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The article focuses on the spread and impact of bluetongue virus (BTV), specifically BTV serotype 3 (BTV-3), in European ruminants, highlighting its emergence in the Netherlands and subsequent spread to Belgium, the United Kingdom, and Germany by 2023, necessitating regulatory responses under EU guidelines to manage outbreaks.

Model forecasts of the spatiotemporal occurrence dynamics of diseases are necessary and can help understand and thus manage future disease outbreaks. In our study, we used ecological niche modelling to assess the impact of climate on the vector suitability for bluetongue disease, a disease affecting livestock production with important economic consequences. Specifically, we investigated the relationship between the occurrence of bluetongue outbreaks and the environmental suitability of each of the four vector species studied. We found that the main vector for bluetongue disease, Culicoides imicola, a typically tropical and subtropical species, was a strong predictor for disease outbreak occurrence in a region of southern Portugal from 2004 to 2021. The results highlight the importance of understanding the climatic factors that might influence vector presence to help manage infectious disease impacts. When diseases impact economically relevant species, the impacts go beyond mortality and have important economic consequences. [ABSTRACT FROM AUTHOR]

Simple Summary: This study aimed to describe the epidemiological, clinicopathological, and therapeutic aspects of allergic dermatitis caused by Culicoides spp. bites in donkeys. Clinical signs included restlessness and severe itching. Skin lesions were observed across various parts of the body and included areas of alopecia with crusts and serosanguinous exudates. Overall, 378 Culicoides insects were collected and Culicoides ocumarensis Ortiz was identified as the most common species. These findings suggest an association between allergic dermatitis and Culicoides. Additionally, a combination of copaiba oil and a multivitamin emulsion showed therapeutic potential. To date, this is the first study on allergic dermatitis in donkeys in Brazil. An allergy to bites from Culicoides (Diptera: Ceratopogonidae) occurs because of a hypersensitivity reaction caused by the inoculation of insect salivary antigens during the bite, resulting in immune-mediated dermatitis. To the best of our knowledge, no previous studies have focused on allergic dermatitis in donkeys in Brazil. Therefore, this study aimed to describe the epidemiological, clinicopathological, and therapeutic aspects of allergic dermatitis in donkeys and to identify the insects involved in its epidemiology. This study reported the occurrence of dermatitis in 17 animals. The clinical signs were restlessness and severe itching. Skin lesions were found on the head, depigmented areas of the muzzle and cheeks, flanks, pelvic and thoracic limbs, and the scrotal sac. The lesions were characterized by areas of alopecia with crusts accompanied by serosanguineous exudates. Histologically, the lesions were characterized as moderate superficial dermatitis with irregular epidermal acanthosis and pronounced diffuse orthokeratotic hyperkeratosis. In total, 378 Culicoides specimens were collected, with Culicoides ocumarensis Ortiz being the most abundant species. The combined application of copaiba oil and a multivitamin emulsion exhibited potential for topical treatment of allergic dermatitis caused by insect bites in donkeys. Our study revealed an association between allergic dermatitis in donkeys and Culicoides. [ABSTRACT FROM AUTHOR]

Background: Culicoides is a genus of ubiquitous biting midges (Ceratopogonidae). Female midges have blood‐sucking habit. They not only bite and harass humans and animals but also may be an important vector of disease transmission. Therefore, building an animal allergy model caused by Culicoides biting is very beneficial for studying its pathogenesis and exploring the therapeutic methods. Material and method: Kunming mice were used in this study to build the model and sensitised by two‐step injection of midge extracts. Scratching behaviour and histological examination were used to check the immediate and delayed responses. Immunoglobulin E (IgE) and Immunoglobulin G (IgG) were detected using indirect enzyme‐linked immunosorbent assay (ELISA) assay. Splenic cell proliferation and cytokine production were determined using 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) and ELISA assays. The response of cytokine gene expression to midge stimulation was analysed through quantitative real‐time polymerase chain reaction (qPCR). Results: Behavioural results revealed a significant increase in scratching frequency among the midge‐sensitised animals (p < 0.05). Histological examination showed more inflammatory cytokine infiltration at the injection site of midge‐sensitised mice comparing to the ones in the control group. The serum levels of IgE and IgG1 antibodies in the midge‐sensitised group were significantly elevated (p < 0.05). After splenocytes were stimulated in vitro with midge extracts, the midge‐sensitised group's splenocyte count significantly increased in comparison to the control group. The midge‐sensitised group's qPCR data revealed a down‐regulation of tumor necrosis factor alpha (TNF‐α) expression and an increase in the expression of interleukin (IL)‐4, IL‐5, IL‐10 and IL‐13 but not in the control group (p < 0.05). Conclusions: In this study, an animal model of Culicoides–mouse sensitisation was successfully constructed using a two‐step method. The mode of administration of the model was in good agreement with the natural immune pathway, and the immune response induced by the sensitisation of the model was similar to that produced by the bite of a midge. [ABSTRACT FROM AUTHOR]

Simple Summary: The number of leishmaniasis cases caused by autochthonous Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis has been continuously growing in Southeast Asia over the decades, particularly in Thailand. Recent evidence has suggested that Culicoides biting midges are the most likely natural vectors of autochthonous leishmaniasis in Thailand. Nevertheless, the epidemiology of vector infection and the genetic variation of Leishmania parasites in endemic areas have remained largely unknown. In this study, we report the high infection prevalence, sympatric circulation, and regional genetic diversity of two Leishmania (Mundinia) parasites in Culicoides spp. caught from the affected community and Tham Phra cave in Chiang Rai Province, Northern Thailand. These results provide us with a more complete understanding of the complexity of leishmaniasis transmission, which will be useful for establishing effective management and control measures for this neglected disease, especially in endemic areas of Northern Thailand. In this research, we elucidated the species composition of Culicoides biting midges, infection prevalence, and genetic diversity of Leishmania parasites circulating in the affected community in Chiang Rai Province, being the most endemic area in Northern Thailand. A total of 146 parous and gravid females, belonging to at least twelve Culicoides species in five subgenera and one species group, were trapped from three collection sites with an overall Leishmania prevalence of 26.7% (39/146). Leishmania was detected, using ITS1-PCR, in C. mahasarakamense (15), C. guttifer (11), C. (Trithecoides) spp. (8), C. jacobsoni (2), C. oxystoma (2), and C. orientalis (1). The evidence of Leishmania infection in these last five species represents new records in Northern Thailand. Given a high infection rate in cavernicolous specimens, this indicates an increased risk of parasite exposure when visiting the cave. Using the nanopore amplicon sequencing, L. martiniquensis was ubiquitously identified in all positives, and more than half of these were also co-infected with L. orientalis. The genetic diversity analysis revealed 13 and 17 unique haplotypes for L. martiniquensis and L. orientalis, respectively. Higher haplotype diversity and relatively low nucleotide diversity were observed in both parasite populations, suggesting recent population divergence. Neutrality tests (Tajima's D and Fu and Li's D) showed to be significantly negative, indicating rapid population growth or a selective sweep. Moreover, dominant haplotypes of both Leishmania species were 100% identical to those in all leishmaniasis patients previously reported from Northern Thailand, strongly supporting the imperative role of Culicoides spp. in disease transmission. Essentially, this research provides the first entomological surveillance data representing the sympatric existence, transmission dynamics, and genetic complexity of two autochthonous Leishmania (Mundinia) parasites in several Culicoides species in the endemic area of Northern Thailand. This would contribute to a more complete understanding of the epidemiology of vector infection and facilitate the development of vector control programs to effectively reduce the transmission of this neglected tropical disease in endemic areas of Northern Thailand. [ABSTRACT FROM AUTHOR]

