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.

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.

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.

Effects of Coleus amboinicus L. Essential Oil and Ethanolic Extracts on Planktonic Cells and Biofilm Formation of Microsporum canis Isolated from Feline Dermatophytosis.

Microsporum canis is an important zoonotic fungus that causes dermatophytosis in domestic animals and their owners. Domestic cats are the primary reservoir for M. canis. Antifungal drugs frequently produce adverse effects on the host animal, increasing the demand for novel alternative treatments derived from nature. We evaluated the antifungal activity of Coleus amboinicus essential oil (CEO) and ethanolic extracts (CEE) against M. canis in planktonic and biofilm growth. Twelve clinical isolates of M. canis were identified in feline dermatophyte samples. Using GC-MS, 18 compounds were identified in CEO, with carvacrol being the major constituent. HPLC analysis of CEE revealed that it contained rosmarinic acid, apigenin, and caffeic acid. The planktonic growth of all M. canis isolates was inhibited by C. amboinicus extracts. The minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC50) was 128 µg/mL (32-256 µg/mL) for both CEO and CEE. The MIC90 values of CEO and CEE were 128 and 256 µg/mL, respectively. CEO at MIC (128 µg/mL) and 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of weak, moderate, and strong biofilm-producing M. canis. CEE at 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of all isolates. Overall, C. amboinicus extracts inhibited planktonic growth and exhibited a significant antibiofilm effect against M. canis. Thus, C. amboinicus is a potential source of natural antifungal compounds.

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.

Contact and Fumigant Activities of Citrus aurantium Essential Oil against the Stable Fly Stomoxys calcitrans (Diptera: Muscidae).

The stable fly, Stomoxys calcitrans (L.), is a cosmopolitan hematophagous fly of medical and veterinary importance. It is widely considered a major livestock pest that can cause significant economic losses. This study aimed to evaluate the insecticidal activity of Citrus aurantium (L.) essential oil against S. calcitrans based on contact and fumigant toxicity tests. Chemical analysis by gas chromatography-mass spectrometry of the essential oil showed the dominance (93.79%) of limonene in the total essential oil composition. Furthermore, the insecticidal test results showed that the mortality of flies increased with concentration and time within 24 h of exposure. In the contact toxicity test, the median lethal dose was 105.88 µg/fly, while the 90% lethal dose was 499.25 µg/fly. As for the fumigant toxicity test, the median lethal concentration was 13.06 mg/L air, and the 90% lethal concentration was 43.13 mg/L air. These results indicate that C. aurantium essential oil exhibits insecticidal activity against S. calcitrans. Therefore, it can be used as an alternative to synthetic insecticides for achieving stable fly control.

Insecticidal Activity of Plectranthus amboinicus Essential Oil against the Stable Fly Stomoxys calcitrans (Diptera: Muscidae) and the Horse Fly Tabanus megalops (Diptera: Tabanidae).

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), and the horse fly, Tabanus megalops (Diptera: Tabanidae), are important ectoparasites of livestock in Thailand. These species affect animal health and cause economic losses. This study investigated the insecticidal activity of Plectranthus amboinicus essential oil against S. calcitrans and T. megalops through contact and fumigant toxicity tests and evaluated the effects of the essential oil on these flies through histopathological and scanning electron microscopic (SEM) studies. The results of the contact toxicity test indicated that the median lethal dose against S. calcitrans and T. megalops was 12.05 and 131.41 µg/fly, and the 90% lethal dose was 45.53 and 200.62 µg/fly, respectively. The results of the fumigant toxicity test showed that the median lethal concentration against S. calcitrans and T. megalops was 1.34 and 7.12 mg/L air, and the 90% lethal concentration was 4.39 and 30.37 mg/L air, respectively. Histopathology revealed neuronal degeneration in the brain of S. calcitrans and interstitial neuronal edema of the brain and ovarian necrosis in T. megalops. No external morphological changes were observed via SEM. Given its insecticidal properties against S. calcitrans and T. megalops, P. amboinicus essential oil could be developed into a natural insecticide to control these fly species.

Landmark Data to Distinguish and Identify Morphologically Close Tabanus spp. (Diptera: Tabanidae).

Tabanus spp., also known as horse flies (Diptera: Tabanidae), are important vectors of several animal pathogens. Adult females of Tabanus megalops and Tabanus striatus, which are members of the T. striatus complex, are morphologically similar and hence difficult to distinguish using morphological characteristics. In addition, molecular identification by DNA barcoding is also unable to distinguish these species. These two species can occur sympatrically with Tabanus rubidus, which is morphologically similar to T. megalops and T. striatus. Wing geometric morphometrics has been widely used in various insects to distinguish morphologically similar species. This study explored the effectiveness of landmark-based geometrics at distinguishing and identifying T. megalops, T. rubidus, and T. striatus in Thailand. Specimens were collected from different geographical regions of Thailand, and only unambiguously identified specimens were used for geometric morphometric analyses. Left wings of females of T. megalops (n = 160), T. rubidus (n = 165), and T. striatus (n = 85) were photographed, and 22 wing landmarks were used for the analysis. Wing shape was able to distinguish among species with high accuracy scores, ranging from 94.38% to 99.39%. We showed that morphologically very close species of Tabanus can be reliably distinguished by the geometry of their wing venation, and we showed how our experimental material could be used as a reference to tentatively identify new field collected specimens.

