Seroprevalence study in humans and molecular detection in Rhipicephalus sanguineus ticks of severe fever with thrombocytopenia syndrome virus in Thailand.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus with a mortality rate of up to 30%. First identified in China in 2009, it was later reported in other Asian countries, including Thailand in 2020. SFTSV has been detected in several tick species, including Rhipicephalus sanguineus, known for infesting dogs. We conducted a seroprevalence study of SFTSV in Bangkok and Nong Khai, Thailand, by analyzing 1162 human samples collected between 2019 and 2023. The testing method relied on IgG detection using ELISA and confirmed though a virus seroneutralization test. The results indicated that out of the participants, 12 (1.1%) tested positive for anti-SFTSV IgG antibodies; however, none exhibited positive results in the seroneutralization assay. Additionally, molecular detection of SFTSV, Crimean-Congo hemorrhagic fever (CCHF), Coxiella spp., Bartonella spp., and Rickettsia spp. was performed on 433 Rh. sanguineus ticks collected from 49 dogs in 2023 in Chachoengsao Province, Thailand. No evidence of these pathogens was found in ticks. These findings highlight the importance of exploring viral cross-reactivity. Furthermore, it is important to conduct additional studies to isolate SFTSV from animals and ticks in order to identify the potential transmission routes contributing to human and animal infections in Thailand.

Demographic inference from the mt-DNA COI gene and wing geometry of Culex gelidus (Diptera: Culicidae), an important vector of Japanese encephalitis in Thailand.

Culex gelidus (Diptera: Culicidae), an important vector of the Japanese encephalitis virus (JEV), contributes to human viral encephalitis in many Asian countries, including Thailand. This study represents the first investigation of the demographic patterns of Cx. gelidus populations in Thailand using cytochrome c oxidase subunit I (COI) gene analysis and wing geometric morphometrics (GM). Mosquitoes were collected from 10 provinces across six regions of Thailand in 2022. Analysis of the COI sequences (n = 182) indicated high haplotype diversity (0.882) and low nucleotide diversity (0.006), with 72 haplotypes identified. The haplotype network demonstrated no profound splits among the geographic populations. Neutral tests, including Tajima's D and Fu's Fs, displayed negative values, with a significant result observed for Fu's Fs (-33.048, p < 0.05). The mismatch distribution analysis indicated that the population does not statistically deviate from a model of sudden population expansion (SSD = 0.010, p > 0.05; Rg = 0.022, p > 0.05). The estimations suggest that the Cx. gelidus population in Thailand began its expansion approximately between 459,243 and 707,011 years ago. The Mantel test showed no significant relationship between genetic and geographic distances (r = 0.048, p > 0.05). Significant phenotypic differences (based on wing shape) were observed among most populations. Additionally, in this study, we found no significant relationships between phenotypic and genetic distances (r = 0.250, p > 0.05). Understanding the genetic and morphological dynamics of Cx. gelidus is vital for developing targeted surveillance and vector control measures. This knowledge will also help to predict how future environmental changes might affect these populations, thereby informing long-term vector management strategies.

Island mosquitoes of Thailand: an update on species diversity and DNA barcoding.

Mosquitoes (Diptera: Culicidae) are among the most medically significant insects, with several species acting as vectors for human pathogens. Although there are frequent reports of mosquito-borne diseases in the border island areas of Thailand, comprehensive data on the diversity and DNA barcoding of these mosquito species remain limited. This study investigated mosquito diversity in two main archipelagos in Thailand-the Trat archipelago (comprising Chang Island and Kood Island) and the Ranong archipelago (comprising Chang Island and Phayam Island)-and generated DNA barcode data from the mosquitoes found there. The survey across these islands discovered a total of 41 species, highlighting the presence of several species known to be vectors for human diseases. Thirty-seven mosquito species from the island areas were documented to provide reference DNA barcode sequences for mosquitoes in Thailand's island regions. Two species, Aedes fumidus and Finlaya flavipennis, have been added as new COI sequence records in the database. DNA barcoding was highly effective in classifying almost all species by identifying barcoding gaps, except for Anopheles baimaii and Anopheles dirus, which could not be distinguished. Additionally, the study noted that geographical variations might influence certain mosquito species, such as Anopheles barbirostris A3 and Mansonia dives, causing them to be split into two distinct subgroups. The findings of this study are crucial, as they aid in classifying mosquito species using molecular techniques and expand our knowledge of disease vectors in these biodiverse regions.

