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.

An Integrated Pest Management Strategy Approach for the Management of the Stable Fly Stomoxys calcitrans (Diptera: Muscidae).

Stable flies, Stomoxys calcitrans, stand as formidable pests with a global impact, inflicting significant economic losses on the livestock sector. Larval development occurs in diverse substrates, including decomposing plant material and manure, while emerged adults pose a threat through blood-feeding on both animals and humans. Conventional chemical control methods, predominantly reliant on insecticides, not only pose environmental risks but also face challenges of resistance among stable fly populations. To address this pressing issue, we propose an integrated pest management (IPM) strategy for stable fly control. This approach involved a combination of sanitary-cultural practices, animal protection, the release of natural enemies targeting immature stages, and a specialized trapping system for adults. The Stomoxycc® trap, designed for mass trapping of adult Stomoxys, was employed alongside the release of the predatory mite Macrocheles robustulus and two wasp parasitoids, Spalangia cameroni and Muscidifurax raptor (under the commercial brands Biomite® and Biowasp®) on animal bedding as a key component of this IPM strategy. The implementation of this initiative has been undertaken at a significant sanctuary for donkeys and mules in western Spain. In this publication, we present the application and results of the IPM strategy utilized and provide insights into its use as a sustainable and environmentally friendly option for controlling stable fly populations.

Wing Phenotypic Variation among Stomoxys calcitrans (Diptera: Muscidae) Populations in Thailand.

Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae) is a cosmopolitan hematophagous ectoparasite of veterinary and medical importance. It is an important mechanical vector of several animal pathogens and can cause significant economic losses. However, the morphological variation of this species remains unknown. This study aimed to investigate the phenotypic variation in the wing size and shape of S. calcitrans populations in Thailand based on a landmark-based geometric morphometric approach. Specimens were collected from five populations in five geographical regions in Thailand. A total of 490 left wings of S. calcitrans (245 female and 245 male individuals) were used for geometric morphometric analysis. Wing size differences were detected between some populations of S. calcitrans, whereas wing shape differences were found among populations. Therefore, the phenotypic variation in S. calcitrans populations indicated that these populations are adaptive responses to local environmental pressures, suggesting the presence of phenotypic plasticity in this species.

Comparison of blue cotton and blue polyester fabrics to attract hematophagous flies in cattle farms in Thailand.

Tabanids and stable flies are important nuisances to livestock and sometimes humans. Nzi, Vavoua, and Biconical traps or insecticide-impregnated blue screens are commonly used to attract and catch these flies. Such devices are made of a specific cotton or cotton-polyester phthalogen blue fabric acting as a visual attractant. However, the cost of cotton fabrics is high, and they are no longer available due to toxic dyes. The present study compared four blue polyester fabrics produced in Thailand with a reference blue cotton-polyester fabric made in France by TDV® to attract hematophagous flies. Vavoua traps and blue screens covered with a sticky film made with the five different blue fabrics were compared. The TDV® had the highest trapping scores; however, there was no significant difference between TDV® and some polyester fabrics. Among the tested polyester fabrics, CR Solon No.41 was nearly as effective as the TDV® in attracting biting flies. The mean attractivity indices of CR Solon No.41, NS No.1469, Globe 2000 No.21, Globe 2000 No.34 were 0.86, 0.79, 0.69, and 0.39, respectively. Thus, we recommend that CR Solon No.41 would be the appropriate fabric for the further development of low-cost and optimized screens and traps in Thailand and other countries.

A new species of Haematobosca Bezzi (Diptera: Muscidae) from Thailand.

A new species of biting-fly from northern Thailand is described as Haematobosca aberrans sp. nov. (Muscidae). It differs from all known species of Haematobosca by the absence of the anterior katepisternal seta.

Attractiveness and Specificity of Different Polyethylene Blue Screens on Stomoxys calcitrans (Diptera: Muscidae).

