Molecular detection of blood protozoa and identification of black flies of the Simulium varicorne species group (Diptera: Simuliidae) in Thailand.

Species of the Simulium varicorne group in Thailand have veterinary significance as vectors of haemosporidian parasites. Accurate identification is, therefore, critical to the study of vectors and parasites. We used morphology and molecular markers to investigate cryptic genetic lineages in samples identified as Simulium chumpornense Takaoka & Kuvangkadilok, 2000. We also tested the efficiency of the nuclear internal transcribed spacer 2 (ITS2) marker for the identification of species in this group. Morphological examinations revealed that S. chumpornense lineage A is most similar to S. khelangense Takaoka, Srisuka & Saeung, 2022, with minor morphological differences. They are also genetically similar based on mitochondrial cytochrome c oxidase I (COI) sequences. Geographically, the sampling site where paratypes of S. khelangense were originally collected is <50 km from where S. chumpornense lineage A was collected. We concluded that cryptic lineage A of S. chumpornense is actually S. khelangense. COI sequences could not differentiate S. kuvangkadilokae Pramual and Tangkawanit, 2008 from S. chumpornense and S. khelangense. In contrast, ITS2 sequences provided perfect accuracy in the identification of these species. Molecular analyses of the blood protozoa Leucocytozoon and Trypanosoma demonstrated that S. khelangense carries L. shoutedeni, Leucocytozoon sp., and Trypanosoma avium. The Leucocytozoon sp. in S. khelangense differs genetically from that in S. asakoae Takaoka & Davies, 1995, signaling the possibility of vector-parasite specificity.

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.

Diversity, Abundance and Host Blood Meal Analysis of Culicoides Latreille (Diptera: Ceratopogonidae) from Cattle Pens in Different Land Use Types from Thailand.

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.

Population Genetic Structure and Population History of the Biting Midge Culicoides mahasarakhamense (Diptera: Ceratopogonidae).

Biting midges of the genus Culicoides Latreille are significant pests and vectors of disease agents transmitted to humans and other animals. Understanding the genetic structure and diversity of these insects is crucial for effective control programs. This study examined the genetic diversity, genetic structure, and demographic history of Culicoides mahasarakhamense, a possible vector of avian haemosporidian parasites and Leishmania martiniquensis, in Thailand. The star-like shape of the median joining haplotype network, a unimodal mismatch distribution, and significant negative values for Tajima's D and Fu's FS tests indicated that populations had undergone recent expansion. Population expansion time was estimated to be 2000-22,000 years ago. Population expansion may have been triggered by climatic amelioration from cold/dry to warm/humid conditions at the end of the last glaciations, resulting in the increased availability of host blood sources. Population pairwise FST revealed that most (87%) comparisons were not genetically different, most likely due to a shared recent history. The exception to the generally low level of genetic structuring is a population from the northern region that is genetically highly different from others. Population isolation in the past and the limitation of ongoing gene flows due to large geographic distance separation are possible explanations for genetic differentiation.

Population structure and population history of the black fly Simulium chumpornense (Diptera: Simuliidae) from Thailand.

Understanding genetic structure and diversity of insect vectors is crucial for disease epidemiology. In this study, mitochondrial cytochrome c oxidase I sequences were used to infer genetic diversity, genetic structure and population history of the black fly, Simulium chumpornense Takaoka and Kuvangkadilok, a suspected vector of blood protozoa of the genus Leucocytozoon and Trypanosoma. High intraspecific genetic divergence (max. 3.76%) was found among 142 specimens obtained from 19 locations across Thailand. A median joining network revealed two genetic lineages (A and B) that were geographically associated. Lineage A is representative of central and northeastern regions. Lineage B represents specimens from diverse locations in northern, western, and southern Thailand, including the type locality. Mismatch distribution and the neutrality tests provided signals of past population expansions in both lineages. The expansion time dating back to the end of last glaciations at 12,000 - 15,000 years ago is possibly related to increasing of precipitation at the end of last glacial period. Despite recent population history, population pairwise FST analysis revealed that almost all population comparisons were genetically significantly different. The high level of genetic structuring is possibly a result of historical isolation of the population that survived in different refugia sites during the dry conditions during glaciations.

DNA barcoding of tropical black flies (Diptera: Simuliidae) in Thailand: One decade of progress.

