Integrative morphological and DNA barcoding analysis of Forcipomyia (Lasiohelea) midges: cryptic diversity and first molecular detection of multiple Leishmania species in a leishmaniasis-endemic focus in Southern Thailand.

backgroundCeratopogonid midges of the genus Forcipomyia (subgenus Lasiohelea) are small hematophagous insects widely distributed across tropical regions. In Australia, developmental stages of Leishmania (Mundinia) macropodum have been observed in Forcipomyia (Lasiohelea), suggesting that biting midges may play a role in Leishmania transmission beyond traditional sand fly vectors. However, in Southeast Asia-where leishmaniasis caused by Mundinia species is an emerging autochthonous disease in humans-fundamental information on Forcipomyia (Lasiohelea) diversity and its association with Leishmania remains limited.

methodsAn integrated morphological-molecular approach was employed to characterize Forcipomyia (Lasiohelea) midges collected using sweep nets from a leishmaniasis-endemic area in Nakhon Si Thammarat Province, Southern Thailand, near the residence of a patient with locally acquired cutaneous leishmaniasis, during January and June 2025. Morphological examination of mandibular dentition, sensory pits, cibarial armature, and spermathecal structure was combined with mitochondrial cox1 barcoding, Bayesian and maximum likelihood phylogenetic analyses, and species delimitation methods (ASAP and mPTP). Because females of the subgenus Lasiohelea possess a single spermatheca, only single-spermatheca females were selected for species identification. Specimens identified as Lasiohelea were subsequently screened for Leishmania using 18S rRNA-qPCR and ITS1-PCR, followed by nanopore-based ITS1 metabarcoding for species-level identification. Vertebrate blood meal sources were also characterized using vertebrate cox1 metabarcoding.

resultsFrom 264 collected midges, 72 female specimens with a single spermatheca were selected for analysis. Integrated morphological and molecular data identified seven Lasiohelea specimens forming four genetic clusters, comprising F. (L.) parvitas (n = 2) and three lineages closely related to F. (L.) peditata (n = 2), F. (L.) humilavolita (n = 2), and F. (L.) taiwana (n = 1). These were clearly separated from non-Lasiohelea taxa, including F. (Euprojoannisia) fuscimana and two unclassified Ceratopogonidae lineages. Phylogenetic and species delimitation analyses revealed cryptic genetic diversity despite morphological similarity. Leishmania DNA was detected in six of seven Lasiohelea specimens. Nanopore ITS1 metabarcoding identified autochthonous species (L. (Mundinia) martiniquensis and L. (M.) orientalis) and additional Leishmania species (L. (Leishmania) amazonensis and L. (L.) major), including mixed-species detections in four specimens. A single engorged specimen contained DNA from red junglefowl (Gallus gallus spadiceus).

conclusionsThis study provides the first integrated characterization of Forcipomyia (Lasiohelea) diversity and associated Leishmania detection in Southeast Asia. The results identified several putatively distinct species within Lasiohelea and provide evidence of natural exposure of these midges to multiple Leishmania species, suggesting a complex parasite-midge association and parasite co-circulation in the local environment. Although vector competence was not assessed, these findings suggest that Forcipomyia (Lasiohelea) may be involved in the circulation of Leishmania parasites and may represent promising candidates for further investigation of vector competence. Future studies focusing on host associations, parasite development, and experimental transmission are needed to clarify their epidemiological significance.