Stomoxys calcitrans is considered as a major pest of livestock worldwide. Insecticides have been extensively used to control this pest but resistance to these chemical compounds is now reported in many countries. Therefore, a more sustainable and efficient control is needed. Seven different types of blue screens, with reflectances around 460 nm, were tested during summer 2016 in southwestern France to evaluate their attractiveness and their specificity for stable flies. Height of the screen and orientation (east or west) of a blue screen were also considered. High levels of S. calcitrans captures were recorded during this study (from 141 to 7301 individuals per blue screen and per day) whereas the numbers of tabanids and pollinator insects remained extremely low (less than 10 individuals per screen and per day). No significant difference in attractiveness has been shown between the different types of blue screens. The lower half of the blue screens caught significantly more stable flies (70%) than the higher half (30%). The "east" side of the screen attracted 60% of stable flies but this was not significantly different from the west side. These results are highlighting the interest in these blue polyethylene screens for controlling stable flies in cattle farms, in comparison with more expensive blue fabrics.

Molecular Identification and Geometric Morphometric Analysis of Haematobosca aberrans (Diptera: Muscidae).

The genus Haematobosca Bezzi, 1907 (Diptera: Muscidae) contains haematophagous flies of veterinary importance. A new fly species of this genus was recognised from northern Thailand based on morphological characters and described as Haematobosca aberrans Pont, Duvallet & Changbunjong, 2020. In the present study, the mitochondrial cytochrome c oxidase I (COI) gene was used to confirm the morphological identification of H. aberrans. In addition, landmark-based geometric morphometrics was used to determine sexual dimorphism. The molecular analysis was conducted with 10 COI sequences. The results showed that all sequences were 100% identical. The sequence was not highly similar to reference sequences from GenBank and did not match any identified species from Barcode of Life Data Systems (BOLD). Phylogenetic analysis clearly differentiated this species from other species within the subfamily Stomoxyinae. For geometric morphometric analysis, a total of 16 wing pictures were analysed using the landmark-based approach. The results showed significant differences in wing shape between males and females, with a cross-validated classification score of 100%. The allometric analysis showed that wing shape has no correlation with size. Therefore, the COI gene is effective in species identification of H. aberrans, and geometric morphometrics is also effective in determining sexual dimorphism.

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 Composition and Abundance of Stomoxys spp. (Diptera: Muscidae) in Peninsular Thailand.

Stable fly collections were conducted to study the species composition and daytime activity of Stomoxys spp. (Diptera: Muscidae) in Peninsular Thailand (Songkhla, Trang, Pattalung, Nakon Si Thammarat, and Satun provinces). Vavoua traps were used for fly collections in wet and dry seasons each year. Four Stomoxys species were found, S. calcitrans (L.) (Diptera: Muscidae) being the most common with 2,512 specimens (87.43% of captures), followed by S. indicus (Picard) (Diptera: Muscidae) with 306 specimens (10.65%), S. sitiens (Rondani) (Diptera: Muscidae) with 44 specimens (1.53%), and S. uruma (Shinonaga and Kano) (Diptera: Muscidae) with 11 specimens (0.39%). Subsequently, Songkhla Province was further selected to be a potential collection site to conduct a 1-yr abundance study, due to mixed crop-livestock farming in one place. Stomoxys calcitrans was predominant from 0800 to 1000 h with 4,921 specimens (93.6%) while S. indicus with 317 specimens (6%) and S. sitiens with 19 specimens (0.4%) were numerically dominant from 0600 to 0800 and 1600 to 1800 h. A proportion of male and female of each species was also established. This overall finding could be used as an ideal for nationwide species distribution status for the guidance of appropriate fly control. This information can lighten the way for the future control measures program activity of stable flies in Thailand.

Stomoxyinae Flies in Thailand: A Précis, with Abridged Taxonomic Key to the Adult Species.

Stomoxyinae flies (Diptera: Muscidae) are cosmopolitan obligate hematophagous insects of medical and veterinary importance. The subfamily Stomoxyinae includes 51 known species within 10 genera. Five genera, Stomoxys, Haematobosca, Haematobia, Haematostoma, and Stygeromyia, represent important livestock pests causing substantial economic losses in the commercial animal industry. The majority of Stomoxyinae research and control efforts have focused on Stomoxys species. Following the earliest descriptions of Stomoxyinae in Asia in the beginning of the past century, the first published list of species identified in Thailand did not occur until 1978. Currently, there are 11 confirmed species in the country with 3 others suspected present. This includes six species of Stomoxys with Stomoxys calcitrans the most common and widespread in the country. Since 1978, 20 publications, 17 of which since 2006, have covered original research on Stomoxyinae fly biology, species distribution, patterns of daily and seasonal activity, gene flow, and phylogenetics in Thailand. All Stomoxyinae-related published research from Thailand through 2017 is reviewed herein, and include an updated identification key to the Stomoxyinae genera and all known or suspected species, both male and female adults, present in Thailand.