(1) Background: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are orbiviruses that cause hemorrhagic disease (HD) with significant economic and population health impacts on domestic livestock and wildlife. In the United States, white-tailed deer (Odocoileus virginianus) are particularly susceptible to these viruses and are a frequent blood meal host for various species of Culicoides biting midges (Diptera: Ceratopogonidae) that transmit orbiviruses. The species of Culicoides that transmit EHDV and BTV vary between regions, and larval habitats can differ widely between vector species. Understanding how midges are distributed across landscapes can inform HD virus transmission risk on a local scale, allowing for improved animal management plans to avoid suspected high-risk areas or target these areas for insecticide control. (2) Methods: We used occupancy modeling to estimate the abundance of gravid (egg-laden) and parous (most likely to transmit the virus) females of two putative vector species, C. stellifer and C. venustus, and one species, C. haematopotus, that was not considered a putative vector. We developed a universal model to determine habitat preferences, then mapped a predicted weekly midge abundance during the HD transmission seasons in 2015 (July–October) and 2016 (May–October) in Florida. (3) Results: We found differences in habitat preferences and spatial distribution between the parous and gravid states for C. haematopotus and C. stellifer. Gravid midges preferred areas close to water on the border of well and poorly drained soil. They also preferred mixed bottomland hardwood habitats, whereas parous midges appeared less selective of habitat. (4) Conclusions: If C. stellifer is confirmed as an EHDV vector in this region, the distinct spatial and abundance patterns between species and physiological states suggest that the HD risk is non-random across the study area. [ABSTRACT FROM AUTHOR]

Yunnan province in China shares its borders with three neighboring countries: Myanmar, Vietnam, and Laos. The region is characterized by a diverse climate and is known to be a suitable habitat for various arthropods, including midges which are notorious for transmitting diseases which pose significant health burdens affecting both human and animal health. A total of 431,100 midges were collected from 15 different locations in the border region of Yunnan province from 2015 to 2020. These midges were divided into 37 groups according to the collection year and sampling site. These 37 groups of midges were then homogenized to extract nucleic acid. Metatranscriptomics were used to analyze their viromes. Based on the obtained cytochrome C oxidase I gene (COI) sequences, three genera were identified, including one species of Forcipomyia, one species of Dasyhelea, and twenty-five species of Culicoides. We identified a total of 3199 viruses in five orders and 12 families, including 1305 single-stranded positive-stranded RNA viruses (+ssRNA) in two orders and seven families, 175 single-stranded negative-stranded RNA viruses (−ssRNA) in two orders and one family, and 1719 double-stranded RNA viruses in five families. Six arboviruses of economic importance were identified, namely Banna virus (BAV), Japanese encephalitis virus (JEV), Akabane virus (AKV), Bluetongue virus (BTV), Tibetan circovirus (TIBOV), and Epizootic hemorrhagic disease virus (EHDV), all of which are capable, to varying extents, of causing disease in humans and/or animals. The survey sites in this study basically covered the current distribution area of midges in Yunnan province, which helps to predict the geographic expansion of midge species. The complexity and diversity of the viral spectrum carried by midges identified in the study calls for more in-depth research, which can be utilized to monitor arthropod vectors and to predict the emergence and spread of zoonoses and animal epidemics, which is of great significance for the control of vector-borne diseases. [ABSTRACT FROM AUTHOR]

Culicoides are responsible for a number of diseases transmitted to the animals causing severe health conditions and great economic losses. The outbreak of bluetongue virus in Albania in 2014 and lumpy skin disease in 2016 strongly indicated the establishment of a sentinel herd surveillance in the country ruminant farms. Considering all evidence for Albania, investigation of species composition and abundance of Culicoides on livestock farms was performed in 10 sentinel sites. Ultraviolet-light traps (OVI-traps) were placed in selected sites for collection of Culicoides starting from April 15 to July 30, 2021. A total of 37,111 Culicoides midges from 18 different species were collected during the investigation. The most abundant species identified were C. newsteadi and C. obsoletus. Moreover, three species were described for the first time in Albania (C. collessi, C. chiopterus and C. immaculatus). Presence and abundance of the collected Culicoides spp. are associated with potential outbreak of diseases (i.e. bluetongue, ephemeral fever) linked with their transmission from Culicoides. [ABSTRACT FROM AUTHOR]

Background: Culicoides, also known as biting midges, carry pathogens which include Mansonella perstans. Mansonella perstans is a nematode parasite implicated in a number of disease outcomes. Even though a high prevalence of about 75% M. perstans infection has been recorded in some communities in the middle belt of Ghana, and a wide diversity of Culicoides species has been identified, the exact Culicoides species transmitting M. perstans in Ghana has not yet been deciphered. This study therefore aimed at assessing the species diversity of Culicoides and their role in the transmission of M. perstans in the middle belt of Ghana. Methods: Culicoides species were sampled from 11 communities in the Asante-Akim North and Sene West districts in the middle belt of Ghana. Centre for Disease Control (CDC) UV light traps, as well as human bait (i.e. human landing catch and engorged catch) methods were used to assess the species abundance and diversity of Culicoides in the study communities in the wet and dry season. A colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was performed to assess the vector competence of the various Culicoides species. Results: A total of 4810 Culicoides from 6 species were sampled. These included Culicoides inornatipennis, C. milnei, C. schultzei, C. grahamii, C. neavei, and C. imicola. Culicoides imicola was the most abundant species (56%) followed by C. grahamii (16%). Light traps sampled the most diverse species (6 species). Human landing catch and engorged catch methods identified three anthropophilic species, C. grahamii, C. milnei, and C. inornatipennis, with C. grahamii being the most anthropophilic with a peak biting time between the hours of 5 p.m. to 6 p.m. Generally, there was relatively higher species abundance in the wet than dry season. LAMP assay identified C. grahamii as the potential vector for M. perstans transmission in the middle belt of Ghana. Conclusions: For the first time, we have demonstrated that C. grahamii is the potential competent vector for M. perstans transmission in the middle belt of Ghana. It is more abundant in the rainy season and has a peak biting time between the hours of 5 and 6 p.m. [ABSTRACT FROM AUTHOR]

Deciphering the population structure and variations of vector species is indispensable for effective disease management programs. The population genetic structure of a species is an attribute of its vector competence. In India, Culicoides oxystoma Kieffer, 1910 (Diptera: Ceratopogonidae) has recently been implicated in the transmission of BTV-1. We studied the genetic structure of C. oxystoma obtained from various physiographic regions of India and evaluated it based on cytochrome c oxidase subunit-1 (COX-1). Genetic similarity was assessed through median-joining networks based on haplotypes and FST analysis. Major findings reveal that the geographic clustering is aligned with the Palearctic-Indomalayan-Australian cluster which is distinct from the Senegal haplotypes. Pairwise FST showed significant genetic differentiation among the haplotypes of the Palearctic, Indomalayan, Australian and African regions except between China and Japan. Tests of neutrality illustrated an expanding population within the Indian subcontinent. Calculated values of coefficient of difference of morphological parameters across various physiographic regions suggest an absence of subspecies, which, however, requires further validation. In summary, our study indicates there is presence of gene flow in C. oxystoma across various physiographic regions of India. However, homogeneity in the population structure of this vector species, C. oxystoma, indicates that it poses a significant risk of outbreaks in multiple localities in a conducive environment. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host–pathogen interactions with implications for transmission and evolution. [ABSTRACT FROM AUTHOR]