The pests of a pest: A systematic review of ectoparasitic fauna among synanthropic rodents in the 21st century with meta-analysis.

Synanthropic rodents are important urban pests that frequently carry hematophagous ectoparasites. These blood-sucking pests are capable of transmitting epizootic and zoonotic pathogens by landing on one host after feeding on an infected animal. This study aimed to estimate the prevalence of ectoparasites carried by synanthropic rodents and discuss the pathogens that are associated with these external parasites. We searched relevant literatures using predefined criteria in the following databases: EMBASE, PUBMED, Web of Science and Scopus from January 2000 to June 2020. Quality of studies was evaluated using Newcastle-Ottawa scale (NOS). Of 35 included studies from 15 countries in Africa, America, Asia, Europe and Oceania, black rats (R. rattus), brown rats (R. norvegicus), pacific rats (R. exulans) and house mice (Mus musculus) were common synanthropic rodents. Mites (Mesostigmata, Sarcoptiformes and Trombidiformes) were the most prevalent (42.6%, 95% CI 26-59.2), followed by ticks (Ixodida) (21.5%, 95% CI 10.5-32.6), lice (Phthiraptera) (17.8%, 95% CI 7.7-27.9) and fleas (Siphonaptera) (14.1%, 95% CI 10.1-18.1). Heterogeneity (I2>96%) across studies was statistically significant. The ectoparasitic fauna was shared considerably by different urban rodent species and appeared to be more diverse in R. rattus and R. norvegicus. Nonetheless, pathogens carried by these ectoparasites were rarely investigated. In conclusion, ectoparasites are ubiquitous in urban-dwelling rodents but our understanding of the epidemiology and the associated pathogens of these parasites remains limited. Further studies are warranted to unravel the pathogen landscape found in rodent-associated ectoparasites.

New records and DNA barcoding of deer flies, Chrysops (Diptera: Tabanidae) in Thailand.

Chrysops spp. or deer flies (Diptera: Tabanidae) are hematophagous flies of medical and veterinary importance and some species are important vectors of Trypanosoma evansi, the causative agent of surra in Thailand. However, data regarding deer fly species and their molecular identification are limited. Accurate species identification will indicate the appropriate control measures. In this study, an entomological survey of deer flies from different sites in Thailand between May 2018 and June 2019 were conducted. In addition, mitochondrial cytochrome oxidase subunit I (COI) barcoding region was used for species identification. A total of 82 females were collected and 6 species were identified. Of these, three species are new records for Thailand: C. designatus, C. fuscomarginalis and C. vanderwulpi bringing the species total found in Thailand to nine. The COI sequences revealed an intraspecific divergence of 0.0%-2.65% and an interspecific divergence of 7.03%-13.47%. Phylogenetic analysis showed that all deer fly species were clearly separated into distinct clusters according to morphologically identified species. These results indicated that COI barcodes were capable in discriminating between deer fly species on the basis of the barcoding gap and phylogenetic analysis. Therefore, DNA barcoding is a valuable tool for species identification of deer flies in Thailand.

Species identification of horse flies (Diptera: Tabanidae) in Thailand using DNA barcoding.

Horse flies (Diptera: Tabanidae) are of medical and veterinary importance because they are known to transmit pathogens. Approximately 80 species of horse flies have been reported in Thailand. Monitoring the distribution of horse fly species is important to control the spread of diseases transmitted by them. Currently, the species identification of horse flies is based on their morphology; this requires considerable skills and taxonomic expertise, and it may be difficult to identify morphologically similar species. DNA-based identification methods are increasingly being developed for rapid and accurate identification of various insect species. In this study, we used mitochondrial cytochrome oxidase subunit I (COI) for species identification of horse flies in Thailand. A 658 bp fragment of COI was amplified from 145 adult horse flies belonging to 48 morphologically distinct species and sequenced. Sequence analysis revealed an intraspecific divergence of 0.0%-4.4% and an interspecific divergence of 0.0%-16.2%. Our results showed that COI barcodes were effective in discriminating a majority of horse flies in Thailand on the basis of the barcoding gap and phylogenetic analyses. However, COI barcodes were unable to distinguish among members of the Tabanus striatus complex and some species within the T. ceylonicus group.

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.

Molecular epidemiology of bovine Babesia spp. and Theileria orientalis parasites in beef cattle from northern and northeastern Thailand.

