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.

Integrating wing morphometrics and mitochondrial DNA analysis to assess the filaria vector Mansonia uniformis (Diptera: Culicidae) populations in Thailand.

Mansonia uniformis (Diptera: Culicidae) is recognized as a vector of Brugia malayi and has been reported to transmit Wuchereria bancrofti, both causing lymphatic filariasis in humans. This study employed geometric morphometrics (GM) to investigate wing shape variation and analyzed genetic diversity through cytochrome c oxidase subunit 1 (COI) gene analyses in Ma. uniformis populations across Thailand. Wing GM analyses indicated significant differences in wing shape based on Mahalanobis distances among nearly all population pairs (p < 0.05), with no significant correlation between wing shape and geographic distance (r = 0.210, p > 0.05). Genetic analyses identified 63 haplotypes and 49 polymorphic sites, with the overall population exhibiting a nucleotide diversity of 0.006 (± 0.001) and a haplotype diversity of 0.912 (± 0.017). Deviations from neutrality, as indicated by Tajima's D and Fu's FS tests for the overall Ma. uniformis populations in Thailand, were statistically significant and negative, suggesting population expansion (both p < 0.05). Analysis of molecular variance revealed no significant genetic structure when all populations were categorized based on collection sites and geographic regions. However, significant differences in FST values were observed between some populations. These findings enhance our understanding of the geographical and genetic factors influencing Ma. uniformis populations, which are crucial for developing effective control strategies in Thailand.

Encapsulation of Eucalyptus largiflorens Essential Oil by Mesoporous Silicates for Effective Control of the Cowpea Weevil, Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae).

Although the use of synthetic chemicals is the principal method for insect pest management, their widespread application has led to numerous side effects, including environmental pollution and threats to human and animal health. Plant essential oils have been introduced as promising natural substitutes for synthetic insecticides. However, high volatility and/or low durability are the main limiting factors for essential oil application for control of insect pests. Accordingly, along with an evaluation of the fumigant toxicity of Eucalyptus largiflorens essential oil against the cowpea weevil, Callosobruchus maculatus, essential oil was nanoencapsulated by two mesoporous silicates, MCM-41 and zeolite 3A, to enhance fumigant persistence and toxicity. The chemical profile of essential oil was also analyzed through gas chromatographic-mass spectrometry. E. largiflorens essential oil showed significant concentration-dependent toxicity against insect pests; a concentration of 5.16 µL/L resulted in 100% mortality after 48 h. The toxicity of essential oil could be attributed to the presence of various insecticidal terpenes, such as spathulenol (15.6%), cryptone (7.0%), and 1,8-cineole (5.8%). Fumigant persistence was increased from 6 days to 19 and 17 days for pure and capsulated essential oil with MCM-41 and Zeolite 3A, respectively. The insect mortality also increased from 99 insects in pure essential oil to 178 and 180 insects in MCM-41 and Zeolite 3A encapsulated formulations, respectively. Therefore, the encapsulation of E. largiflorens essential oil by MCM- 41 and Zeolite 3A is a beneficial method for enhancing its persistence and toxicity against C. maculatus.

Molecular detection and genetic diversity of bovine Babesia spp., Theileria orientalis, and Anaplasma marginale in beef cattle in Thailand.