Influence of insular conditions on wing phenotypic variation in two dominant mosquito vectors, Aedes albopictus and Armigeres subalbatus (Diptera: Culicidae), in the border archipelagos of Thailand.

Insects geographically separated into island and mainland populations often exhibit phenotypic variations, a phenomenon known as insular conditions. These conditions can lead to rapid evolutionary changes that affect the morphological characteristics of mosquito vectors. Nevertheless, studies that specifically examine phenotype differences between island and mainland mosquito populations have been limited. In this study, wing variation in size and shape was investigated using the geometric morphometric (GM) technique in two dominant mosquito vectors, Aedes albopictus and Armigeres subalbatus, in the Ranong and Trat archipelagos of Thailand. Significant differences in average wing centroid size (CS) were found in 6 out of 15 population pairs for Ae. albopictus (p < 0.05) and in 5 population pairs for Ar. subalbatus (p < 0.05). After removing the allometric effect, canonical variate analyses (CVA) based on wing shape analysis revealed overlap across all populations for both Ae. albopictus and Ar. subalbatus. However, the statistical analysis indicated that Ar. subalbatus exhibited wing shape differences across all populations (p < 0.05), and most Ae. albopictus populations also displayed distinct wing shapes (p < 0.05), except for the populations from Chang Island and the mainland of Ranong, which showed no significant differences (p > 0.05). These findings enhance our understanding of mosquito adaptability in island regions and provide valuable data for the surveillance and monitoring of vector evolution.

Geometric morphometrics to differentiate species and explore seasonal variation in three Mansonia species (Diptera: Culicidae) in central Thailand and their association with meteorological factors.

Mansonia mosquito species are recognised as a significant vector of human pathogens, primarily transmitting the filarial nematode, Brugia malayi. In central Thailand, the three most prevalent Mansonia species are Mansonia annulifera, Mansonia indiana and Mansonia uniformis. This study explored the influence of seasonal changes on the phenotypic variation of these Mansonia species in central Thailand using the geometric morphometrics (GM). To ensure accurate species identification, we integrated GM techniques with DNA barcoding, examining distinctions in both phenotype and genotype among the species. The intraspecific genetic divergence ranged from 0.00% to 1.69%, whereas the interspecific genetic divergence ranged from 10.52% to 16.36%. The clear distinction between intra- and interspecific distances demonstrated the presence of a barcoding gap, confirming the successful differentiation of the three Mansonia mosquito species through DNA barcoding. Similarly, the interspecies GM assessment for classifying Mansonia species demonstrated a high degree of accuracy, with an overall performance of 98.12%. Exploring seasonal variation in the three Mansonia species revealed wing variations across different seasons, and pronounced variations appearing in the cool season. Regarding their association with meteorological factors, Ma. annulifera and Ma. uniformis showed significant positive correlations with temperature (p < 0.05), and Ma. uniformis also displayed a significant negative correlation with atmospheric pressure (p < 0.05). The insights from this study will deepen our understanding of the adaptive patterns of Mansonia mosquitoes in Thailand's central region, paving the way for enhanced disease surveillance related to these vectors.

Insights into the mitochondrial cytochrome oxidase I (mt-COI) gene and wing morphometrics of Anopheles baimaii (Diptera: Culicidae) in malaria-endemic islands of Thailand.

