Biting midges of the genus Culicoides Latreille are significant pests and vectors that transmit pathogens to humans and other animals. Cattle are among the important livestock that can potentially be severely affected by Culicoides. In this study, we examined the species diversity, abundance, and host blood meal identification of biting midges in cattle pens located in three different land use types: villages, agricultural areas, and the forest edge. A total of 12,916 biting midges were collected, and most of these were from cattle pens located in villages (34%) and agricultural land (52%). Morphological identification revealed 29 Culicoides species. The most common species were C. oxystoma, C. mahasarakhamense, C. peregrinus, and C. shortti; taken together, these species represented >80% of all specimens collected. Despite midges being less numerous (14% of the total collection), cattle pens located near the forest showed greater diversity (23) than those from villages and agricultural areas. More diverse immature habitats and host blood sources from wildlife in nearby forests possibly explain the greater diversity in the cattle pens near the forest edge. Host blood meal analysis revealed that most (65%) biting midges had fed on buffalo despite the fact that this animal was much less numerous than cows or chickens. Relatively larger size and black-colored skin could be factors that make buffalo more attractive to biting midges than other host species. In this study, we also provided 67 DNA barcoding sequences of 13 species, three of which (C. flaviscutatus, C. geminus, and C. suzukii) were first reported from Thai specimens. DNA barcode analysis indicated cryptic diversity within C. hegneri and C. flavescens in Thailand, and thus, further investigation is required to resolve their species status.

Adult black flies were collected from diverse habitats in five provinces of Laos. In total, 4659 adults (4570 females and 89 males) were collected. Morphological identification, supplemented with the mitochondrial cytochrome c oxidase I gene, revealed 12 species. The most abundant taxa were Simulium khelangense/S. chumpornense and S. asakoae/S. myanmarense with 58% (2628 of 4476) and 38% (1720 of 4476) of the total specimens, respectively. Simulium asakoae/S. myanmarense was the most geographically widespread taxon (65% of sites) from 174 to 1219 m above sea level. The remaining species were far less abundant (<2%) and found in only one or a few locations. Molecular identifications of vertebrate blood meals indicated that most hosts (57 of 66) were chickens. Five taxa, S. asakoae, S. khelangense, S. myanmarense, S. aureohirtum, and S. striatum group, fed on chickens, representing the first hosts records for the latter three taxa. Simulium daoense fed on water buffalo, marking the first record of this species in Laos and the first report of its blood hosts. The abundance and frequent use of domestic chickens as hosts suggests that S. khelangense and S. asakoae could be significant pests and might serve as vectors of avian blood parasites in Laos.

We conducted a comparative chromosomal analysis of 10 nominal species and 3 unidentified species in the Simulium striatum group from six countries. A total of 66 chromosomal rearrangements were found, of which 78.8% were inversions. The group is defined by 11 fixed inversions, of which 6 are unique, supporting the monophyletic status previously indicated by morphological and molecular characters. Only 1 of the 13 taxa had a unique fixed chromosomal rearrangement. Although the group demonstrates significant macrogenomic reorganization, subsequent speciation occurred largely without chromosomal rearrangement. The results conflict with the pattern seen in all other species groups of Simuliidae, in which one or more diagnostic rearrangements are typically expressed among species in the same group. The chromosomes provide limited evidence that four entities are valid species but no evidence for the nine others. The weight of evidence from combined chromosomal, molecular, and morphological data, in addition to the practical considerations made for insular species, supports the species status of seven of these nominal species; the remaining taxa require further study. The S. striatum group, accordingly, is either over-divided into nominal species or deficient in chromosomal discriminators. If most or all nominal species and unnamed species are valid, all but one are homosequential, an unprecedented condition in Simuliidae. This group illustrates the need for the integration of multiple character sets for discovering and delimiting species.

Many biting midge species of the genus Culicoides Latreille are significant pests and vectors that transmit diverse parasites to humans and other animals. However, knowledge of these hematophagous insects in Laos remains limited, with the most recent information reported more than four decades ago. In this study, we investigated Culicoides species diversity, DNA barcoding and host blood sources using specimens collected across seven provinces in northern, central, and southern Laos. A total of 4592 specimens were collected, comprising 3095 females and 1497 males. Morphological identification, complemented by DNA barcode analysis, revealed 26 species (24 named and 2 unnamed), including five (three named and two unnamed) new country records. Culicoides peregrinus was the most abundant species, representing 25.7% (1179 individuals), followed by C. oxystoma at 23.8% (1093 individuals), and C. arakawae/C. mahasarakhamense, which together comprised 18.5% (849 individuals) of the total specimens. DNA barcode analysis demonstrated that this genetic marker is effective for species identification of Culicoides in Laos. Of the 115 COI sequences, 103 (89.6%) were successfully matched with conspecifics in the BOLD database. Cryptic genetic diversity was detected in three species, C. clavipalpis, C. palpifer, and C. huffi, with two, two, and three divergent lineages, respectively. Host blood meal analysis revealed that chickens and domestic water buffalo were the most common blood sources for the investigated Culicoides species. These findings provide important baseline information for future studies on the pest and vectorial roles of Culicoides in Laos.

Species of Culicoides Latreille (Diptera: Ceratopogonidae) are important biting pests and vectors of pathogens affecting humans and animals. However, information on Culicoides–parasite associations in Laos is lacking. We used molecular methods to screen 237 specimens representing 27 Culicoides species from Laos for filarial nematodes, avian haemosporidians, Trypanosoma spp., and Leishmania spp. Overall, 59 specimens representing 19 Culicoides species were positive for at least one parasite. Only filarial nematodes and avian haemosporidians were detected. Filarial parasites were found in four Culicoides species (C. actoni, C. guttifer, C. huffi, and C. mahasarakhamense). Analysis of 12S rRNA sequences revealed at least two filarial taxa, one closely related to Onchocerca lienalis and the other to an unidentified onchocercid previously detected in Culicoides. Avian haemosporidians were more prevalent. Leucocytozoon spp. were detected in 40 specimens from 18 Culicoides species, with cyt b sequences identifying Leucocytozoon caulleryi and an unidentified Leucocytozoon sp., the latter accounting for 39 of the 40 positive specimens. Plasmodium juxtanucleare was detected in 27 specimens (11.4%), whereas Haemoproteus spp. were found in only two specimens (0.8%), which represented two distinct lineages previously reported from wild birds. No Trypanosoma or Leishmania DNA was detected. Culicoides mahasarakhamense and C. huffi had the highest infection frequencies. Their high abundance and documented feeding on livestock, particularly chickens and cattle, suggest that they may play important roles in pathogen transmission and should be prioritized for future vector surveillance in Laos.