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Background Bat flies belong to the order Diptera and superfamily Hippoboscoidea. They can be divided into two families, Streblidae and Nycteribiidae, which collectively encompass 239 and 280 species worldwide, respectively. In Thailand, 43 species of Nycteribiidae and 16 species of Streblidae have been documented. Despite their diversity, the molecular characteristics and host-parasite interactions of these ectoparasites remain poorly understood. Methods During a bat survey conducted between 2019 and 2022, bat flies were collected across eight sites in three provinces of Thailand. Morphological identification was performed using identification keys and a bat fly checklist endemic to Thailand. DNA barcoding targeted to the mitochondrial Cox1 and nuclear 28S rRNA genes was utilized. Infestation patterns were analyzed in relation to host sex, sampling site, and physiological status. Species identification was confirmed via BLASTN searches, and species delimitation was conducted using the ASAP algorithm under three substitution models. Phylogenetic relationships were inferred using Maximum Likelihood methods, while genetic variation was assessed through TCS haplotype network analysis. Tripartite network analysis was employed to examine site-host-parasite associations. Results A total of 1,042 bats, representing 28 species, were captured during the study, of which 298 individuals (28.59%) were infested with bat flies. In total, 773 bat flies were collected, comprising 737 from the family Streblidae and 36 from Nycteribiidae. Morphological and molecular analyses identified three genera— Raymondia , Brachytarsina , and Nycteribia —along with seven hypothetical species. Phylogenetic reconstruction using mitochondrial ( Cox 1) and nuclear (28S rRNA) gene markers revealed distinct clades within each genus, underscoring substantial genetic diversity. Haplotype analyses identified 18 haplotypes in Raymondia , six in Brachytarsina , and two in Nycteribia , with evidence of site-specific host-parasite associations. Infestation rates varied by host species, sex, and location, with larger bat populations demonstrating higher infestation intensities. Raymondia sp. 1 is the most frequently encountred species an predominantly infested Hipposideros gentilis . Conclusions This study provides the first molecular characterization of bat fly diversity in Thailand, revealing their genetic complexity, taxonomy, host specificity, and ecological interactions. The findings establish a crucial foundation for further research concerning the biodiversity, host-parasite dynamics, and zoonotic risks associated with bat flies. Graphical Abstract

Arthropod vectors play a crucial role in the transmission of hemotropic mycoplasmas, small bacteria that infect red blood cells in a wide range of animals and humans globally, leading to intravascular infections. Traditional Giemsa-stained thin blood smears, used for diagnosing hemotropic mycoplasmas through microscopic examination, have low sensitivity and are effective only when bacteremia levels are high. This study aimed to employ molecular methods to detect and genetically characterize hemotropic mycoplasmas in goats as well as investigate the potential role of fleas as vectors. Blood and flea samples were collected concurrently from goats on 16 farms across seven provinces in Thailand from January 2017 to October 2023. The 16 S rRNA, 23 S rRNA, and rnpB genes of hemoplasmas were amplified and sequenced. All fleas were identified morphologically and molecularly through DNA barcoding of the cytochrome oxidase I gene. A total of 78 out of 500 goats (15.6%), three pooled flea samples (3/6, 50%), and one individual flea (1/49, 2.04%) tested positive for hemoplasmas and all fleas were identified as Ctenocephalides orientis . BLASTN searches utilizing the three genetic markers revealed that the hemoplasmas detected in this study showed 97.81–100% similarity to Mycoplasma ovis and Candidatus Mycoplasma haemovis, which have been previously reported in sheep, goats, and humans, suggesting their zoonotic potential. The sequences were grouped into 28 unique nucleotide sequence types (ntSTs) based on minor variations in the 16 S rRNA gene. Hemotropic mycoplasma infection was significantly associated with farm locations and seasonality of sample collection ( p  < 0.0001), indicating that farm management practices or environmental conditions may play a critical role in the epidemiology of these infections. This study represents the first report of hemotropic mycoplasmas in goats in Thailand, confirms their presence in fleas, and provides valuable insights for farm management, such as guiding the rational use of insecticides and antibiotics.