Anopheles baimaii (Diptera: Culicidae) significantly contributes to the transmission of parasites causing malaria in Southeast Asia and South Asia. This study examined the morphological (wing shape) and molecular (mitochondrial gene) variations of An. baimaii in four of Thailand's border islands, and also investigated the presence of Plasmodium parasites in these mosquitoes. No Plasmodium infections were detected in the samples. Significant differences in wing shape were observed in most island populations (p < 0.05). A single-linkage tree, constructed using Mahalanobis distances, clustered the populations into two groups based on geographical locations. Genetic variation in An. baimaii was also analyzed through cytochrome c oxidase subunit I (COI) gene sequences. This analysis identified 22 segregating sites and a low nucleotide diversity of 0.004. Furthermore, 18 distinct haplotypes were identified, indicating a high haplotype diversity of 0.825. Neutrality tests for the overall population revealed a significantly negative Fu's Fs value (-5.029), indicating a population expansion. In contrast, Tajima's D yielded a negative value (-1.028) that did not reach statistical significance. The mismatch distribution analysis exhibited a bimodal pattern, and the raggedness index was 0.068, showing no significant discrepancy (p = 0.485) between observed and expected distributions. Pairwise genetic differentiation assessments demonstrated significant differences between all populations (p < 0.05). These findings provide valuable insights into the COI gene and wing morphometric variations in An. baimaii across Thailand's islands, offering critical information for understanding the adaptations of this malaria vector and guiding future comprehensive research.

Population genetic structure and wing geometric morphometrics of the filarial vector Armigeres subalbatus (Diptera: Culicidae) in Thailand.

Armigeres subalbatus (Diptera: Culicidae) is a mosquito species of significant medical and veterinary importance. It is widely distributed across Southeast and East Asia and is commonly found throughout Thailand. This study assessed the genetic diversity and population structure of Ar. subalbatus in Thailand using the cytochrome c oxidase subunit I (COI) gene sequences. Additionally, wing shape variations among these populations were examined using geometric morphometrics (GM). Our results demonstrated that the overall haplotype diversity (Hd) was 0.634, and the nucleotide diversity (π) was 0.0019. Significant negative values in neutrality tests (p < 0.05) indicate that the Ar. subalbatus populations in Thailand are undergoing a phase of expansion following a bottleneck event. The mismatch distribution test suggests that the populations may have started expanding approximately 16,678 years ago. Pairwise genetic differentiation among the 12 populations based on Fst revealed significant differences in 32 pairs (p < 0.05), with the degree of differentiation ranging from 0.000 to 0.419. The GM analysis of wing shape also indicated significant differences in nearly all pairs (p < 0.05), except for between populations from Nakhon Pathom and Samut Songkhram, and between those from Chiang Mai and Mae Hong Son, suggesting no significant difference due to their similar environmental settings. These findings enhance our understanding of the population structure and phenotypic adaptations of mosquito vectors, providing vital insights for the formulation of more efficacious vector control strategies.

Ctenocephalides orientis and Ctenocephalides felis in Thailand: Head geometry by species, sex and geography.

Fleas in the genus Ctenocephalides serve as biological vectors or intermediate hosts of microorganisms such as bacteria, rickettsia, protozoa and helminths. Ctenocephalides felis has a worldwide distribution, while C. orientis has long been considered as a subspecies of C. felis in Asia. To help the morphological recognition of these two species and further explore their differences, we used the geometric morphometric approach applied to the head. Both sexes were examined. Five anatomical landmarks of the head were used, and to capture the curvature of the front head, 10 semilandmarks were added. There was a consistent difference in species classification accuracy when considering landmarks only versus their combination with semilandmarks, suggesting the importance of the curve of the head as a taxonomic signal. Using or not the labels in the reclassification analyses, the head shape allowed by itself almost perfect recognition of the two species, in both sexes, even after adjustment for prior probabilities. The same approach disclosed a high level of sexual size and shape dimorphism in both species. The contribution of size variation to the discrimination by shape was much more important between sexes (from 27% to 45%) than between species (from 0.7% to 7.1%). Nevertheless, in our data, size never could represent a way to reliably recognise the sex of an individual, even less its species. Geographical variation in head shape could only be explored for the C. orientis sample. No significant correlation of morphometric variation with geography could be detected, which would be consistent with gene flow between Thai provinces. The geometric morphometric approach of the flea head, when it incorporates head curves, is a promising tool for rapid, economical, and accurate species and sex identification. It is, therefore, a useful tool for future epidemiological and demographic studies.

Wing geometric morphometrics and DNA barcoding to distinguish three closely related species of Armigeres mosquitoes (Diptera: Culicidae) in Thailand.