Frequency of kdr and kdr-his Alleles in Stable Fly (Diptera: Muscidae) Populations From the United States, Costa Rica, France, and Thailand.

Anecdotal evidence of pyrethroid insecticide product failure for the control of stable fly [Stomoxys calcitrans (L.)] populations in the United States and worldwide prompted us to evaluate the frequency of knockdown resistance (kdr)-type polymorphisms within the voltage-sensitive sodium channel (Vssc) gene of field collected specimens from the United States, France, Costa Rica, and Thailand. The kdr-his allele (L1014H), associated with permethrin resistance, was detected in stable flies from the 10 states sampled in the United States, as well as from Costa Rica and France (Toulouse). Field collections of stable flies from California (Modesto) and New York (Cliffton Springs) exhibited reduced susceptibility upon exposure to a diagnostic permethrin concentration of 10× LC99, but survival did not appear to strictly associate with frequency of the kdr-his allele. This suggests that there are additional resistance mechanisms contributing to the phenotype in these states. The kdr allele (L1014F) was detected for the first time in stable flies originating in France and Thailand, and an improved, DNA-based diagnostic assay was developed and validated for use in future screens for kdr and kdr-his allele frequencies from field collections. The absence of kdr in United States and Costa Rica populations suggests that the allele is currently restricted to Europe and Asia.

Horse flies and their seasonal abundance in El Tarf Province of northeastern Algeria.

The horse flies of El Tarf Province, northeastern Algeria comprise 15 species within two subfamilies (Chrysopsinae and Tabaninae) belonging to five genera (Atylotus, Chrysops, Dasyrhamphis, Haematopota, and Tabanus). The genera Chrysops, Dasyrhamphis, and Haematopota are represented by two species for each, while the genera Atylotus and Tabanus are represented by four and five species, respectively. Seasonal abundance was assessed from mid-March until the end of September, 2017 for the trapped species. Haematopota pluvialis and Tabanus autumnalis appeared in April and lasted until September with a peak in May. Atylotus agrestis appeared in May with the highest density and lasted until September. Atylotus fulvus, Atylotus kroberi, and Haematopota italica emerged in April with peaks in May, while Chrysops flavipes appeared in March, reaching its peak in May and June, declining in July, and disappearing thereafter. Atylotus flavoguttatus, Chrysops mauritanicus, and Dasyrhamphis tomentosus appeared in April with a peak in June, then disappearing in August. Tabanus nemoralis started to appear in April with a peak in May and disappearance after July. Dasyrhamphis algirus, Tabanus bromius, and Tabanus eggeri started to appear in May. Both Tabanus eggeri and Dasyrhamphis algirus peaked in June and July, whereas T. eggeri lasted until the end of September. Tabanus tinctus was observed in June with a peak in August and lasted until September.

Phenotypic susceptibility to pyrethroids and organophosphate of wild Stomoxys calcitrans (Diptera: Muscidae) populations in southwestern France.

