Population genetics of the solanum fruit fly, Bactrocera latifrons (Hendel) (Diptera: Tephritidae).

The solanum fruit fly, Bactrocera latifrons (Hendel), is an important pest species of commercial plants in the family Solanaceae. In this study, the population genetic structure of B. latifrons was investigated using mitochondrial cytochrome c oxidase I sequences. A mitochondrial DNA haplotype network revealed no major genetic break, but haplotypes from recently invaded areas in Japan, Tanzania, and Kenya were genetically divergent. The overall haplotype network is approximately star-shaped, characteristic of recent demographic expansion of populations. This is also supported by large negative values of neutrality tests. Despite the overall pattern of recent population history, genetic structure analysis revealed considerable genetic structuring with 33% of pairwise comparisons being significantly different. Populations that were genetically different from the others usually possess low genetic diversity, suggesting that genetic drift is potentially a factor driving genetic differentiation. Local extinction and recolonization processes related to the availability of host plants are most likely responsible for a founder effect and subsequent genetic drift in a population.

DNA barcode variability and host plant usage of fruit flies (Diptera: Tephritidae) in Thailand.

The objectives of this study were to examine the genetic variation in fruit flies (Diptera: Tephritidae) in Thailand and to test the efficiency of the mitochondrial cytochrome c oxidase subunit I (COI) barcoding region for species-level identification. Twelve fruit fly species were collected from 24 host plant species of 13 families. The number of host plant species for each fruit fly species ranged between 1 and 11, with Bactrocera correcta found in the most diverse host plants. A total of 123 COI sequences were obtained from these fruit fly species. Sequences from the NCBI database were also included, for a total of 17 species analyzed. DNA barcoding identification analysis based on the best close match method revealed a good performance, with 94.4% of specimens correctly identified. However, many specimens (3.6%) had ambiguous identification, mostly due to intra- and interspecific overlap between members of the B. dorsalis complex. A phylogenetic tree based on the mitochondrial barcode sequences indicated that all species, except for the members of the B. dorsalis complex, were monophyletic with strong support. Our work supports recent calls for synonymization of these species. Divergent lineages were observed within B. correcta and B. tuberculata, and this suggested that these species need further taxonomic reexamination.

Cytogenetic and Molecular Evidence of Additional Cryptic Diversity in High Elevation Black fly Simulium feuerborni (Diptera: Simuliidae) Populations in Southeast Asia.

Simulium feuerborni Edwards is geographically widespread in Southeast Asia. Previous cytogenetic study in Thailand revealed that this species is a species complex composed of two cytoforms (A and B). In this study, we cytologically examined specimens obtained from the Cameron Highlands, Malaysia, and Puncak, Java, Indonesia. The results revealed two additional cytoforms (C and D) of S. feuerborni. Specimens from Malaysia represent cytoform C, differentiated from other cytoforms by a fixed chromosome inversion on the long arm of chromosome III (IIIL-5). High frequencies of the B chromosome (33-83%) were also observed in this cytoform. Specimens from Indonesia represent the cytoform D. This cytoform is differentiated from others by a fixed chromosome inversion difference on the long arm of chromosome II (IIL-4). Mitochondrial DNA sequences support genetic differentiation among cytoforms A, B, and C. The pairwise F(ST) values among these cytoforms were highly significantly consistent with the divergent lineages of the cytoforms in a median-joining haplotype network. However, a lack of the sympatric populations prevented us from testing the species status of the cytoforms.

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.

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.

Discovery of the Larvae and Pupae of the Black Fly Simulium (Gomphostilbia) khelangense and Breeding Habitats of Potential Pest Species of the S. (G.) chumpornense Subgroup (Simuliidae).

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.

Preconcentration of Heterocyclic Aromatic Amines in Edible Fried Insects Using Surfactant-Assisted Hydrophobic Deep Eutectic Solvent for Homogeneous Liquid-Liquid Microextraction prior to HPLC.