Beef cattle production represents the largest cattle population in Thailand. Their productivity is constrained by tick-borne diseases such as babesiosis and theileriosis. In this study, we determined the prevalence of Babesia bigemina, Babesia bovis and Theileria orientalis using polymerase chain reaction (PCR). The genetic markers that were used for detection of the above parasites were sequenced to determine identities and similarity for Babesia spp. and genetic diversity of T. orientalis. Furthermore the risk factors for the occurrence of the above protozoan parasites in beef cattle from northern and northeastern parts of Thailand were assessed. A total of 329 blood samples were collected from beef cattle in 6 provinces. The study revealed that T. orientalis was the most prevalent (30.1%) parasite in beef cattle followed by B. bigemina (13.1%) and B. bovis (5.5%). Overall, 78.7% of the cattle screened were infected with at least one of the above parasites. Co-infection with Babesia spp. and T. orientalis was 30.1%. B. bigemina and T. orientalis were the most prevalent (15.1%) co-infection although triple infection with the three parasites was observed in 3.0% of the samples. Sequencing analysis revealed that B. bigemina RAP1 gene and B. bovis SBP2 gene were conserved among the parasites from different cattle samples. Phylogenetic analysis showed that the T. orientalis MPSP gene from parasites isolated from cattle in north and northeast Thailand was classified into types 5 and 7 as reported previously. Lack of tick control program was the universal risk factor of the occurrence of Babesia spp. and T. orientalis infection in beef cattle in northern and northeastern Thailand. We therefore recommend training of farmers on appropriate tick control strategies and further research on potential vectors for T. orientalis and elucidate the effect of co-infection with Babesia spp. on the pathogenicity of T. orientalis infection on beef in northern and northeastern Thailand.

Seasonal abundance and potential of Japanese encephalitis virus infection in mosquitoes at the nesting colony of ardeid birds, Thailand.

OBJECTIVE: To investigate the abundance and seasonal dynamics of mosquitoes, and to detect Japanese encephalitis virus (JEV) in these mosquitoes at the nesting colony of ardeid birds. METHODS: Mosquitoes were collected bimonthly from July 2009 to May 2010 by Centers for Disease Control. Light traps and dry ice, as a source of CO2, were employed to attract mosquitoes. Mosquitoes were first identified, pooled into groups of upto 50 mosquitoes by species, and tested for JEV infection by viral isolation and reverse transcriptase polymerase chain reaction. RESULTS: A total of 20 370 mosquitoes comprising 14 species in five genera were collected. The five most abundant mosquito species collected were Culex tritaeniorhynchus (95.46%), Culex vishnui (2.68%), Culex gelidus (0.72%), Anopheles peditaeniatus (0.58%) and Culex quinquefasciatus (0.22%). Mosquito peak densities were observed in July. All of 416 mosquito pools were negative for JEV. CONCLUSIONS: This study provides new information about mosquito species and status of JEV infection in mosquitoes in Thailand. Further study should be done to continue a close survey for the presence of this virus in the ardeid birds.

Distribution and abundance of Stomoxyini flies (Diptera: Muscidae) in Thailand.

Stomoxyini flies (Diptera: Muscidae) include species of parasitic flies of medical and veterinary importance. The adult flies feed on the blood of mammals and may transmit several parasites and pathogens. We conducted an entomological survey of Stomoxyini flies from different sites in Thailand. Stomoxyini flies were collected at four major types of sites: zoos, livestock farms, wildlife conservation areas and a national park using vavoua traps between November 2010 and April 2011. A total of 3,314 Stomoxyini flies belonging to the genera Stomoxys, Haematobosca, Haematostoma and Haematobia were collected. Eight species were identified: S. calcitrans (46.6%), S. uruma (26.8%), S. pulla (4.3%), S. indicus (0.7%), S. sitiens (0.1%), H. sanguinolenta (11.2 %), H. austeni (0.5%) and H. irritans exigua (9.8%). The diversity of Stomoxyini flies in the livestock farms was higher than the other sites. Altitude correlated with the number of flies. This study provides information that may be useful for Stomoxyini flies control.

Occurrence of ectoparasites on rodents in Sukhothai Province, northern Thailand.

A survey of ectoparasites on rodents was carried out bimonthly from April 2008 to March 2009 in 3 districts of Sukhothai Province, northern Thailand. A total of 130 rodents comprising 8 species of hosts were captured and examined for ectoparasites. The hosts examined were Bandicota indica, Bandicota savilei, Rattus losea, Rattus rattus, Rattus exulans, Rattus norvegicus, Menetes berdmorei and Tamiops mcclellandii. Ninety-seven ectoparasites were collected: 1 species of tick (Hemaphysalis bandicota), 2 species of mites (Laelaps nuttali and Laelaps echidninus), and 1 species of flea (Xenopsylla cheopis) were identified. The infestation rates by ticks, mites and fleas on the rodents were 0.77, 5.38 and 6.15%, respectively. Monitoring the rodent population and their ectoparasites is important for future planning of prevention and control of zoonotic diseases in the area.