Simple Summary: Haemoproteus parasites are one of the most studied avian blood parasites; however, their natural vectors, Culicoides biting midges, have been identified for only a small portion of them. The main reason for that might be due to the existence of a few research groups working with an integrative approach that allows not only the identification of parasite DNA in the insects, but also confirms the presence of the parasite infective stage (sporozoites) using microscopy. In this study, we aimed to identify the natural vectors of Haemoproteus parasites and to determine their prevalence in Culicoides biting midges in four different localities in Lithuania. Almost 2000 parous Culicoides females belonging to 11 species previously registered in the country were processed. The most abundant one was C. pictipennis. Parasite DNA was found in 7.9% of the analyzed biting midges, and sporozoites were present in almost 30% of these insects. The Botanical Garden was the locality with the highest abundance of biting midges and parasite species, as well as the place with the highest number of biting midges containing sporozoites. One new Haemoproteus vector (C. reconditus) was confirmed, and 12 new interactions between Haemoproteus parasite lineages and Culicoides biting midges of different species were identified. This study helps to understand the relationship between Haemoproteus parasites and Culicoides biting midges in the wild. Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors. [ABSTRACT FROM AUTHOR]

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-transmitted viral disease and a World Organization for Animal Health (WOAH)-listed disease of domestic and wild ruminants since 2008. EHDV is transmitted among susceptible animals by a few species of midges of genus Culicoides. During the fall of 2021, a large outbreak caused by the epizootic hemorrhagic disease virus (EHDV), identified as serotype 8, was reported in Tunisian dairy and beef farms with Bluetongue virus (BTV)-like clinical signs. The disease was detected later in the south of Italy, in Spain, in Portugal and, more recently, in France, where it caused severe infections in cattle. This was the first evidence of EHDV-8 circulation outside Australia since 1982. In this study, we analyzed the epidemiological situation of the 2021–2022 EHDV outbreaks reported in Tunisia, providing a detailed description of the spatiotemporal evolution of the disease. We attempted to identify the eco-climatic factors associated with infected areas using generalized linear models (GLMs). Our results demonstrated that environmental factors mostly associated with the presence of C. imicola, such as digital elevation model (DEM), slope, normalized difference vegetation index (NDVI), and night-time land surface temperature (NLST)) were by far the most explanatory variables for EHD repartition cases in Tunisia that may have consequences in neighboring countries, both in Africa and Europe through the spread of infected vectors. The risk maps elaborated could be useful for disease control and prevention strategies. [ABSTRACT FROM AUTHOR]

The emergence of culicoid-transmitted bluetongue and Schmallenberg viruses in several European countries demonstrated the ability of indigenous biting midge species to transmit pathogens. Entomologic research programs identified members of the Obsoletus Group (Culicoides subgenus Avaritia) as keyplayers in disease epidemiology in Europe. However, morphological identification of potential vectors is challenging due to the recent discovery of new genetic variants (haplotypes) of C. obsoletus sensu stricto (s.s.), forming distinct clades. In this study, 4422 GenBank entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene of subgenus Avaritia members of the genus Culicoides were analyzed to develop a conventional multiplex PCR, capable of detecting all vector species and clades of the Western Palearctic in this subgenus. Numerous GenBank entries incorrectly assigned to a species were identified, analyzed and reassigned. The results suggest that the three C. obsoletus clades represent independent species, whereas C. montanus should rather be regarded as a genetic variant of C. obsoletus s.s. Based on these findings, specific primers were designed and validated with DNA material from field-caught biting midges which achieved very high diagnostic sensitivity (100%) when compared to an established reference PCR (82.6%). [ABSTRACT FROM AUTHOR]

Forcipomyia Meigen, 1818, is the second-largest genus of the family Ceratopogonidae, having medico veterinary and agricultural importance. A few mitogenomes of Ceratopogonidae have previously been available. Here, we aimed to characterize the whole mitogenome of Forcipomyia pulchrithorax Edwards, 1924, collected from Central Anatolia Region of the Türkiye using NGS to contribute to the genetic diversity within this family. The mitogenome of F. pulchrithorax (OR666457) consisted of a circular DNA molecule spanning 15,930 bp and containing 72.7% AT content. It includes 13 proteincoding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 1 control region. All PCGs exhibited typical ATN start codons and followed the conventional TAN stop codons, except COX1, COX2, COX3, ND1, and ND5, which ended with incomplete codons. The 22 tRNA genes demonstrated the ability to form a cloverleaf structure, except tRNA-Ser1, which lacked DHU arm, similar to most insect mitogenomes. The ML phylogenetic analyses clearly delineated the species of Culicomorpha into well supported monophyletic clades. Forcipomyia pulchrithorax was clustered with the Ceratopogonidae species constituting an outer taxon among the Culicomorpha. The comprehensive mitogenome of F. pulchrithorax will be valuable for future studies focused on the phylogenetic characterization and diversity of Ceratopogonidae within the Culicomorpha. [ABSTRACT FROM AUTHOR]

Background Bluetongue is a non-contagious viral disease that afects both domestic and wild ruminants. It is transmitted primarily by small hematophagous Diptera belonging to the genus Culicoides (Diptera: Ceratopogonidae). The current study represents the frst molecular investigation into the potential role of Culicoides imicola, Culicoides paolae, Culicoides newsteadi, Culicoides spp., and Culicoides circumscriptus as bluetongue virus (BTV) vectors in Morocco. Additionally, the study aimed to evaluate the vectorial activity of midges during the survey seasons. Methods Parous females of these species were captured from several regions of Morocco (6 out of 12) from 2018 to 2021 using Onderstepoort Veterinary Institute (OVI) traps. A total of 2003 parous female specimens were grouped into 55 batches. The midge body of each batch was dissected into three regions (head, thorax, and abdomen), and these regions were analyzed separately using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results BTV RNA was detected in 45 out of the 55 batches tested, indicating a positivity rate of 81.8%. The RT-qPCRpositive pools of the studied Culicoides species exhibited high levels of BTV positivity in each body part (head, thorax, and abdomen), confrming the successful replication of the virus within midge bodies. The BTV circulation was substantial across all three survey seasons (spring, summer, and autumn). High infection rates, calculated using the minimum infection rate (MIR) and maximum likelihood estimation (MLE), were observed during the collection seasons, particularly in autumn and spring, and for all investigated Culicoides species, most notably for C. imicola and C. newsteadi. These increased infection rates underscore the signifcant risk of Culicoides transmitting the BTV in Morocco. Conclusions The detection of BTV positivity in Culicoides spp. (lacking wing spots that allow their diferentiation according to morphological identifcation keys) suggested that other Culicoides species are competent for BTV transmission in Morocco. The study results indicated, for the frst time at the molecular level, that C. imicola and C. newsteadi are the primary potential vectors of BTV in Morocco and that C. paolae and C. circumscriptus are strongly implicated in the propagation of bluetongue at the national level. [ABSTRACT FROM AUTHOR]