Babesia spp., Theileria orientalis, and Anaplasma marginale are significant tick-borne pathogens that affect the health and productivity of cattle in tropical and subtropical areas. In this study, we used PCR to detect the presence of Babesia bovis, Babesia bigemina, and T. orientalis in 279 beef cattle from Western Thailand and A. marginale in 608 beef cattle from the north, northeastern, and western regions. The PCRs were performed using species-specific primers based on the B. bovis spherical body protein 2 (BboSBP2), B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. orientalis major piroplasm surface protein (ToMPSP), and A. marginale major surface protein 4 (AmMSP4) genes. To determine the genetic diversity of the above parasites, amplicons of B. bovis and B. bigemina ITS1-5.8s rRNA gene-ITS2 regions (B. bovis ITS, B. bigemina ITS), ToMPSP, and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the prevalence of B. bovis, B. bigemina, T. orientalis, and A. marginale in the Western region was 11.1, 12.5, 7.8, and 39.1 %, respectively. Coinfections of two or three parasites were observed in 17.9 % of the animals sampled. The study revealed that the prevalence of A. marginale in the western region was higher than in the north and northeastern regions (7 %). Sequence analysis showed the BboSBP2 gene to be more conserved than B. bovis ITS in the different isolates and, similarly, the BbiRAP-1a was more conserved than B. bigemina ITS. In the phylogenetic analysis, T. orientalis MPSP sequences were classified into types 3, 5, and 7 as previously reported. A. marginale MSP4 gene sequences shared high identity and similarity with each other and clustered with isolates from other countries. This study provides information on the prevalence and genetic diversity of tick-borne pathogens in beef cattle and highlights the need for effective strategies to control these pathogens in Thailand.

Landmark and outline-based geometric morphometrics analysis of three Stomoxys flies (Diptera: Muscidae).

Adult flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae), especially S. pullus Austen, 1909, S. uruma Shinonaga et Kano, 1966 and S. indicus Picard, 1908, are morphologically similar and sometimes difficult to distinguish when using external morphological characteristics. These species may act as vectors and/or potential vectors of many pathogens (virus, bacteria and protozoa). Their correct identification is important to target the vectors involved in the transmission of the pathogens and also helps in the fly control program.The aim of the present study was to distinguish three species which are difficult to separate using traditional diagnostic characters for species of Stomoxys such as colour patterns and body proportions. Modern morphometrics, both landmark and outline-based, was used to access wing geometry of S. pullus, S. uruma and S. indicus. A total of 198 and 190 wing pictures were analysed for landmark- and outline-based approaches, respectively. Wing shape was able to separate species and sexes of the three Stomoxys flies with highly significant difference of Mahalanobis distances. The cross-validated classification scores ranged from 76% to 100% for landmark and 77% to 96% for outline-based morphometrics. The geometry of wing features appears to be a very useful, low-cost tool to distinguish among the vectors S. pullus, S. uruma and S. indicus.

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.

Evaluation of the wing cell contour to distinguish between Stomoxys bengalensis and Stomoxys sitiens (Diptera: Muscidae) using outline-based morphometrics.

The blood-sucking flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae) are significant ectoparasites that can cause irritation and transmit pathogens to both animals and humans. Within the genus Stomoxys, two species, Stomoxys bengalensis and Stomoxys sitiens, have similar morphology and coexist in the same habitat. Accurate species identification of these flies is crucial for understanding disease vectors and implementing effective control measures. In this study, we assessed the effectiveness of outline-based geometric morphometrics (GM) by analyzing the wing cell contour of the first posterior cell (R5) to distinguish between species and sexes of S. bengalensis and S. sitiens. Our results demonstrate that the outline-based GM method is highly effective in distinguishing between species and sexes of these flies based on contour shape, with accuracy scores ranging from 90.0% to 97.5%. Therefore, outline-based GM emerges as a promising alternative to landmark-based GM or as a supplementary tool in conjunction with traditional morphology-based methods for species identification.

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.

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.

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.

Spatio-temporal patterns of stomoxyine flies (Diptera: Muscidae) in a forested area of Thailand.