Thermal processing techniques are often accompanied by the production of many harmful compounds such as heterocyclic aromatic amines (HAAs). To protect human health, an efficient and environmentally friendly method, namely, homogeneous liquid-liquid microextraction (HLLME), was investigated. This method is based on a surfactant-assisted hydrophobic deep eutectic solvent for the determination of HAAs in edible fried insect samples prior to their analysis by high-performance liquid chromatography coupled with UV detection. A hydrophobic deep eutectic solvent (as extraction solvent) was synthesized using decanoic acid as a hydrogen bond donor and tetrabutylammonium bromide (TBABr) as a hydrogen bond acceptor and then characterized by Fourier transform infrared (FTIR) spectroscopy. The surfactant was used as the emulsifier and induces mass transfer, resulting in an increasing extraction efficiency of the proposed method. Various factors affecting the extraction performance were investigated and optimized. A matrix-match calibration method was used to analyze HAAs in high heat-treated edible fried insect samples. Under optimized conditions, the proposed method showed good linearity (R2 ≥ 0.99) with satisfactory limits of detection and satisfactory reproducibility with relative standard deviation of less than 10.0%. Furthermore, the procedure greenness was assessed using the Analytical Eco-Scale. This paper represents the first application of HLLME based on a surfactant-assisted hydrophobic deep eutectic solvent to analyze HAAs in edible fried insect samples.

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.

Fast-evolving nuclear genes as barcoding markers for black flies (Diptera: Simuliidae) in Thailand.

Rapid and accurate identification is a prerequisite for the study of all aspects of species, particularly for pests and vectors. Black flies are economically significant blood-sucking insects, as many species are pests and vectors that transmit parasites to humans and other animals. We examined the efficiency of two fast-evolving nuclear genes, elongator complex protein 1 (ECP1) and big zinc finger (BZF), for identifying 13 nominal species in three species-groups of black flies, the Simulium multistriatum, S. striatum, and S. tuberosum groups, in Thailand where the mitochondrial cytochrome c oxidase I (COI) gene has not been successful for differentiating many nominal species. ECP1 gene sequences were highly effective for identification, with >96% (181 of 188) of the specimens correctly identified. Unsuccessful identifications based on ECP1 were between S. nakhonense and S. chiangmaiense, which are members of the S. striatum species-group, whereas all identifications of nominal species of the S. multistriatum and S. tuberosum species-groups were successful. In contrast, BZF had successful rates for the S. striatum species-group, with >93% (71 of 76) of the specimens correctly identified. This gene also successfully assigned unknown larvae of the S. striatum group to species. Phylogenetic analyses and molecular species delimitations based on the BZF gene uncovered cryptic diversity in two nominal species, S. nakhonense and S. wangkwaiense, which will require resolution through further study.

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.

Integrated cytogenetic, ecological, and DNA barcode study reveals cryptic diversity in Simulium (Gomphostilbia) angulistylum (Diptera: Simuliidae).

An integrated approach based on cytogenetics, molecular genetics, and ecology was used to examine diversity in the black fly Simulium angulistylum Takaoka & Davies in Thailand. Cytological analysis revealed three cytoforms (A, B, and C) of S. angulistylum differentiated by fixed chromosome inversions. Distributions of these cytoforms were associated with ecology. Cytoforms A and B were found in low-altitude habitats (<600 m above sea level), whereas cytoform C occurred at high altitudes (>1000 m above sea level). Mitochondrial DNA sequences of the cytochrome oxidase subunit I barcoding region revealed significant genetic differentiation among the cytoforms. The mitochondrial DNA haplotype network revealed divergent lineages within cytoforms, indicating additional hidden diversity. Therefore, integrated approaches are necessary for fully understanding black fly biodiversity. Population genetic analysis revealed high genetic structuring that could be due to the habitat preferences of S. angulistylum. Phylogeographic analyses indicated population demographic expansion at the mid-Pleistocene (900 000 years ago), which is older than for other black flies and insects in the Southeast Asian mainland. The high level of genetic structure and diversity, therefore, could also be due to the long demographic history of S. angulistylum.

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.

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.

Spotted fever group Rickettsia, Anaplasma and Coxiella-like endosymbiont in Haemaphysalis ticks from mammals in Thailand.

Ticks are ectoparasites of vertebrates and vectors of various pathogenic microorganisms. In this study, the presence of bacteria and protozoa was evaluated by PCR and DNA sequencing in 233 mammal ticks collected from 8 provinces in Thailand. Sequence and phylogenetic analyses of partial rickettsial ompA, ompB, sca4 and partial Coxiella 16S rRNA, GroEL, rpoB genes clearly revealed, for the first time, a co-infection of SFG Rickettsia belonging to R. massiliae subgroup and Coxiella-like endosymbiont (CLE), Cox-hein, in a male of Haemaphysalis heinrichi tick infesting Burmese ferret-badger in Loei province. Moreover, a male of H. hystricis tick infesting the same host was infected with another CLE, Cox-hys. Based on the 16S rRNA gene sequence, Anaplasma sp., closely related to Anaplasma bovis was also detected in a male of H. heinrichi infesting the same Burmese ferret-badger. In addition, the third CLE, Cox-asia, found in H. asiatica collected from Asian palm civet in Chiang Rai province, was different from both Cox-hein and Cox-hys. This study provided important data and broadened our knowledge on tick-borne pathogens and endosymbionts in Thailand and Southeast Asia.