Two new species of biting midges are described: Forcipomyia (Lepidohelea) buccina sp. n. from Lepchakha (Queen of Dooars), the northern part of West Bengal at the Indo-Bhutan border and Forcipomyia (Lepidohelea) biharinathensis sp. n. from Biharinath Hill, highest peak of Bankura district of West Bengal and a remnant of Chota Nagpur Plateau. The discovery of these two new species elevates the total number of species in India to 80. A key to the Oriental species of the subgenus Lepidohelea Kieffer is also designed. [ABSTRACT FROM AUTHOR]

An updated checklist of Mexican non-biting midges (Chironomidae) is presented. A total of 110 species of Chironomidae are known for Mexico: 52 species in 25 genera belong to the subfamily Chironominae, 30 species in 13 genera to Orthocladiinae, 21 species in nine genera to Tanypodinae, five species in two genera to Telmatogetoninae, and two species in one genus to Diamesinae. In addition, 41 genera without identified species are listed. The highest number of species (29) is recorded from the state of Campeche, while 19 species have been found in Veracruz and 15 in Nuevo León. Few or no records exist for states in Central and Northern Mexico, or those on the Pacific coast. The type localities for 34 species are in Mexico; of these, 27 species (25% of the total number of species recorded in the country) are endemic. Twenty-nine species recorded in Mexico have a Neotropical distribution, 15 a Nearctic distribution, and 39 species are distributed in both the Neotropical and Nearctic regions or more widely. It has been suggested that as many as 1000 species might occur in Mexico; so only a little more than 10% of the expected diversity has so far been recorded. [ABSTRACT FROM AUTHOR]

Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) are Orbiviruses primarily transmitted by their biological vector, Culicoides spp. Latreille, 1809 (Diptera: Ceratopogonidae). These viruses can infect a diverse range of vertebrate hosts, leading to disease outbreaks in domestic and wild ruminants worldwide. This study, conducted at the Belo Horizonte Municipal Parks and Zoobotany Foundation (FPMZB-BH), Minas Gerais, Brazil, focused on Orbivirus and its vectors. Collections of Culicoides spp. were carried out at the FPMZB-BH from 9 December 2021 to 18 November 2022. A higher prevalence of these insects was observed during the summer months, especially in February. Factors such as elevated temperatures, high humidity, fecal accumulation, and proximity to large animals, like camels and elephants, were associated with increased Culicoides capture. Among the identified Culicoides spp. species, Culicoides insignis Lutz, 1913, constituted 75%, and Culicoides pusillus Lutz, 1913, 6% of the collected midges, both described as competent vectors for Orbivirus transmission. Additionally, a previously unreported species in Minas Gerais, Culicoides debilipalpis Lutz, 1913, was identified, also suspected of being a transmitter of these Orbiviruses. The feeding preferences of some Culicoides species were analyzed, revealing that C. insignis feeds on deer, Red deer (Cervus elaphus) and European fallow deer (Dama dama). Different Culicoides spp. were also identified feeding on humans, raising concerns about the potential transmission of arboviruses at the site. In parallel, 72 serum samples from 14 susceptible species, including various Cervids, collected between 2012 and 2022 from the FPMZB-BH serum bank, underwent Agar Gel Immunodiffusion (AGID) testing for BTV and EHDV. The results showed 75% seropositivity for BTV and 19% for EHDV. Post-testing analysis revealed variations in antibody presence against BTV in a tapir and a fallow deer and against EHDV in a gemsbok across different years. These studies confirm the presence of BTV and EHDV vectors, along with potential virus circulation in the zoo. Consequently, implementing control measures is essential to prevent susceptible species from becoming infected and developing clinical diseases. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct—cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution. [ABSTRACT FROM AUTHOR]

Two strains of viruses, JC13C644 and JC13C673, were isolated from Culicoides tainanus collected in Jiangcheng County, Yunnan Province, situated along the border area shared by China, Laos, and Vietnam. JC13C644 and JC13C673 viruses can cause cytopathic effect (CPE) in mammalian cells BHK21 and Vero cells, and cause morbidity and mortality in suckling mice 48 h after intracerebral inoculation. Whole-genome sequencing was performed, yielding complete sequences for all 10 segments from Seg-1 (3942nt) to Seg-10 (810nt). Phylogenetic analysis of the sub-core-shell (T2) showed that the JC13C644 and JC13C673 viruses clustered with the Epizootic Hemorrhagic Disease Virus (EHDV) isolated from Japan and Australia, with nucleotide and amino acid homology of 93.1% to 98.3% and 99.2% to 99.6%, respectively, suggesting that they were Eastern group EHDV. The phylogenetic analysis of outer capsid protein (OC1) and outer capsid protein (OC2) showed that the JC13C644 and JC13C673 viruses were clustered with the EHDV-10 isolated from Japan in 1998, with the nucleotide homology of 98.3% and 98.5%, and the amino acid homology of 99.6% and 99.6–99.8%, respectively, indicating that they belong to the EHDV-10. Seroepidemiological survey results demonstrated that JC13C644 virus-neutralizing antibodies were present in 29.02% (177/610) of locally collected cattle serum and 11.32% (89/786) of goat serum, implying the virus's presence in Jiangcheng, Yunnan Province. This finding suggests that EHDV-10 circulates not only among blood-sucking insects in nature but also infects local domestic animals in China. Notably, this marks the first-ever isolation of the virus in China and its discovery outside of Japan since its initial isolation from Japanese cattle. In light of these results, it is evident that EHDV Serotype 10 exists beyond Japan, notably in the natural vectors of southern Eurasia, with the capacity to infect local cattle and goats. Therefore, it is imperative to intensify the surveillance of EHDV infection in domestic animals, particularly focusing on the detection and monitoring of new virus serotypes that may emerge in the region and pose risks to animal health. [ABSTRACT FROM AUTHOR]

Tuberose (Agave amica (Medikus) Thiede and Govaerts) is a hardy and economically important flower crop in India. Tuberose growers have been facing severe loss in flower yield due to the large-scale deformities and rotting of buds caused by midge fly attacks. Morphology and DNA barcoding have confirmed the identity of this midge species as Contarinia maculipennis Felt (Diptera: Cecidomyiidae). Maggot feeding causes deformation and rotting of the infested buds and imparts an ugly appearance to the tuberose florets. Midge fly infestation ranged from 5.67% to 88% from July to September 2022 in different tuberose-growing areas of Maharashtra, Telangana and Andhra Pradesh in India. The bioecology of this midge species and the nature of damage to tuberose were studied to develop eco-friendly management methods. This study offers novel insights into the nature and extent of midge damage to tuberose and how midge symptoms differ from tuberose melody symptoms caused by the foliar nematode. [ABSTRACT FROM AUTHOR]

Background: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. 'hongkongensis'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott's test (MKT). Results: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. 'hongkongensis'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. Conclusions: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT' small and portable MinION™ means that such methods could be deployed for field use. [ABSTRACT FROM AUTHOR]