Understanding the distribution patterns of vector populations is crucial for comprehending the dynamics of vector-borne diseases. However, data on vector composition and abundance in areas of forest and wildlife-human interface in Thailand remain limited. This research aimed to investigate the spatio-temporal distribution and species diversity of stomoxyine flies (Diptera: Muscidae) in Salakpra Wildlife Sanctuary, Thailand's first wildlife sanctuary. A longitudinal entomological survey was conducted monthly from May 2022 to April 2023 in four habitats: core forest, grassland forest, a wildlife breeding center, and a local cattle farm. A total of 11,256 stomoxyine flies from four genera were captured. Based on morphological keys, nine species of stomoxyine flies were identified: Stomoxys pullus (29.63%), Stomoxys calcitrans (19.65%), Stomoxys indicus (16.09%), Haematostoma austeni (14.23%), Haematobia irritans exigua (8.22%), Haematobosca sanguinolenta (7.96%), Stomoxys uruma (1.98%), Stomoxys sitiens (1.75%), and Stomoxys bengalensis (0.49%). Heterogeneous variations in abundance across months and habitats were observed, in which abundance increased in the rainy season (June-October), exhibiting bimodal peaks at seasonal transitions. Human-disturbed areas, such as the cattle farm, exhibited the highest density and species diversity of stomoxyine flies. In contrast, areas with minimal human disturbance, like core forest, had low diversity and density but supported unique species, like the abundant Haematostoma austeni, which had minor populations in other types of habitats. The results of this study can be integrated into epidemiological models and lay the groundwork for more comprehensive research on vector-borne diseases at the wildlife-livestock interface to mitigate transmission risks and preserve biodiversity.

Title: Schémas spatio-temporels des stomoxes (Diptera : Muscidae) dans une zone forestière de Thaïlande. Abstract: Comprendre les schémas de distribution des populations de vecteurs est essentiel pour comprendre la dynamique des maladies à transmission vectorielle. Cependant, les données sur la composition et l'abondance des vecteurs dans les zones de forêt et d'interface faune-humain en Thaïlande restent limitées. Cette recherche visait à étudier la distribution spatio-temporelle et la diversité des espèces de stomoxes (Diptera : Muscidae) dans le sanctuaire faunique de Salakpra, le premier sanctuaire faunique de Thaïlande. Une enquête entomologique longitudinale a été menée mensuellement de mai 2022 à avril 2023 dans quatre habitats : forêt centrale, forêt de prairie, un centre d'élevage d'animaux sauvages et une ferme d'élevage locale. Au total, 11 256 stomoxes de quatre genres ont été capturés. Sur la base des clés morphologiques, neuf espèces de stomoxes ont été identifiées : Stomoxys pullus (29,63 %), Stomoxys calcitrans (19,65 %), Stomoxys indicus (16,09 %), Haematostoma austeni (14,23 %), Haematobia irritans exigua (8,22 %), Haematobosca sanguinolenta (7,96 %), Stomoxys uruma (1,98 %), Stomoxys sitiens (1,75 %) et Stomoxys bengalensis (0,49 %). Des variations hétérogènes de l'abondance selon les mois et les habitats ont été observées, l'abondance augmentant pendant la saison des pluies (juin à octobre), présentant des pics bimodaux aux transitions saisonnières. Les zones perturbées par l'homme, comme la ferme d'élevage, présentaient la plus forte densité et la plus grande diversité d'espèces de stomoxes. En revanche, les zones peu perturbées par l'homme, comme la forêt centrale, présentaient une faible diversité et une faible densité, mais abritaient des espèces uniques, comme l'abondant Haematostoma austeni, dont les populations étaient mineures dans d'autres types d'habitats. Les résultats de cette étude peuvent être intégrés dans des modèles épidémiologiques et posent les bases d'une recherche plus complète sur les maladies à transmission vectorielle à l'interface faune-bétail afin d'atténuer les risques de transmission et de préserver la biodiversité.

Genetic and morphometric differentiation between two morphs of Haematobosca sanguinolenta (Diptera: Muscidae) from Thailand.

