detecting bacterial endosymbionts co-occurring with Leishmania spp. in Culicoides biting midges may suggest a potential, but unconfirmed, antagonistic effect on Leishmania . This provides preliminary data that could inform the development of new vector control strategies for diseases transmitted by Culicoides spp. in Thailand.