Armigeres subalbatus, a mosquito species widely found in Thailand and other Asian countries, serves as a vector for filarial parasites, affecting both humans and animals. However, the surveillance of this vector is complicated because of its morphological similarity to two other species, Armigeres dohami and Armigeres kesseli. To differentiate these morphologically similar species, our study employed both wing geometric morphometrics (GM) and DNA barcoding, offering a comprehensive approach to accurately identify these closely related Armigeres species in Thailand. Our GM analyses based on shape demonstrated significant accuracy in differentiating Armigeres species. Specifically, the outline-based GM method focusing on the 3rd posterior cell exhibited an accuracy rate of 82.61%, closely followed by the landmark-based GM method with 81.54%. Both these GM techniques effectively distinguished Ar. subalbatus from Ar. dohami and Ar. kesseli. Regarding DNA barcoding, our investigation of pairwise intra- and interspecific divergences revealed a "barcoding gap". Furthermore, the results of species confirmation using both species delimitation methods including the automatic barcode gap discovery method (ABGD) and the Multi-rate Poisson tree process (mPTP) were consistent with those of morphological identification, sequence comparisons with the GenBank and Barcode of Life Data System (BOLD) databases, and the neighbor-joining tree construction. These consistent results emphasize the efficacy of DNA barcoding in the precise identification of Armigeres species.

Outline-based geometric morphometrics: Wing cell differences for mosquito vector classification in the Tanaosri mountain range, Thailand.

Recent studies have revealed taxonomic signals within the wing cells of certain mosquito species. In our study, wing cell differentiation among mosquito vectors from the Tanaosri mountain range in Thailand was evaluated using the outline-based geometric morphometric (GM) approach. Our focus was on four specific wing cells for GM analysis: the wing contour (external cell), the second submarginal cell (internal cell 1), the first posterior cell (internal cell 2), and the third posterior cell (internal cell 3). Before proceeding with the GM approach, the identity of seven mosquito genera and 21 species was confirmed using molecular techniques. Our validated classification tests demonstrated that the performance of mosquito species classification varies according to genus. Notably, three Aedes species exhibited the highest accuracy for both internal cell 2 and internal cell 3, each registering a score of 93.20 %. In the case of two Mansonia species, the wing contour displayed a remarkable accuracy of 98.57 %. Consequently, we suggest the use of the outline-based GM approach, particularly focusing on the wing contour, for differentiating Mansonia annulifera and Mansonia uniformis. In contrast, the highest accuracy for classifying Culex species was found in internal cell 1, at 75.51 %, highlighting the challenges due to similarities in wing cells within this genus. These findings provide a guideline for future applications of the outline-based GM approach, focusing on wing cells, as an alternative method to classify mosquito vector species.

Molecular characterization and genetic diversity of three Stomoxys flies S. bengalensis, S. calcitrans, and S. sitiens (Diptera: Muscidae) in Central Thailand.

Stomoxys flies (Diptera: Muscidae) are hematophagous ectoparasites of medical and veterinary importance. In this study, three Stomoxys species, i.e. S. bengalensis, S. calcitrans, and S. sitiens, were collected from three provinces in Central Thailand with the aim of estimating the genetic divergence between species, for species identification, as well as within species, for a genetic diversity study based on the cytochrome c oxidase subunit I (COI) gene. Our results showed that the average intraspecific genetic divergences of Stomoxys flies ranged from 0.11% in S. sitiens to 0.98% in S. calcitrans, whereas the average interspecific genetic divergences ranged from 5.24% between S. sitiens and S. bengalensis to 6.69% between S. calcitrans and S. bengalensis. In addition, there was no overlap between the intraspecific and interspecific genetic divergences. The COI sequence analysis revealed a high haplotype diversity and low nucleotide diversity, reflecting a rapid population expansion after past bottlenecks. Moreover, there was no significant difference (P > 0.05) in the pairwise population differentiation (Fst) among Stomoxys flies in Central Thailand, because of the lack of natural barriers, thus allowing genetic exchange between them. The monitoring of the haplotype network revealed that two lineages of S. calcitrans in Central Thailand were distributed in all study areas, including the Nakhon Pathom, Pathum Thani, and Saraburi Provinces. These findings may improve our understanding of the genetic patterns of these three Stomoxys flies, as well as the underlying biological mechanisms, which is knowledge that can be used for further effective control of these flies.