Simple Summary: Black flies are small-sized (<6 mm) blood-sucking insects belonging to the family Simuliidae of the order Diptera. More than 2400 species have been recorded globally, and approximately 6% (145 species) are found in Thailand. Many black fly species are pests to humans and other animals. They also transmit pathogens such as viruses, protozoa and filarial nematodes to humans and other animals, including economically significant livestock. Therefore, knowledge of species the diversity and distribution of wild adults, as well as host blood sources, provides important information required to prevent problems related to black fly biting. In this study, we collected 7706 wild adult black flies over a seven-year period (2017–2023) from diverse natural habitats in Thailand. In total, 16 black fly taxa were recorded; one of them, Simulium yvonneae Takaoka and Low, is a new record for the country. The majority of the specimens were Simulium asakoae Takaoka and Davies complex, contributing 74% of the total number of specimens collected. This species predominantly occurs in forest and urban habitats. The second most abundant species, S. chumpornense Takaoka and Kuvangkadilok (16% of the total specimens collected) was predominant in domestic animal shelters. Host blood meal identification using molecular approaches revealed four vertebrate host species: humans, chickens, turkeys and water buffalo. Understanding the factors associated with the species diversity and distribution of insect vectors is critically important for disease epidemiology. Black flies (Diptera: Simuliidae) are significant hematophagous insects, as many species are pests and vectors that transmit pathogens to humans and other animals. Ecological factors associated with black fly species distribution have been extensively examined for the immature stages but are far less well explored for the adult stage. In this study, we collected a total of 7706 adult black fly specimens from various locations in forests, villages and animal shelters in Thailand. The integration of morphology and DNA barcoding revealed 16 black fly taxa, including Simulium yvonneae, a species first found in Vietnam, which is a new record for Thailand. The most abundant species was the Simulium asakoae complex (n = 5739, 74%), followed by S. chumpornense Takaoka and Kuvangkadilok (n = 1232, 16%). The Simulium asakoae complex was dominant in forest (3786 of 4456; 85%) and village (1774 of 2077; 85%) habitats, while S. chumpornense predominated (857 of 1175; 73%) in animal shelter areas. The Simulium asakoae complex and S. nigrogilvum Summers, which are significant pests and vectors in Thailand, occurred at a wide range of elevations, although the latter species was found mainly in high (>1000 m) mountain areas. Simulium chumpornense, S. nodosum Puri and the S. siamense Takaoka and Suzuki complex occurred predominately in low (<800 m)-elevation areas. Simulium furvum Takaoka and Srisuka; S. phurueaense Tangkawanit, Wongpakam and Pramual; and S. nr. phurueaense were only found in high (>1000 m) mountain areas. A host blood meal analysis revealed that the S. asakoae; S. chamlongi Takaoka and Suzuki; S. nigrogilvum; S. chumpornense; and the S. striatum species group were biting humans. This is the first report of the latter two species biting humans. We also found that S. chumpornense was biting turkeys, and S. chamlongi was biting chickens, which are new host blood sources recorded for these species. In addition, we found that the S. feuerborni Edwards complex was biting water buffalo, which is the first report on the biting habits of this species. [ABSTRACT FROM AUTHOR]

Simple Summary: Vesicular stomatitis virus (VSV) infects cows, horses, and pigs, economically impacting livestock producers due to animal production losses, quarantines, and animal movement/trade restrictions. Typically, VSV is transmitted from animal to animal by direct contact, but it is also transmitted by insects such as Culicoides biting midges. These tiny flies can ingest virus particles when blood feeding on an infected animal, multiply them inside their bodies, and then transmit them to other animals the next time they feed. In addition, midges are also able to pass the virus from one to another with extremely high efficiency when they mate, even though they carry very little virus in their bodies. Through this mechanism, VSV may be maintained and overwinter in midges and appear again in livestock the next summer once the insects start feeding on blood again. Our research shows that one reason midges can transmit VSV to other midges so efficiently is because viruses that come from insect cells have an increased ability to infect more insect cells. This helps explain the midge-to-midge infection efficiency and highlights the importance of Culicoides midges in VSV maintenance and transmission. Vesicular stomatitis virus (VSV) is an arthropod-borne virus affecting livestock. In the United States, sporadic outbreaks result in significant economic losses. During epizootics, Culicoides biting midges are biological vectors and key to the geographic expansion of outbreaks. Additionally, Culicoides may play a role in VSV overwintering because females and males are capable of highly efficient venereal transmission, despite their relatively low virus titers. We hypothesized that VSV propagated within a midge has increased fitness for subsequent midge infections. To evaluate the potential host-specific fitness increase, we propagated three viral isolates of VSV in porcine skin fibroblasts and Culicoides cell lines. We then evaluated the viral infection dynamics of the different cell-source groups in Culicoides sonorensis. Our results indicate that both mammalian- and insect-derived VSV replicate well in midges inoculated via intrathoracic injection, thereby bypassing the midgut barriers. However, when the virus was required to infect and escape the midgut barrier to disseminate after oral acquisition, the insect-derived viruses had significantly higher titers, infection, and dissemination rates than mammalian-derived viruses. Our research suggests that VSV replication in Culicoides cells increases viral fitness, facilitating midge-to-midge transmission and subsequent replication, and further highlights the significance of Culicoides midges in VSV maintenance and transmission dynamics. [ABSTRACT FROM AUTHOR]

Simple Summary: Our study elucidates a genetic differentiation of F. taiwana populations across the central mountain ranges of Taiwan, which stands out as a significant geographical feature shaping these populations' genetic landscape. The mountain range acts as a significant topographic barrier, influencing gene flow and contributing to distinct genetic clusters in eastern and western regions. Despite potential mechanisms for passive dispersal, such as human-mediated transportation and monsoon winds, the genetic divergence remains pronounced. These findings provide crucial insights for understanding the species' molecular ecology and can inform targeted pest management strategies. Future research, involving more detailed genomic analyses, will be pivotal in exploring specific genetic adaptations and further elucidating gene flow across these geographical divisions. Forcipomyia (Lasiohelea) taiwana, a small bloodsucking midge, thrives in moderately moist habitats and is commonly found in grassy and bushy areas at an elevation below 250 m. This species exhibits a diurnal biting pattern and shows a marked preference for human blood. Although not known to transmit arthropod-borne diseases, the bites of F. taiwana can induce severe allergic reactions in some individuals. As a significant nuisance in Taiwan, affecting both daily life and the tourism industry, comprehensive studies on its population genetics across different geographical regions remain scarce. The central mountain ranges in Taiwan, comprising more than two hundred peaks above 3000 m in elevation, extend from the north to the south of the island, creating distinct eastern and western geographical divisions. This study utilizes microsatellite markers to explore the genetic differentiation of F. taiwana populations located in the eastern and western regions of the mountain ranges. Our findings reveal substantial genetic differentiation among populations inhabiting Taiwan's western region compared to those in the eastern region. This indicates that the topographical barriers presented by the mountain ranges significantly restrict gene flow, particularly given the species' limited active flight ability and habitat preferences. Although passive dispersal mechanisms, like wind or human activity, could contribute, this study concludes that the gene flow of F. taiwana between the western and eastern regions is primarily influenced by topographical constraints. [ABSTRACT FROM AUTHOR]