Black flies (Diptera: Simuliidae) are important blood sucking insects because they are the vectors of disease agents transmitted to human and other animals. Rapid and correct species identifications are necessary for all aspects of the study of black flies. DNA barcodes based on mitochondrial cytochrome c oxidase I (COI) have been effectively used for the determination of black fly species. However, the success of this method requires a large and reliable COI sequence library. In this study, 171 DNA barcoding sequences from 17 black fly species were added to NCBI GenBank database, six of these species were reported for the first time. Efficacy of DNA barcodes for species identification was examined using 1,286 sequences representing 89 nominal species of black flies in Thailand. A considerable level of success (90%) was achieved but efficiency of COI sequences for species identification was very low in the following species-groups; Simulium asakoae, S. feuerborni, S. multistriatum and S. striatum. Incomplete lineage sorting or inadequate variation of this genetic marker for differentiation of recently diverged species are the more likely explanations, and thus, more variable genetic markers are needed. Other reasons for unsuccessful DNA barcoding are imperfect taxonomy and the misidentification of sources of reference sequences. Because many new black fly species in Thailand were described recently, a reassessment of the COI sequences reported previously is necessary.

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.

A new species of black fly (Diptera: Simuliidae) in the Simulium (Simulium) multistriatum species-group from Thailand.

A new black fly species of the Simulium multistriatum species-group of the subgenus Simulium Latreille is described from the mountainous area in northeastern Thailand, based on morphology and mitochondrial DNA sequences. The new species is morphologically similar to S. laui Takaoka and Sofian-Azirun and S. lacduongense Takaoka and Ya'cob originally described from Vietnam, S. fenestratum Edwards originally described from Indonesia and S. chaliowae Takaoka and Boonkemtong originally described from Thailand, but can be distinguished in the adult stage by the number of upper eye facets and globular shape of the spermatheca and in the pupal stage by the cocoon and shape of thoracic tubercles. Genetic distance and phylogenetic analyses of mitochondrial cytochrome c oxidase I sequences differentiated the new species from other members of S. multistriatum species-group. All specimens of the new species formed a monophyletic clade with strong support in all phylogenetic analyses. The minimum interspecific genetic distance of 4.9% is considerably greater than the new species maximum intraspecific genetic distance (2.7%).

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).

Genetic diversity and population structure of blue-crested lizard, Calotes mystaceus Duméril & Bibron, 1837 (Squamata: Agamidae) in Thailand.

The blue-crested lizard, Calotes mystaceus Duméril & Bibron, 1837, is listed as a protected wild animal in Thailand. Its population is likely to be dramatically reduced due to massive hunting in several areas in this country. Basic information on its population genetics is therefore needed to facilitate its conservation. Thus, in this study we investigated the mitochondrial cytochrome c oxidase subunit 1 (CO1) sequence variation of 238 individualC.mystaceus from 42 different geographical localities of the north, west, central, east and northeast regions of Thailand. High genetic diversity and genetic differentiation at the intrapopulation and interpopulation levels was observed.We detected 63 unique haplotypes and 12 common/shared haplotypes. The phylogenetic analysis reveals two major lineages, I and II. These two lineages are separated by mountain ranges, which play an important role as natural barriers blocking gene flow. Our finding reveal at least two cryptic lineages represented in C. mystaceus populations in Thailand. However, a comprehensive investigation of the morphology, biology, ecology and genetic diversity of C. mystaceus in other regions within its area of distribution is needed.

DNA barcoding of human-biting black flies (Diptera: Simuliidae) in Thailand.

Black flies (Diptera: Simuliidae) are important insect vectors and pests of humans and animals. Accurate identification, therefore, is important for control and management. In this study, we used mitochondrial cytochrome oxidase I (COI) barcoding sequences to test the efficiency of species identification for the human-biting black flies in Thailand. We used human-biting specimens because they enabled us to link information with previous studies involving the immature stages. Three black fly taxa, Simulium nodosum, S. nigrogilvum and S. doipuiense complex, were collected. The S. doipuiense complex was confirmed for the first time as having human-biting habits. The COI sequences revealed considerable genetic diversity in all three species. Comparisons to a COI sequence library of black flies in Thailand and in a public database indicated a high efficiency for specimen identification for S. nodosum and S. nigrogilvum, but this method was not successful for the S. doipuiense complex. Phylogenetic analyses revealed two divergent lineages in the S. doipuiense complex. Human-biting specimens formed a separate clade from other members of this complex. The results are consistent with the Barcoding Index Number System (BINs) analysis that found six BINs in the S. doipuiense complex. Further taxonomic work is needed to clarify the species status of these human-biting specimens.