Haematobosca is a genus of biting fly within the subfamily Stomoxyinae of the family Muscidae. It is currently recognized to include 16 species worldwide. These species, acting as ectoparasites, are considered to have significant importance in the veterinary and medical fields. To address the color polymorphism related to the genus Haematobosca in Thailand, herein, we focused on the normal (legs mainly black) and yellow (legs mainly yellow) morphs of Haematobosca sanguinolenta and examined them for genetic differences using three molecular markers: the cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) genes from the mitochondrial genome as well as the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA. In addition, we analyzed wing differences between the two morphs using geometric morphometrics (GM). The genetic divergences between the two morphs showed that cytb gene showed the greatest divergence, for which the average distance was 5.6%. This was followed by the combination of cox1-cytb-ITS2, exhibiting an average divergence of 4.5%, ITS2 with a divergence of 4.1%, and finally cox1, showing the lowest divergence of 3.5%. Phylogenetic analyses distinctly separated the two morphs of H. sanguinolenta; this separation was supported by high bootstrap values (97-100%). These results were further corroborated by three species delimitation methods, i.e. assemble species by automatic partitioning (ASAP), automated barcode gap discovery (ABGD), and Poisson tree processes (PTP), all of which suggested that the two morphs likely represent separate species. In addition, a GM study identified a statistically significant difference in wing shape between the two morphs of H. sanguinolenta (P < 0.05). This combination of genetic and morphometric results strongly supports the existence of two distinct species within H. sanguinolenta in Thailand.

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.

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.

Spent Coffee Grounds and Novaluron Are Toxic to Aedes aegypti (Diptera: Culicidae) Larvae.

Aedes aegypti (Diptera: Culicidae) is a vector for mosquito-borne diseases worldwide. Insecticide resistance is a major concern in controlling this mosquito. We investigated the chemical compounds in wet and dry spent coffee grounds (wSCGs and dSCGs) and evaluated the efficacy of dSCGs, wSCGs, and novaluron on the mortality and adult emergence inhibition of Ae. aegypti. We found higher concentrations of chemical compounds in wSCGs than in dSCGs. The wSCGs and dSCGs both contained total phenolic compounds, total flavonoid compounds, caffeic acid, coumaric acid, protocatechuic acid, and vanillic acid. Complete mortality was observed after 48 h of exposure to 50 g/L wSCGs, while similar mortality was found after 120 h of exposure to 10 µg/L of novaluron. The sublethal dose was a concentration of wSCGs (5 g/L) and novaluron (0.01, 0.1, and 1 µg/L) combined that resulted in a larval mortality lower than twenty percent (at 72 h) to determine their synergistic effects. The death rate of larvae exposed in sublethal combination of wSCGs and novaluron was significantly higher than that of its stand-alone. The findings indicate that the combination of wSCGs and novaluron at sublethal concentrations had synergistic effects on the mortality of Ae. aegypti larvae and could be applied as an alternative control measure.

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.

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.

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.

Effects of Piper nigrum L. Fruit Essential Oil Toxicity against Stable Fly (Diptera: Muscidae).

The efficacy of Piper nigrum L. fruit essential oil (EO) against Stomoxys calcitrans (stable fly), a blood-feeding fly distributed worldwide, was investigated. This study aimed to evaluate the insecticidal activity of EO based on contact and fumigant toxicity tests. Chemical analysis of the EO using gas chromatography-mass spectrometry revealed that sabinene (24.41%), limonene (23.80%), ß-caryophyllene (18.52%), and α-pinene (10.59%) were the major components. The results demonstrated that fly mortality increased with increasing EO concentration and time during the first 24 h of exposure. The median lethal dose was 78.37 µg/fly for contact toxicity, while the 90% lethal dose was 556.28 µg/fly. The median lethal concentration during fumigant toxicity testing was 13.72 mg/L air, and the 90% lethal concentration was 45.63 mg/L air. Our findings suggested that essential oil extracted from P. nigrum fruit could be a potential natural insecticidal agent for control of stable fly. To examine the insecticidal properties of P. nigrum fruit EO, further field trials and investigation into the efficacy of nano-formulations are required.