Behavioral manipulation (BM) is a multimodal control approach based on the interference with the stimuli mediating insect perception and interaction with the surroundings. BM could represent a win–win strategy for the management of vector-borne plant pathogens as the bacterium Xylella fastidiosa, since it could reduce the number of vectors alighting on host plants and, consequently, the chances for transmission to occur. In this review, we summarized current knowledge and highlighted gaps in information on (i) how insect vectors of X. fastidiosa in general, and more specifically the meadow spittlebug Philaenus spumarius, locate and accept the host plant; and (ii) how behavioral manipulation techniques could be applied to disrupt the vector–host plant interaction. Finally, we discussed how diverse BM strategies could be combined with other integrated pest management tools to protect olive groves from inoculation with the fastidious bacterium. [ABSTRACT FROM AUTHOR]

Bluetongue (BT) disease is a viral, insect borne, noncontagious illness of small ruminants caused by Orbivirus, impacting huge economic loss worldwide. The existing BT diagnostic techniques are costly, time-consuming and require both specialized equipment and also skilled personnel. So there is need to develop a rapid, sensitive, on site detection assay for diagnosis of BT. This study utilized secondary antibody derivatized Gold nanoprobes for rapid and sensitive detection of BT over lateral flow device (LFD). The detection limit for this assay was found 1.875 µg of BT IgG/ml and a comparison between LFD and indirect ELISA was performed and the sensitivity and specificity was found at 96% and 99.23%, respectively, with observed kappa value of 0.952. This developed LFD may therefore offer a quick, affordable and accurate diagnosis of BT disease at the field level. [ABSTRACT FROM AUTHOR]

Animal motion and flocking are ubiquitous nonequilibrium phenomena that are often studied within active matter. In examples such as insect swarms, macroscopic quantities exhibit power laws with measurable critical exponents and ideas from phase transitions and statistical mechanics have been explored to explain them. The widely used Vicsek model with periodic boundary conditions has an ordering phase transition but the corresponding homogeneous ordered or disordered phases are different from observations of natural swarms. If a harmonic potential (instead of a periodic box) is used to confine particles, then the numerical simulations of the Vicsek model display periodic, quasiperiodic, and chaotic attractors. The latter are scale-free on critical curves that produce power laws and critical exponents. Here, we investigate the scale-free chaos phase transition in two space dimensions. We show that the shape of the chaotic swarm on the critical curve reflects the split between the core and the vapor of insects observed in midge swarms and that the dynamic correlation function collapses only for a finite interval of small scaled times. We explain the algorithms used to calculate the largest Lyapunov exponents, the static and dynamic critical exponents, and compare them to those of the three-dimensional model. [ABSTRACT FROM AUTHOR]

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife and livestock. Understanding the composition and distribution of vector species is crucial for implementing control strategies and preventing the spread of infectious diseases. This study provides a morphological and molecular characterisation of Culicoides caucoliberensis, which represents the first record for Spain, increasing the number of Culicoides species in the country to 85. A total of 213 specimens were collected using Onderstepoort‐ultraviolet down‐draught light traps on a rocky coastline in the Balearic Islands during two sampling periods in 2022. Phylogenetic analysis showed that C. caucoliberensis forms a monophyletic cluster within the Maritimus group. Host preferences were determined for the first time and showed propensity to feed on the European shag (Phalacrocorax aristotelis). The vector role of C. caucoliberensis for haemosporidian transmission remains unclear since molecular detection of Haemosporidians (Haemoproteus and Plasmodium) was negative for all the pools of parous and engorged females analysed. This study emphasises the importance of conducting entomofauna studies in lesser‐known Mediterranean islet landscapes and highlights the need for research on vectors within the One Health framework. [ABSTRACT FROM AUTHOR]

Biting flies (Diptera) transmit pathogens that cause many important diseases in humans as well as domestic and wild animals. The networks of feeding interactions linking these insects to their hosts, and how they vary geographically and in response to human land‐use, are currently poorly documented but are relevant to understanding cross‐species disease transmission. We compiled a database of biting Diptera–host interactions from the literature to investigate how key interaction network metrics vary latitudinally and with human land‐use. Interaction evenness and H2' (a measure of the degree of network specificity) did not vary significantly with latitude. Compared to near‐natural habitats, interaction evenness was significantly lower in agricultural habitats, where networks were dominated by relatively few species pairs, but there was no evidence that the presence of humans and their domesticated animals within networks led to systematic shifts in network structure. We discuss the epidemiological relevance of these results and the implications for predicting and mitigating future spill‐over events. [ABSTRACT FROM AUTHOR]

Simple Summary: Avian haemoproteids are prevalent parasites known to induce pathology or mortality in birds. Culicoides biting midges act as vectors for these parasites. Despite the annual arrival of various haemoproteids to Europe with migrant birds, not all these parasites undergo local transmission. The factors constraining the local spread of these pathogens remain incompletely understood. Our study investigates if the ecological isolation of birds from vectors, either temporally or spatially during the breeding period when transmission occurs, could lead to the cessation of transmission. Biting midges were systematically collected from two distinct habitats between May and September. A total of 1135 parous Culicoides females were identified and examined for the presence of Haemoproteus parasites using both microscopy and molecular tools. Notably, we report the first detection of Haemoproteus asymmetricus sporozoites in the salivary glands of Culicoides festivipennis females. The sporozoites of four Haemoproteus genetic lineages were also identified in Culicoides segnis, C. festivipennis, and C. kibunensis, further validating their status as potential Haemoproteus vectors. While the highest abundance of collected Culicoides females occurred in June, the peak prevalence of Haemoproteus parasites in biting midges was observed in July. Interestingly, the abundance of Culicoides was significantly greater in woodlands compared to reeds throughout the season. Avian haemosporidians (Apicomplexa, Haemosporida) are widespread blood protists, often causing severe haemosporidiosis, pathology, or even mortality in their hosts. Migrant birds regularly bring various haemosporidian parasites from wintering grounds to European breeding areas. Some haemosporidian parasites are prevalent in breeding sites and complete their life cycles in temperate climate zones and can be transmitted, but others do not. The factors altering the spread of these haemosporidians are not fully understood. Culicoides biting midges (Diptera: Ceratopogonidae) play an important role in the transmission of worldwide distributed avian haemosporidian parasites belonging to the genus Haemoproteus, but this information is particularly scarce and insufficient. The key factors limiting the spread of these pathogens in temperate climate zones, which we suspect and aim to study, are the absence of susceptible vectors and the ecological isolation of birds from vectors during the breeding period when transmission occurs. The primary objective of this study was to evaluate how the habitats of biting midges and bird breeding sites influence parasite transmission while also seeking to expand our understanding of the natural vectors for these parasites. Biting midges were collected using UV traps on the Curonian Spit, Lithuania, in different habitats, such as woodland and reeds, from May to September. Parous Culicoides females were identified, dissected, and investigated for the presence of Haemoproteus parasites using both microscopy and PCR-based tools. Among the dissected 1135 parous Culicoides females, the sporozoites of Haemoproteus asymmetricus (genetic lineage hTUPHI01) have been detected for the first time in the salivary glands of Culicoides festivipennis. The sporozoites of four Haemoproteus lineages were detected in Culicoides segnis, C. festivipennis, and Culicoides kibunensis biting midges. PCR-based screening showed that the females of seven Culicoides species were naturally infected with Haemoproteus parasites. The DNA of the parasite of owls, Haemoproteus syrnii (hSTAL2), was detected for the first time in Culicoides punctatus. The highest abundance of collected Culicoides females was in June, but the highest prevalence of Haemoproteus parasites in biting midges was in July. The abundance of Culicoides was higher in the woodland compared with reeds during the season. The acquired findings indicate the varied abundance and diversity of biting midges throughout the season and across distinct habitats. This variability could potentially impact the transmission of Haemoproteus parasites among birds with diverse breeding site ecologies. These outcomes hold the potential to enhance our understanding of the epizootiology of Haemoproteus infections within temperate climatic zones. [ABSTRACT FROM AUTHOR]