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.

Molecular detection of avian haemosporidian parasites in biting midges (Diptera: Ceratopogonidae) from Thailand.

Culicoides biting midges are vectors of many haemosporidian parasite species. These parasites are found in several wild and domestic avian species in Thailand but knowledge of the insect vectors is very limited. In this study, a molecular approach was used to detect haemosporidian parasites in six biting midge species in Thailand. A total of fifteen cytochrome b haplotypes of three haemosporidian parasites were detected from 1,165 specimens of six different Culicoides species. Comparisons of these sequences with those recorded in a public database revealed that they were comprised sequences of three species, Leucocytozoon sp., Plasmodium juxtanucleare and P. gallinaceum. All of these haemosporidian parasite species were detected in Culicoides mahasarakhamense Pramual, Jomkumsing, Piraonapicha, & Jumpato while P. juxtanucleare was also detected in C. huffi Causey and C. guttifer Meijere and Leucocytozoon sp. were also detected in C. guttifer. All of these parasites are commonly found in chickens in agreement with information that these biting midge species will bite chickens. Detection DNA of Leucocytozoon sp. in biting midges reported here provides the second record of this parasitic genus infecting Culicoides. This study also provides the first records of P. juxtanucleare and P. gallinaceum in biting midge species. Further investigation is required to determine whether Culicoides biting midge species are competent vectors of these parasites.

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.

Integrative taxonomy uncovers a new Culicoides (Diptera: Ceratopogonidae) biting midge species from Thailand.

Fully understanding biodiversity often requires an integrated approach especially for small insects because species diagnostic morphological characters are limited. In this study, morphological characters and DNA barcodes were used to examine previously recognized genetically divergent lineages detected in the biting midge, Culicoides arakawae (Arakawa), from Thailand. Morphological examinations revealed that specimens belonging to one lineage are morphologically different from C. arakawae in shape of the paramere in males, and in the leg color pattern of both sexes. Therefore, a formal description is provided for this new species, Culicoides mahasarakhamense sp. nov. Based on morphological characters including a large and shallow palpal pit, one sac like spermatheca and male with parameres bent at base with large basal knob, the new species was assigned into the subgenus Meijerehelea Wirth and Hubert. Morphological differentiation including wing pattern and shape of spermatheca of the new species are discussed and compared with other members of this subgenus. Mitochondrial cytochrome c oxidase I sequence analysis indicated that this new species is different from other members of the subgenus Meijerehelea with minimum interspecific genetic divergence of 3.92%. Automatic Barcode Gap Discovery species delimitation analysis also supported the recognition of a new species. Phylogenetic analyses revealed that the new species is closely related to C. arakawae, consistent with morphological similarity of these species. Results of this study highlight the necessity of using integrated approach for Culicoides taxonomy.

Genetic variation, DNA barcoding and blood meal identification of Culicoides Latreille biting midges (Diptera: Ceratopogonidae) in Thailand.

Biting midges of the genus Culicoides Latreille are blood sucking insects of medical and veterinary importance. Many species are vectors of disease agents transmitted to humans and other animals. Therefore, rapid and accurate species identification is essential for appreciation of all aspects of these insects. In this study, DNA barcode efficacy and molecular identification of host blood sources were examined in biting midges from Thailand. A total of 203 barcoding sequences were obtained from 16 Culicoides taxa. Intraspecific genetic divergence varied from 0.28% to 9.90% for specimens collected in Thailand. Despite this high level of genetic variation, DNA barcode identifications in the Barcoding of Life Data System had a considerable success rate (90%). Phylogenetic analyses and distance-based species delimitation methods indicated the possibility of cryptic species in four taxa, namely, Culicoides actoni Smit, C. arakawae Arakawa, C. huffi Causey and C. jacobsoni Macfie. Further investigations will be required to examine the species status of these lineages. Host blood meal identifications from 42 blood engorged females of 10 Culicoides taxa revealed three animal hosts: chicken, cattle and buffalo. Most of this information agrees with previous knowledge but this is the first report of C. actoni, C. fulvus and C. huffi feeding on chicken.