Molecular and morphometric differentiation of secondary filariasis vector Coquillettidia mosquitoes (Diptera: Culicidae) in Thailand.

Coquillettidia mosquitoes are important nuisance-biting pests and a vector of brugian filariasis in Thailand. However, comprehensive information about these mosquitoes remains unavailable such as molecular and morphometric differences among species. The lack of vector knowledge on Coquillettidia species could affect future disease control. This study aims to investigate differences in molecular variations based on mitochondrial cytochrome oxidase subunit I (COI) gene and wing geometric traits of three Coquillettidia species, namely Cq. crassipes, Cq. nigrosignata, and Cq. ochracea in Thailand. The results of molecular analyses revealed the differences among three Coquillettidia species. The genetic difference measure based on the Kimura two-parameter model among three Coquillettidia species showed low intraspecific distances (0%-3.05%) and large interspecific distances (10.10%-12.41%). The values of intra- and inter-genetic differences of three Coquillettidia species did not overlap which showed the existence of a barcoding gap indicating the efficiency of the identification based on the COI gene. As with molecular analysis, the landmark-based geometric morphometrics approach based on wing shape analysis indicated three distinct species groups which were supported by the high total performance score of cross-validated classification (97.16%). These results provide the first evidence of taxonomic signal based on molecular and wing geometric differences to support species identification and biological variations of Coquillettidia mosquitoes in Thailand for understanding these rare vector mosquitoes in depth and leading to effective further mosquito control.

Wing geometric morphometrics to distinguish and identify Haematobosca flies (Diptera: Muscidae) from Thailand.

The hematophagous flies of the genus Haematobosca Bezzi, 1907 (Diptera: Muscidae) are important ectoparasites in domestic animals and wildlife. Two species of this genus have been recorded in Thailand, viz., Haematobosca sanguinolenta (Austen, 1909) and Haematobosca aberrans (Pont, Duvallet & Changbunjong, 2020). They have a similar morphology and coexist in the same habitat. The correct species identification of these flies is extremely important for understanding disease epidemiology and developing effective control measures. Geometric morphometrics (GM) has been confirmed to be a useful tool for differentiating and identifying morphologically similar insect species. Therefore, GM was used to distinguish and identify H. sanguinolenta and H. aberrans in Thailand. Adult flies of both sexes were collected using Nzi traps, morphologically identified, and analyzed by landmark-based GM of the wing. Results showed that GM was highly effective in distinguishing the two Haematobosca species based on their wing shape, with an overall accuracy score of 99.3%. We also revealed that our study material could be used as reference data to identify new field specimens collected from other geographic locations. We propose that wing GM can be used as a supplement to conventional morphology identification, particularly for Haematobosca specimen that has been damaged or has lost its diagnostic characteristics due to specimen collection and processing in the field.

Species Discrimination of Stomoxys Flies S. bengalensis, S. calcitrans, and S. sitiens (Diptera: Muscidae) Using Wing Geometric Morphometrics.

The flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae), are regarded as pests of veterinary and medical importance. In Thailand, Stomoxys calcitrans (Linnaeus, 1758) is the most abundant species and is widely distributed throughout the country. This Stomoxys species can coexist with two other morphologically similar species: Stomoxys bengalensis Picard, 1908, and Stomoxys sitiens Rondani, 1873. Hence, discriminating using morphological characteristics is difficult, especially if the specimen is damaged or loses its diagnostic characteristics. In this study, we evaluated the effectiveness of the landmark-based geometric morphometric (GM) approach to discriminate among the three Stomoxys spp.: S. bengalensis, S. calcitrans, and S. sitiens. Left-wing images of S. bengalensis (n = 120), S. calcitrans (n = 150), and S. sitiens (n = 155) were used for the GM analyses. The results of the wing shape analyses revealed that the GM approach was highly effective for discriminating three Stomoxys, with high accuracy scores ranging from 93.75% to 100%. This study adds to the evidence that landmark-based GM is an excellent alternative approach for discriminating Stomoxys species.