Bluetongue virus serotype 16 detection in NSW : In coastal New South Wales (NSW), bluetongue virus (BTV) serotypes 1 and 21 are endemic and transmitted in most years without evidence of disease. However, serotype 16 (BTV‐16) infection was detected for the first time in NSW in November 2016 in cattle undergoing testing for export. Retrospective testing of blood samples collected from sentinel cattle as part of the National Arbovirus Monitoring Program (NAMP) established that the first detected transmission of BTV‐16 in NSW occurred in April 2016 in sentinel cattle on the NSW North Coast. Subsequently, until 2022, BTV‐16 has been transmitted in most years and was the predominant serotype in the 2018–2019 transmission season. The data available suggests that BTV‐16 may have become endemic in NSW. Experimental studies: During experimental infection studies with BTV‐16, all sheep were febrile, with the peak of viremia occurring 6–10 days after inoculation. There was nasal and oral hyperaemia in most sheep with several animals developing a nasal discharge and nasal oedema. All sheep developed coronitis of varying severity, with most also developing haemorrhages along the coronary band. There was a high incidence of haemorrhage in the pulmonary artery, epicardial petechiae, extensive pericardial haemorrhages and moderate body cavity effusions including pericardial effusions. Conclusion: Overall, experimental pathogenicity findings suggest moderate disease may occur in sheep in the field. These findings, when combined with climatic variability that could result in an expansion of the range of Culicoides brevitarsis into major sheep‐producing areas of the state, suggest that there is an increasing risk of bluetongue disease in NSW. [ABSTRACT FROM AUTHOR]

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis. [ABSTRACT FROM AUTHOR]

Background: Arthropods transmit a wide range of pathogens of importance for the global health of humans, animals, and plants. One group of these arthropod vectors, Culicoides biting midges (Diptera: Ceratopogonidae), is the biological vector of several human and animal pathogens, including economically important livestock viruses like bluetongue virus (BTV). Like other arthropod-borne viruses (arboviruses), Culicoides-borne viruses must reach and replicate in the salivary apparatus, from where they can be transmitted to susceptible hosts through the saliva during subsequent blood feeding. Despite the importance of the salivary gland apparatus for pathogen transmission to susceptible animals from the bite of infected Culicoides, these structures have received relatively little attention, perhaps due to the small size and fragility of these vectors. Results: In this study, we developed techniques to visualize the infection of the salivary glands and other soft tissues with BTV, in some of the smallest known arbovirus vectors, Culicoides biting midges, using three-dimensional immunofluorescence confocal microscopy. We showed BTV infection of specific structures of the salivary gland apparatus of female Culicoides vectors following oral virus uptake, related visualisation of viral infection in the salivary apparatus to high viral RNA copies in the body, and demonstrated for the first time, that the accessory glands are a primary site for BTV replication within the salivary apparatus. Conclusions: Our work has revealed a novel site of virus-vector interactions, and a novel role of the accessory glands of Culicoides in arbovirus amplification and transmission. Our approach would also be applicable to a wide range of arbovirus vector groups including sand flies (Diptera: Psychodidae), as well as provide a powerful tool to investigate arbovirus infection and dissemination, particularly where there are practical challenges in the visualization of small size and delicate tissues of arthropods. [ABSTRACT FROM AUTHOR]

Chitinases play a crucial role in insect metamorphosis by facilitating chitin degradation. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a typical holometabolous insect and an important hygiene pest that causes myiasis in humans and other mammals and acts as a vector for various parasitic agents, including bacteria, viruses, and parasites. Enhancing the understanding of the metamorphosis in this species has significance for vector control. In this study, we identified a total of 12 chitinase genes in S. peregrina using bioinformatic analysis methods. Based on transcriptome data, SpIDGF2 and SpCht10 were selected for further functional investigation. The down-regulation of these genes by RNA interference led to developmental delays, disruptions in molting, and differences in cuticle composition during the pupal stage. These findings underscore the pivotal role of chitinase genes in the metamorphic process and offer valuable insights for effective control strategies. [ABSTRACT FROM AUTHOR]

The genus Culex has 817 species subdivided into 28 subgenera. It has a cosmopolitan distribution, being most abundant in countries with a tropical climate. Understanding the ecology and diversity of viruses circulating in the species of this genus is important for understanding their role as arbovirus vectors in Brazil. To conduct an integrative review to identify the importance of the Culex genus as arbovirus vectors in Brazil. A search was carried out for scientific papers in the PubMed, BVSalud, Patuá-IEC and International Catalogue of Arboviruses: including certain other viruses of vertebrates databases. 36 publications describing arbovirus detections in Culex mosquitoes collected in the field in Brazil were evaluated. A total of 42 arbovirus species were detected, as well as studies analyzing the vector competence of C. quinquefasciatus for the transmission of four different arboviruses. The study of the Culex genus and its role as a vector of arboviruses in Brazil is essential for understanding transmission cycles, with the main aim of reducing cases of human infection. Thus, entomovirological surveillance guides the implementation of actions to detect circulating arboviruses among vectors to anticipate measures aimed at preventing or reducing the risk of arbovirus outbreaks in the country. [ABSTRACT FROM AUTHOR]

Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) is a diverse genus of tiny biting flies that are both pests and vectors of pathogens of medical, veterinary, and economic importance. Herein, we provide an identification key and wing atlas of adult female biting midges for the 40 Culicoides species that have been recorded or are likely to be present in Ontario, Canada. The key includes: C. albertensis Wirth and Jones, C. alexanderi Wirth and Hubert, C. arboricola Root and Hoffman, C. atchleyi Wirth and Blanton, C. baueri Hoffman, C. bergi Cochrane, C. bickleyi Wirth and Hubert, C. biguttatus (Coquillett), C. chiopterus (Meigen), C. cockerellii (Coquillett), C. crepuscularis Malloch, C. denticulatus Wirth and Hubert, C. downesi Wirth and Hubert, C. flukei Jones, C. footei Wirth and Jones, C. franclemonti Cochrane, C. frohnei Wirth and Blanton, C. furensoides Williams, C. guttipennis (Coquillett), C. haematopotus Malloch, C. jamnbacki Wirth and Hubert, C. nanus Root and Hoffman, C. obsoletus (Meigen), C. paraimpunctatus Borkent, C. parapiliferus Wirth and Blanton, C. piliferus Root and Hoffman, C. sanguisuga (Coquillett), C. snowi Wirth and Jones, C. sphagnumensis Williams, C. spinosus Root and Hoffman, C. stellifer (Coquillett), C. stilobezzioides Foote and Pratt, C. testudinalis Wirth and Hubert, C. travisi Vargas, C. utowana Jamnback, C. variipennis (Coquillett), C. venustus Hoffman, C. villosipennis Root and Hoffman, C. wisconsinensis Jones, and C. yukonensis Hoffman. We provide a pictorial key based on novel and existing published observations of the morphological features observed from these 40 species, and include a pictorial wing atlas. This key will allow for the identification of Culicoides from Ontario down to the lowest taxonomic level possible including: one subgenus (with three species), one species group (with 11 species), and 26 separate species. [ABSTRACT FROM AUTHOR]

