Diversity, Distribution and Host Blood Meal Analysis of Adult Black Flies (Diptera: Simuliidae) from Thailand.

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 (<800 m)-elevation areas. Simulium furvum Takaoka and Srisuka; S. phurueaense Tangkawanit, Wongpakam and Pramual; and S. nr. phurueaense were only found in high (>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.

Characterization of the bacterial microbiota of cattle ticks in northeastern Thailand through 16S rRNA amplicon sequencing.

Ticks are vectors of a variety of pathogens that can infect humans and animals. Ticks also harbor non-pathogenic microbiota. This study characterized the microbiota of the ticks infesting beef cattle in Thailand. Two species of ticks; Rhipicephalus microplus (n = 15) and Haemaphysalis bispinosa (n = 5), were collected in seven provinces in northeastern Thailand. Microbial community profile of ticks was examined based on sequences of the V3-V4 region of 16S rRNA gene. Proteobacteria (Pseudomonadota) was the most abundant phylum, followed by Firmicutes (Bacillota), and Actinobacteriota. Coxiella-like endosymbiont was the most abundant bacterial taxon overall (49% of sequence reads), followed by Anaplasma (8.5%), Corynebacterium (5.5%), Ehrlichia (3.9%), and Castellaniella (3.4%). Co-infections of the pathogenic bacteria Ehrlichia and Anaplasma were detected in 19/20 (95%) female ticks. The tick with the lowest number of bacteria had the lowest abundance of the Coxiella-like endosymbiont, and the pathogenic bacteria Anaplasma and Ehrlichia were absent. This study provides baseline information of the microbiota of cattle ticks in northeastern Thailand, suggesting that ticks carry a few dominant bacterial taxa that are primarily non-pathogenic but can co-occur with pathogenic microorganisms. The information obtained is useful for monitoring disease outbreaks in the future and informing prevention and control strategies against cattle tick-borne diseases.

Genetic diversity of tick (Acari: Ixodidae) populations and molecular detection of Anaplasma and Ehrlichia infesting beef cattle from upper-northeastern Thailand.

Genetic diversity, genetic structure and demographic history of the ticks infesting beef cattle in Thailand were examined based on mitochondrial cytochrome c oxidase I (cox1) sequences. Tick samples were collected in 12 provinces in upper-northeastern Thailand. Three species were found; Rhipicephalus microplus, R. sanguineus, and Haemaphysalis bispinosa. Of these, R. microplus was by far the most abundant species in beef cattle and was widely distributed throughout the area. No cox1 sequence variation was found in the R. sanguineus or H. bispinosa specimens collected. Low nucleotide diversity but high haplotype diversity was observed in R. microplus. All collected R. microplus specimens belonged to lineage A. Mismatch-distribution analysis, as well as Tajima's D and Fu's Fs tests, provided evidence of recent demographic expansion. A subsample of tick specimens was investigated for presence of Anaplasma and Ehrlichia using a fragment of the 16S rRNA gene. Three species of Anaplasma were detected from R. microplus; Anaplasma marginale (19.08%), Anaplasma platys (1.97%) and unidentified Anaplasma strain (0.66%). The infection rate of Ehrlichia was 7.24% (two ticks were infected with E. minasensis (1.97%) and eight with an unidentified Ehrlichia strain (5.26%). No infections were found in R. sanguineus or H. bispinosa. This is the first report of A. platys and E. minasensis in cattle ticks in Thailand, providing information for future epidemiological surveys and control strategies in this region.

Effect of Larval Diets on House Fly (Diptera: Muscidae) Production and Host Suitability for a Pupal House Fly Parasitoid (Spalangia gemina) (Hymenoptera: Pteromalidae).