Species Identification of the Major Japanese Encephalitis Vectors within the Culex vishnui Subgroup (Diptera: Culicidae) in Thailand Using Geometric Morphometrics and DNA Barcoding.

Japanese encephalitis (JE) is a viral infection of the brain caused by the Japanese encephalitis virus, which spreads globally, particularly in 24 countries of Southeast Asia and the Western Pacific region. In Thailand, the primary vectors of JE are Cx. pseudovishnui, Cx. tritaeniorhynchus, and Cx. vishnui of the Cx. vishnui subgroup. The morphologies of three mosquito species are extremely similar, making identification challenging. Thus, geometric morphometrics (GM) and DNA barcoding were applied for species identification. The results of cross-validation reclassification revealed that the GM technique based on wing shape analysis had relatively high potential for distinguishing Cx. pseudovishnui, Cx. tritaeniorhynchus, and Cx. vishnui (total performance = 88.34% of correctly assigned individuals). While the DNA barcoding yielded excellent results in identifying these Culex species based on the DNA barcode gap (average intraspecific genetic distance = 0.78% ± 0.39% and average interspecific genetic distance = 6.14% ± 0.79%). However, in the absence of the required facilities for DNA barcoding, GM techniques can be employed in conjunction with morphological methods to enhance the reliability of species identification. Based on the results of this study, our approach can help guide efforts to identify members of the Cx. vishnui subgroup, which will be useful for the effective vector control of JE in Thailand.

Evaluation of Modern Techniques for Species Identification of Lutzia Mosquitoes (Diptera: Culicidae) in Thailand: Geometric Morphometrics and DNA Barcoding.

There are four species of Lutzia mosquitoes in Thailand, including Lutzia chiangmaiensis, Lt. fuscana, Lt. halifaxii, and Lt. vorax. The accurate species identification of adult Lutzia mosquitoes based on morphological features requires many body parts, including the abdominal terga and wing. However, species identification is difficult in the case of damaged specimens when some of their morphological character is missing due to transit or gathering in the field. Thus, we evaluated the efficacy of the landmark-based geometric morphometric (GM) approach for the discrimination of Lutzia species in Thailand. In addition, DNA barcoding was also used in parallel with the GM approach to identify the species. Larvae of Lutzia were collected, raised into adults, and identified based on their morphological characteristics. The validated reclassification test results clearly demonstrated that wing shape resulted in a high level of success in identification (correct identifications ranged from 92.50% to 100%); however, based on the DNA barcoding analyses, our results showed that it was poorly effective in identifying Lt. fuscana and Lt. halifaxii based on an overlap between the intraspecific and interspecific divergence. Moreover, our survey results provide updates on the distribution of Lt. chiangmaiensis and Lt. vorax in Thailand. This research will help medical entomologists more efficiently identify mosquitoes in the genus Lutzia, resulting in more effective mosquito control and surveillance.

Genetic Diversity, Haplotype Relationships, and kdr Mutation of Malaria Anopheles Vectors in the Most Plasmodium knowlesi-Endemic Area of Thailand.

Plasmodium knowlesi, a malaria parasite that occurs naturally in long-tailed macaques, pig-tailed macaques, and banded leaf monkeys, is currently regarded as the fifth of the human malaria parasites. We aimed to investigate genetic diversity based on the cytochrome c oxidase subunit I (COI) gene, detect Plasmodium parasites, and screen for the voltage-gated sodium channel (VGSC)-mutation-mediated knockdown resistance (kdr) of Anopheles mosquitoes in Ranong province, which is the most P. knowlesi-endemic area in Thailand. One hundred and fourteen Anopheles females belonging to eight species, including An. baimaii (21.05%), An. minimus s.s. (20.17%), An. epiroticus (19.30%), An. jamesii (19.30%), An. maculatus s.s. (13.16%), An. barbirostris A3 (5.26%), An. sawadwongporni (0.88%), and An. aconitus (0.88%), were caught in three geographical regions of Ranong province. None of the Anopheles mosquitoes sampled in this study were infected with Plasmodium parasites. Based on the sequence analysis of COI sequences, An. epiroticus had the highest level of nucleotide diversity (0.012), followed by An. minimus (0.011). In contrast, An. maculatus (0.002) had the lowest level of nucleotide diversity. The Fu's Fs and Tajima's D values of the Anopheles species in Ranong were all negative, except the Tajima's D values of An. minimus (0.077). Screening of VGSC sequences showed no presence of the kdr mutation of Anopheles mosquitoes. Our results could be used to further select effective techniques for controlling Anopheles populations in Thailand's most P. knowlesi-endemic area.