Stomoxys calcitrans (Diptera: Muscidae), is an important vector of lumpy skin disease and bovine besnoitiosis in Europe. Control of this biting fly could represent a keystone in the containment of this emerging disease. Reports of insecticide resistance in S. calcitrans are scarce in Europe. Therefore, the purpose of this study was to evaluate the phenotypic susceptibility to deltamethrin, cypermethrin and phoxim of five wild S. calcitrans populations from southwestern France, where transmission of bovine besnoitiosis is very prevalent. Adult S. calcitrans were caught at each study site and exposed to insecticide-impregnated filter papers under laboratory conditions. Quantities of active ingredients on filter papers corresponded to the recommended doses proposed by the manufacturers (37.5 mg a.i./m2 of cattle's skin, 125 mg a.i./m2 and 750 mg a.i./m2 for deltamethrin, cypermethrin and phoxim respectively) were tested. Knock-down effects (KD) (1 h after the onset of exposure) and mortality rates (24 h and 48 h after exposure) were evaluated. Phoxim showed a rapid and full efficacy in all populations. However, the KD effects (37.5 to 97.5%) and the mortality rates at 48 h (10 to 91.25%) induced by the exposure to pyrethroids varied greatly according to the study site but none of the populations showed full susceptibility. Therefore, the current recommended doses of these pyrethroids are probably less efficient than expected in the field and should be considered with caution in the control of bovine besnoitiosis in France.

Distribution and abundance of hematophagous flies (Glossinidae, Stomoxys, and Tabanidae) in two national parks of Gabon.

In order to minimize risks of pathogen transmission with the development of ecotourism in Gabon, a seasonal inventory has been performed in five contrasted biotopes in Ivindo (INP) and Moukalaba-Doudou (MDNP) National Parks. A total of 10,033 hematophagous flies were captured. The Glossinidae, with six different species identified, was the most abundant group and constitutes about 60% of the captured flies compared to the Stomoxys (6 species also identified) and Tabanidae with 28% and 12%, respectively. The Glossinidae showed a higher rate of capture in primary forest and in research camps. In INP, the Stomoxys showed a higher rate of capture in secondary forest and at village borders, whereas in MDNP the Stomoxys were captured more in the savannah area. Thus, each fly group seemed to reach maximum abundance in different habitats. The Glossinidae were more abundant in primary forest and near research camps while Stomoxys were more abundant in secondary forest and savannah. The Tabanidae did not show a clear habitat preference.

Tabanids: neglected subjects of research, but important vectors of disease agents!

Tabanids are nuisance pests for people and livestock because of their painful and irritating bite, persistent biting behavior, and blood ingestion. About 4400 tabanid species have been described; they are seasonally present in all kinds of landscapes, latitudes, and altitudes. High populations have a significant economic impact on outdoor activities, tourism, and livestock production. Tabanids are also vectors of animal disease agents, including viruses, bacteria and parasites. However, tabanids have received little attention in comparison with other hematophagous Diptera. Here, we highlight the many direct and indirect impacts of tabanids and provide a brief summary of tabanid morphology, biology, and life cycle. Impacts include pathogen transmission, parasite transportation (Dermatobia hominis), biological transmission (Loa loa), and mechanical transmission of viruses, such as equine infectious anemia virus, protozoa, such as Trypanosoma evansi and Besnotia besnoiti, and bacteria, such as Bacillus anthracis and Anaplasma marginale. We discuss parameters of mechanical transmission and its mathematical modeling. Control methods for tabanid populations are also summarized; these include trapping, the use of insecticides, repellents, and livestock protection. Lastly recommendations are provided for the direction of future research.

Tick repellents for human use: prevention of tick bites and tick-borne diseases.

Ticks are arthropods and the most important vectors of major human diseases after mosquitoes. Due to their impact on public health, in vitro and in vivo assays have been developed to identify molecules with repellent activities on ticks. Repellents are useful to reduce tick bite exposure and the potential transmission of pathogens; they can be used topically or in impregnated clothing. Presently, mainly synthetic molecules are commercialized as skin repellents, e.g., N,N-diethyl-meta-toluamide (DEET), IR3535, picaridin or KBR 3023, and para-menthanediol. Permethrin is largely used for fabric impregnation. Intensive research has been conducted to identify new molecules with repellent activity and more recently, plant-derived molecules, as an alternative to synthetic molecules.

Transmission of pathogens by Stomoxys flies (Diptera, Muscidae): a review.

