Understanding the factors associated with the species diversity and distribution of insect vectors is critically important for disease epidemiology. Black flies (Diptera: Simuliidae) are significant hematophagous insects, as many species are pests and vectors that transmit pathogens to humans and other animals. Ecological factors associated with black fly species distribution have been extensively examined for the immature stages but are far less well explored for the adult stage. In this study, we collected a total of 7706 adult black fly specimens from various locations in forests, villages and animal shelters in Thailand. The integration of morphology and DNA barcoding revealed 16 black fly taxa, including Simulium yvonneae, a species first found in Vietnam, which is a new record for Thailand. The most abundant species was the Simulium asakoae complex (n = 5739, 74%), followed by S. chumpornense Takaoka and Kuvangkadilok (n = 1232, 16%). The Simulium asakoae complex was dominant in forest (3786 of 4456; 85%) and village (1774 of 2077; 85%) habitats, while S. chumpornense predominated (857 of 1175; 73%) in animal shelter areas. The Simulium asakoae complex and S. nigrogilvum Summers, which are significant pests and vectors in Thailand, occurred at a wide range of elevations, although the latter species was found mainly in high (>1000 m) mountain areas. Simulium chumpornense, S. nodosum Puri and the S. siamense Takaoka and Suzuki complex occurred predominately in low (1000 m) mountain areas. A host blood meal analysis revealed that the S. asakoae; S. chamlongi Takaoka and Suzuki; S. nigrogilvum; S. chumpornense; and the S. striatum species group were biting humans. This is the first report of the latter two species biting humans. We also found that S. chumpornense was biting turkeys, and S. chamlongi was biting chickens, which are new host blood sources recorded for these species. In addition, we found that the S. feuerborni Edwards complex was biting water buffalo, which is the first report on the biting habits of this species.

Genetic diversity and ecology are two important aspects of vector species crucial to a full understanding of disease epidemiology. In this study, we examined genetic diversity, genetic structure and the physiochemical parameters of the breeding habitats of the two significant black fly vector species, Simulium chumpornense Takaoka and Kuvangkadilok and S. nodosum Puri, from Laos. Genetic diversity of S. chumpornense in Laos was relatively high with maximum genetic divergence of 3.05% but no significant genetic differentiation between populations. Comparisons with conspecific populations from Thailand also found no genetic differentiation between the two countries. This possibly related to the recent history of this species, as a very recent (31,000 years ago) historical population expansion was detected. Physicochemical parameters of the breeding habitats suggest the ability to utilize diverse stream sizes from small flows (1 m wide) to huge rivers (290 m or more wide). Populations of S. nodosum from Laos had low genetic diversity with maximum genetic divergence of 2.56% and no genetic structuring among populations. Comparisons with those reported from other countries revealed five genetically divergent lineages (I–V) with minimum genetic divergence of 1.36%. The majority (42 of 52) of specimens from Laos belonged to lineage I and the remaining (10) comprised lineage II. Lineage I is the largest and representative of specimens from Thailand, Myanmar, Vietnam, Taiwan and Laos. Population history analysis revealed that lineage I had undergone recent demographic expansion dating back to 7000 years ago. This very recent population expansion resembles others reported from Thailand and possibly indicates a response to increasing human and domestic animals following the agricultural revolution. Breeding sites of S. nodosum are diverse in respect to elevation, velocity, water conductivity and streambed particle size. The ability to utilize a wide variety of breeding sites could promote the production of a large adult population, which can be a pest for humans and other animals.

Black flies (Diptera: Simuliidae) are significant pests and vectors transmitting pathogens to humans and other animals. However, knowledge of species diversity as well as their role as pests and vectors in Laos is limited, despite the country having abundant suitable lotic habitats, that is, running water. In this study, we collected wild adult black fly specimens from Laos. Both morphology and genetic data supported that they were Simulium khelangense Takaoka, Srisuka and Saeung, and therefore, a new distribution record for this species. Screening of the blood protozoa from 106 females detected Leucocytozoon (n = 7) and Trypanosoma (n = 9). Identifications of Leucocytozoon based on cytochrome b sequences in MalAvi database indicated that 5 specimens had identical sequences to those of 4 lineages (GALLUS17 [n = 2], GALLUS35 [n = 1], GALLUS37 [n = 1], and GALLUS41 [n = 1]) of unidentified species (i.e., Leucocytozoon sp.). The remaining 2 had no identical sequences but were very similar (99%) to GALLUS34 and GALLUS41 lineages. Identifications of Trypanosoma based on the small subunit rRNA indicated that all were T. avium. The results presented here further support the role of S. khelangense as a potential vector of Leucocytozoon and Trypanosoma as well as being a possible pest of domestic avian species.

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.

Two species of black flies (Simuliidae) in Thailand, Simulium chumpornense Takaoka and Kuvangkadilok, 2000, and S. khelangense Takaoka, Srisuka & Saeung, 2022, are potent vectors of avian blood protozoa of the genera Leucocytozoon and Trypanosoma and are pests of domestic avian species. Although the adults are abundant throughout Thailand, information on their breeding habitats is limited, and the immature stages of S. khelangense are unknown. We collected the larvae and pupae of S. khelangense from the Mekong River, the first-ever record of Simuliidae from this large continental river. Mitochondrial cytochrome c oxidase I and internal transcribed spacer 2 were used to associate the larvae and pupae with known adults. Both genetic markers strongly supported their identity as S. khelangense. The larvae and pupa of S. khelangense are described. The pupal gill filaments, larval abdominal protuberances, and setae distinguish this species from other members of the S. varicorne species group. The immature stages of S. chumpornense inhabit a wide variety of flowing waters, from small streams (3 m wide) to enormous continental rivers (400 m wide); thus, S. chumpornense is a habitat generalist. In contrast, S. khelangense was found only in the large Mekong River and is, therefore, a habitat specialist. Both species can exploit their principal habitats and produce abundant adult populations.

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.