Phylogeography of the black fly Simulium aureohirtum (Diptera: Simuliidae) in Thailand.

The black fly Simulium aureohirtum Brunetti (Diptera: Simuliidae) is geographically widespread and utilizes diverse habitats. Previous studies revealed high genetic diversity, suggesting that this species could be a species complex. In this study, we used mitochondrial cytochrome c oxidase II sequences to examine genetic structure and demographic history of S. aureohirtum in Thailand. High level of genetic variation was observed due to the existence of genetically divergent lineages (A, B, and C). Lineages A and C were geographically widespread, while lineage B was geographically restricted to the eastern part of Thailand. The geographic localization of lineage B suggested limitations in gene flow, which is most likely a result of ecologically different habitats. Large genetic differences between individuals of lineages A and C, but geographically sympatric distributions of the members of these lineages, are considered to represent an admixture of the populations that have evolved allopatrically. Historical environmental change during the Pleistocene glaciations is possibly the factor that drove the lineage divergence. Population demographic history analyses revealed recent population expansions in the three lineages dating back to the last glaciations. These observations further highlight the significance of the Pleistocene climatic change on current genetic structure and diversity of living organisms of the Southeast Asian mainland.

Molecular phylogeny of black flies in the Simulium tuberosum (Diptera: Simuliidae) species group in Thailand.

Black flies are medically and ecologically significant insects. They are also interesting from an evolutionary standpoint regarding the role of chromosomal change and ecological adaptation. In this study, molecular genetic markers based on multiple gene sequences were used to assess genetic diversity and to infer phylogenetic relationships for a group of cytologically highly diverse black flies of the Simulium tuberosum species group in Thailand. Ecological affinities of the species were also investigated. High levels of genetic diversity were found in cytological species complexes, S. tani and S. doipuiense, and also in S. rufibasis, which was cytologically nearly monomorphic. The results highlight the necessity of integrating multilevel markers for fully understanding black fly biodiversity. Phylogenetic relationships based on multiple gene sequences were consistent with an existing dendrogram inferred from cytological and morphological data. Simulium tani is the most distinctive taxa among the members of the S. tubersosum species group in Thailand based on its divergent morphological characters. Molecular data supported the monophyletic status of S. tani, S. weji, and S. yuphae, but S. doipuiense and S. rufibasis were polyphyletic, most likely due to incomplete lineage sorting and inadequate phylogenetic signals. Ecological analyses revealed that members of the S. tuberosum species group have clearly different ecological niches. The results thus supported previous views of the importance of ecology in black fly evolution.

Cryptic biodiversity and phylogenetic relationships revealed by DNA barcoding of Oriental black flies in the subgenus Gomphostilbia (Diptera: Simuliidae).

Understanding the medical, economic, and ecological importance of black flies relies on correct identification of species. However, traditional taxonomy of black flies is impeded by a high degree of morphological uniformity, especially the presence of cryptic biodiversity, historically recognized by details of chromosomal banding patterns. We assess the utility of DNA barcoding, based on cytochrome c oxidase subunit 1 (COI) sequences, for identifying 13 species of Oriental black flies in the subgenus Gomphostilbia. Samples of larvae fixed in Carnoy's solution were used to gather molecular and chromosomal data from the same individual. We found that larvae refrigerated in Carnoy's fixative for as long as 11 years can be used for DNA study. Levels of intraspecific genetic divergence, based on the Kimura-2 parameter, range from 0% to 9.28%, with a mean of 2.75%, whereas interspecific genetic divergence ranges from 0.34% to 16.05%. Values of intraspecific and interspecific genetic divergence overlap in seven species owing to incomplete lineage sorting and imperfect taxonomy, implying that DNA barcoding to identify these species will be ambiguous. Despite a low level of success, we found that DNA barcoding is useful in revealing cryptic biodiversity, potentially facilitating traditional taxonomy. Phylogenetic analyses indicate that species groups currently recognized on morphological criteria are not monophyletic, suggesting a need to reevaluate the classification of the subgenus Gomphostilbia.