A larval medium for house fly (Musca domestica L.) suitable for house fly parasitoid production was developed from locally available and inexpensive food ingredients. Biological parameters and life table parameters were estimated for house flies treated with five different diets. It was found that percentage survival of 1st-3rd instar larvae of house fly, percentage of pupation, percentage of hatching adults, growth index, and life table parameters (net reproductive rate [R0] the cohort generation time [T], intrinsic rate of increase [r], and finite rate of increase [λ]) were significantly highest on larvae produced on diet 5 (composed of rice barn and chicken feed). Diet composition and performance of house fly larvae in larval medium are discussed, based on the results. The results of Spalangia gemina Boucek (Hymenoptera, Pteromalidae) parasitization on house fly pupae revealed that the number of total parasitized pupa and number of parasitoids hatched from pupa reared with diet 5 (rice bran and chicken feed) were greater than with diet 1 (rice bran, powdered milk, dry yeast, fish meal, soybean meal, and chicken feed). Additionally, S. gemina offspring from diet 5 treatment had a higher proportion of females. Our results indicated that pupal size resulting from larval diet was an important factor for parasitization.

Seasonal population dynamics and a role as natural vector of Leucocytozoon of black fly, Simulium chumpornense Takaoka & Kuvangkadilok.

The black fly, Simulium chumpornense Takaoka & Kuvangkadilok, is suspected to be a natural vector of Leucocytozoon. In this study, seasonal variation in abundance of the adult fly and molecular detection of Leucocytozoon in S. chumpornense and in domestic chicken was investigated in northeastern Thailand. A total of 10,416 female adults were obtained by monthly collection for one year at six sampling sites. There was a peak of abundance in the dry season (March - May) when more than 74% of the specimens were collected. An increased number of suitable habitats for immature stages during the dry season is potentially a factor driving dry season abundance. Molecular genetic investigations revealed that S. chumpornense harbored the infectious stage of Leucocytozoon sp. indicating that it is a natural vector of this parasite. Blood smear screening for the parasite in domestic chickens found a considerable prevalence (52%) of Leucocytozoon spp. infections. The cytochrome b sequences of this parasite in domestic chickens comprised two lineages. One lineage was closely related to Leucocytozoon sp. found in S. chumpornense and another matched with L. schoutedeni. Therefore, S. chumpornense is a vector of Leucocytozoon sp. while L. schoutedeni is possibly transmitted by other ornithophilic black fly species.

Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as leucocytozoon vectors.

Identification of the host blood meal of hematophagous insects can provide significant information regarding host preferences and the possibility of the transmission of disease agents. Currently, this knowledge is limited for black flies in the Oriental region. In this study, we used cytochrome b gene sequences for identification of blood meal sources of two black fly taxa, the Simulium asakoae complex and S. chumpornense Takaoka & Kuvangkadilok in Thailand. A total of 4,260 wild adult females were visually screened revealing 24 blood-engorged females from which cytochrome b was successfully amplified in 19 individuals. Comparisons in GenBank database revealed that all are closest to chicken (Gallus gallus) with sequence similarity of >98%. Therefore, these black fly species are feeding on chickens. We also molecularly investigated the hemosporidian blood protozoa genus Leucocytozoon in black flies and found 13 of 19 blood-engorged females positive for this protozoon. Sequence analysis revealed that this Leucocytozoon DNA could be assigned into two previously recognized groups, one with a Leucocytozoon reported from domestic chickens and black flies in Thailand, and another close to the L. schoutedeni. Our results indicate a high possibility that the S. asakoae complex and S. chumpornense are natural vectors of Leucocytozoon.

Molecular detection of Trypanosoma (Kinetoplastida: Trypanosomatidae) in black flies (Diptera: Simuliidae) from Thailand.

Trypanosomes are parasitic flagellate protozoans that are important disease causing agents in humans and animals including economically significant livestock. Many blood-sucking insects are known to be vectors of trypanosomes but there is no data for the haematophagous black fly species in Asia. In this study, a molecular approach based on the small subunit ribosomal RNA (SSU rRNA) gene was used to detect trypanosomes in black flies from Thailand. A total of 470 wild-caught adult black flies representing nine morphological species were examined. Ten (2%) specimens of two ornithophilic black fly species, Simulium asakoae complex (n = 4) and S. chumpornense (n = 6), were positive for trypanosomes. The SSU rRNA sequences revealed that all trypanosome DNA found in black flies from Thailand is closely related to Trypanosoma avium with >99% sequence similarity. This is also supported by a phylogenetic analysis in which all trypanosomes from Thai black flies were resolved in the clade of T. avium. This is the first report for trypanosomes in Asian black flies and it is suspected that these insects are potential vectors of avian Trypanosoma.