Geometric morphometric and molecular techniques for discriminating among three cryptic species of the Anopheles barbirostris complex (diptera: culicidae) in Thailand.

Anopheles members of the Barbirostris complex are important vectors of malaria in Thailand. However, they are morphologically indistinguishable because they are closely related species. In this study, wing geometric morphometrics (GM) and DNA barcoding based on the cytochrome c oxidase subunit 1 (C O I) gene were applied to differentiate cryptic species of the Barbirostris complex in Thailand. Three cryptic species of the Barbirostris complex, Anopheles dissidens (19.44%), Anopheles saeungae (24.54%), and Anopheles wejchoochotei (56.02%) were initially identified using the multiplex polymerase chain reaction assay. DNA barcoding analyses showed low intraspecific distances (range, 0.27%-0.63%) and high interspecific distances (range, 1.92%-3.68%), consistent with the phylogenetic analyses that showed clear species groups. While wing size and shape analyses based on landmark-based GM indicated differences between three species (p < 0.05). The cross-validated reclassification revealed that the overall efficacy of wing size analysis for species identification of the Barbirostris complex was less than the wing shape analysis (56.43% vs. 74.29% total performance). Therefore, this study's results are guidelines for applying modern techniques to identify members within the Barbirostris complex, which are still difficult to distinguish by morphology-based identification and contribute to further appropriate malaria control.

Intraspecific variation in wing geometry among Tabanus rubidus (Diptera: Tabanidae) populations in Thailand.

Tabanus rubidus (Wiedemann, 1821) (Diptera: Tabanidae) is a hematophagous insect of veterinary and medical importance and is the predominant Tabanus spp. in Thailand. It is a potential mechanical vector of Trypanosoma evansi, which causes surra in domestic and wild animals. Wing geometric morphometrics is widely used as morphological markers for species identification and to assess the insect population structure. Herein, we investigated the intraspecific variation in wing geometry among T. rubidus populations in Thailand using landmark-based geometric morphometric analysis. Tabanus rubidus females were collected from five populations in four geographical regions in Thailand. The left wings of 240 specimens were removed and digitized using 22 landmarks for analysis. While wing size variations were found between some populations, wing shape variations were detected in all. These intraspecific variations in T. rubidus populations indicate an adaptive response to the local environmental conditions.

Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand.

The correct identification of mosquito species is important for effective mosquito vector control. However, the standard morphological identification of mosquito species based on the available keys is not easy with specimens in the field due to missing or damaged morphological features during mosquito collections, often leading to the misidentification of morphologically indistinguishable. To resolve this problem, we collected mosquito species across Thailand to gather genetic information, and evaluated the DNA barcoding efficacy for mosquito species identification in Thailand. A total of 310 mosquito samples, representing 73 mosquito species, were amplified using mitochondrial cytochrome c oxidase subunit I (COI) primers. The average maximum intraspecific genetic variation of the 73 mosquito species was 1% ranged from 0-5.7%. While, average minimum interspecific genetic variation (the distance to the nearest neighbour) of the 73 mosquito species was 7% ranged from 0.3-12.9%. The identification of success rates based on the "Best Match," "Best Close Match," and "All Species Barcodes" methods were 97.7%, 91.6%, and 81%, respectively. Phylogenetic analyses of Anopheles COI sequences demonstrated a clear separation between almost all species (except for those between An. baimaii and An. dirus), with high bootstrap support values (97%-99%). Furthermore, phylogenetic analyses revealed potential sibling species of An. annularis, An. tessellatus, and An. subpictus in Thailand. Our results indicated that DNA barcoding is an effective molecular approach for the accurate identification of mosquitoes in Thailand.