Stomoxys flies are mechanical vectors of pathogens present in the blood and skin of their animal hosts, especially livestock, but occasionally humans. In livestock, their direct effects are disturbance, skin lesions, reduction of food intake, stress, blood loss, and a global immunosuppressive effect. They also induce the gathering of animals for mutual protection; meanwhile they favor development of pathogens in the hosts and their transmission. Their indirect effect is the mechanical transmission of pathogens. In case of interrupted feeding, Stomoxys can re-start their blood meal on another host. When injecting saliva prior to blood-sucking, they can inoculate some infected blood remaining on their mouthparts. Beside this immediate transmission, it was observed that Stomoxys may keep some blood in their crop, which offers a friendly environment for pathogens that could be regurgitated during the next blood meal; thus a delayed transmission by Stomoxys seems possible. Such a mechanism has a considerable epidemiological impact since it allows inter-herd transmission of pathogens. Equine infectious anemia, African swine fever, West Nile, and Rift Valley viruses are known to be transmitted by Stomoxys, while others are suspected. Rickettsia (Anaplasma, Coxiella), other bacteria and parasites (Trypanosoma spp., Besnoitia spp.) are also transmitted by Stomoxys. Finally, Stomoxys was also found to act as an intermediate host of the helminth Habronema microstoma and may be involved in the transmission of some Onchocerca and Dirofilaria species. Being cosmopolite, Stomoxys calcitrans might have a worldwide and greater impact than previously thought on animal and human pathogen transmission.

The repellency of lemongrass oil against stable flies, tested using video tracking.

Lemongrass oil (Cymbopogon citratus) is an effective repellent against mosquitoes (Diptera: Culicidae) and house flies (Diptera: Muscidae). In this study, its effectiveness was assessed on stable flies (Diptera: Muscidae) in laboratory conditions. First, we demonstrated that lemongrass oil is an active substance for antennal olfactory receptor cells of Stomoxys calcitrans as indicated by a significant increase in the electroantennogram responses to increasing doses of lemongrass oil. Feeding-choice tests in a flight cage with stable flies having access to two blood-soaked sanitary pads, one of which was treated with lemongrass oil, showed that stable flies (n = 24) spent significantly more time in the untreated zone (median value = 218.4 s) than in the treated zone (median value = 63.7 s). No stable flies fed on the treated pad, whereas nine fed on the untreated pad. These results suggest that lemongrass oil could be used as an effective repellent against stable flies. Additional studies to confirm its spatial repellent and feeding deterrent effects are warranted.

Phylogenetic analyses of mitochondrial and nuclear data in haematophagous flies support the paraphyly of the genus Stomoxys (Diptera: Muscidae).

The genus Stomoxys Geoffroy (Diptera; Muscidae) contains species of parasitic flies that are of medical and economic importance. We conducted a phylogenetic analysis including 10 representative species of the genus including multiple exemplars, together with the closely related genera Prostomoxys Zumpt, Haematobosca Bezzi, and Haematobia Lepeletier & Serville. Phylogenetic relationships were inferred using maximum likelihood and Bayesian methods from DNA fragments from the cytochrome c oxidase subunit I (COI, 753bp) and cytochrome b (CytB, 587bp) mitochondrial genes, and the nuclear ribosomal internal transcribed spacer 2 (ITS2, 426bp). The combination of mitochondrial and nuclear data strongly supports the paraphyly of the genus Stomoxys because of the inclusion of Prostomoxys saegerae Zumpt. This unexpected result suggests that Prostomoxys should be renamed into Stomoxys. Also, the deep molecular divergence observed between the subspecies Stomoxys niger niger Macquart and S. niger bilineatus Grünbreg led us to propose that they should rather be considered as distinct species, in agreement with ecological data. Bayesian phylogenetic analyses support three distinct lineages within the genus Stomoxys with a strong biogeographical component. The first lineage consists solely of the divergent Asian species S. indicus Picard which appears as the sister-group to all remaining Stomoxys species. The second clade groups the strictly African species Stomoxys inornatus Grünbreg, Stomoxys transvittatus Villeneuve, Stomoxys omega Newstead, and Stomoxys pallidus Roubaud. Finally, the third clade includes both African occurring and more widespread species such as the livestock pest Stomoxys calcitrans Linnaeus. Divergence time estimates indicate that the genus Stomoxys originated in the late Oligocene around 30 million years ago, with the major lineages diversifying in the Early Miocene between 20 and 15 million years ago at a time when temperate forests developed in the Northern Hemisphere.