Who is biting you? DNA barcodes reveal cryptic diversity in human-biting black flies (Diptera: Simuliidae).

Black flies (Simuliidae) are important biting insects and vectors of diseases agents of humans and livestock. Thus, understanding the taxonomy and biodiversity of these insects is crucial for control and management of these diseases. In this study, we used mitochondrial cytochrome c oxidase I sequences to examine genetic diversity of three human-biting and possible vector black fly taxa; the Simulium asakoae species-complex, S. chamlongi and S. nigrogilvum. High levels of genetic diversity (>3.5% intraspecific genetic divergence) were found in all three taxa. Phylogenetic analyses indicated that the S. asakoae complex can be divided into seven groups with the largest group consisting of specimens from Thailand, Malaysia and Myanmar. This group most likely represents true S. asakoae. The remaining haplotypes formed groups with conspecific haplotypes or with other closely related species. Among these groups, one including S. monglaense and another including S. myanmarense suggest that certain specimens identified as S. asakoae most likely belong to those species. Therefore, they constitute new locality records for Thailand and also represent new records of anthropophily. Members of S. chamlongi are not monophyletic as its clade also included S. hackeri. A median joining network revealed strong geographic associations of the haplotypes of S. nigrogilvum suggesting limitation of gene flow. Because this species occurs mainly in high elevation habitats, low land areas could present a barrier to gene flow.

Molecular detection of Leucocytozoon (Apicomplexa: Haemosporida) in black flies (Diptera: Simuliidae) from Thailand.

Information regarding vector-parasite association is necessary for fully understanding the epidemiology of vector borne diseases yet, this information is lacking in the case of Leucocytozoonosis in the Oriental region, despite a high incidence of the disease. In this study, we used a molecular approach based on mitochondrial cytochrome b (cyt b) sequence to detect the parasite, Leucocytozoon, in potential black fly (Simuliidae) vectors in Thailand. A total of 404 wild caught black flies representing six morphological species of two subgenera were examined.- Gomphostilbia (Simulium asakoae complex, S. chumpornense) and Simulium (S. chamlongi, S. nodosum, S. nigrogilvum). Forty-four black fly specimens from two species of the Gomphostilbia were positive for Leucocytozoon. Most (35) of these were found in a village where high numbers of domestic chicken were kept, consistent with the possibility that chickens are a host of Leucocytozoon species found in black flies. Sixteen haplotypes were identified among 44 cyt b sequences. Comparisons of the sequences with previous reports revealed that the 11 haplotypes obtained in this study were identical or very similar to unknown Leucocytozoon found in infected domestic chickens. Four haplotypes are genetically similar to L. schoutedeni and one haplotype is genetically very different from existing cyt b sequences in public database. Our results indicate that two black fly species of the subgenus Gomphostilbia in Thailand are possible vectors of Leucocytozoon transmitted among poultry and wild birds in the country.

A new black fly (Diptera: Simuliidae) species of the subgenus Asiosimulium Takaoka Choochote from Thailand.

A new black fly species of the subgenus Asiosimulium Takaoka Choochote of the genus Simulium was recognized from northeastern Thailand based on morphology, mitochondrial DNA and ecology. This black fly species has similar morphological characteristics to Simulium oblongum Takaoka and Choochote that was also described from the same geographic region in all life stages. However, this new species could be distinguished at the adult stage by coloration of the maxillary palp and in the larval stage by the presence of a pigmented subesophageal ganglion that is lacking in S. oblongum. Genetic distance and phylogenetic analyses based on mitochondrial cytochrome c oxidase I sequences clearly differentiated the two species with minimum genetic distance of 3.51%. These species are also ecologically isolated as S. oblongum is found only at low elevation (<650 m above sea level) but the new species occurs only at high elevation (>1,100 m above sea level).