Recent molecular approaches toward insect taxonomy are revealing cryptic diversity within biting midge genus Culicoides Latreille, 1809. A new biting midge species was collected from highly restricted area of Dokdo Island, East Sea of the Republic of Korea, and was identified as Culicoides dokdoensis, sp. nov., based on morphological characters and DNA barcoding. The specimens collected from the island were previously known as Culicoides circumscriptus Kieffer, 1918, a cosmopolitan blood‐feeder, due to its morphological similarity. However, the specimens from Dokdo Island can be distinguished from other congeners by the following characters: more curved arms in male aedeagus, wing spot pattern, sensory pit depth, and absence of a spermatheca neck in female. In addition, mitochondrial cytochrome c oxidase I sequence exhibited substantial genetic difference compared to C. circumscriptus populations from the Korean Peninsula, sufficiently supporting the genetic differentiation between the two species. The discovery of this cryptic species highlights the necessity of integrated taxonomy for Culicoides. [ABSTRACT FROM AUTHOR]

This paper presents the first results of 1-year survey of aquatic Diptera, carried out in two Moroccan High Atlas rivers, Assif Ahançal and Assif n'Aït Bouguemaz, which are registered as Ramsar sites and drain a large part of the mountainous Geopark of Mgoun. This survey, which consists in a first study of the longitudinal distribution of these insects, occurs during the period September 2019–July 2020, at high and medium altitudes (1086–2408 m). Limited to immature stages, this study permitted to collect 561 specimens of Diptera from 16 river sections, mainly using a Surber-net sampler. Among the collected material, we were able to identify 38 genera and 11 species, belonging to five families. This fauna is relatively rich in new taxa for Morocco or for the High Atlas mountains. Indeed, two Chironomidae species, Macropelopia notata (Meigen, 1818) and Psectrocladius (Allopsectrocladius) flavus (Johannsen, 1905), and one Tipulidae, Tipula (Yamatotipula) pierrei, Tonnoir, 1921, are newly recorded in Morocco, as well as five genera: three Chironomidae (Krenopelopia, Fittkau; Natarsia, Fittkau; and Psilometriocnemus, Saether), one Empididae (Chelifera, Macquart), and one Ceratopogonidae (Atrichopogon, Kieffer). Two other species, Parachironomus frequens (Johannsen) and Heterotrissocladius marcidus (Walker), and seven genera are reported as new taxa in the High Atlas. The two rivers have similar richness (21 taxa each), mainly due to Chironomidae (94%), the most abundant and diversified family. The Empididae (4.8%) are relatively under-represented in the two rivers, as well as the other families (Ceratopogonidae, Tipulidae, and Tabanidae), which do not exceed 1% of the total dipteran abundance. The vertical distribution of the taxa revealed significant influence of the altitude on community compositions and distributions. In waiting to collect adults, immature dipteran stages prove the high abundance and diversity of the benthic entomological assemblages, which give hope that a more deep study of the dipteran fauna in the two Ramsar sites will provide remarkable novelties, in both systematic and ecological domains. It is also expected that this study will permit to assess the aquatic biodiversity of these ecosystems and its sensitivity to the increasing human disturbances. [ABSTRACT FROM AUTHOR]

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis. [ABSTRACT FROM AUTHOR]

Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is a common cause of traumatic myiasis in livestock in Mediterranean countries as well as in Central and Eastern Europe. The current study investigated the utility of external body genitalia and mitochondrial and ribosomal DNA characteristics for identifying this species. W. magnifica was successfully identified morphologically and molecularly based on partial sequences of three DNA fragments, mitochondrial cytochrome oxidase I (mtCOI), 12S ribosomal DNA (rDNA), and 16S rDNA. A BLAST search for samples containing 12S and 16S rDNA sequences yielded 100 hits, with just one from W. magnifica (accession number: KU578263.1). More W. magnifica sequences were discovered for the mtCOI, including Egyptian isolates of W. magnifica cytochrome oxidase subunit I sequence (accession numbers: MN241041 and MN241043). As a result, we believe that mtCOI performed the best in terms of species identification. Phylogenetic analysis of mtCOI, 12S rDNA and 16S rDNA showed that W. magnifica identified in the current study was most closely related to W. magnifica retrieved from GenBank. [ABSTRACT FROM AUTHOR]

South America, particularly within its tropical belt, is renowned for its unparalleled high levels of species richness, surpassing other major biomes. Certain neotropical areas harbor fragmented knowledge of insect diversity and face imminent threats from biodiversity loss and climate change. Hence, there is an urgent need for rapid estimation methods to complement slower traditional taxonomic approaches. A variety of algorithms for delimiting species through single‐locus DNA barcodes have been developed and applied for rapid species diversity estimates across diverse taxa. However, tree‐based and distance‐based methods may yield different group assignments, leading to potential overestimation or underestimation of putative species. Here, we investigate the performance of different DNA‐based species delimitation approaches to rapidly estimate the diversity of Polypedilum (Chironomidae, Diptera) in South America. Additionally, we test the hypothesis that significant differences exist in the community structure of Polypedilum fauna between South America and its neighboring regions, particularly the Nearctic. Our analysis encompasses a dataset of 1492 specimens from 598 locations worldwide, with a specific focus on South America. Within this region, we analyzed a subset of 247 specimens reported from 37 locations. Using various methods including the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), multi‐rate Poisson tree processes (mPTP), single‐rate Poisson tree processes (sPTP), and generalized mixed Yule coalescent (sGMYC), we identify molecular operational taxonomic units (MOTUs) ranging from 267 to 520. Our results indicate that the sGMYC method is the most suitable for estimating putative species in our dataset, resulting in the identification of 75 species in the Neotropical region, particularly in South America. Notably, this region exhibited higher species richness in comparison to the Palearctic and Oriental realms. Additionally, our findings suggest potential differences in species composition of Polypedilum fauna between the Neotropical and the adjacent Nearctic realms, highlighting high levels of endemism and species richness in the first. These results support our hypothesis that there are substantial differences exist in species composition between the Polypedilum fauna in South America and the neighboring regions. [ABSTRACT FROM AUTHOR]

Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus. [ABSTRACT FROM AUTHOR]

Black flies are blood-sucking insects of public health importance, and they are effective vectors of pathogens and parasites, such as filarial nematodes of the genus Onchocerca. Our previous surveys have shown that individuals of Simulium turgaicum are annoying pests of humans and livestock in the Aras River Basin of Iran. In the present study, adult black flies of S. turgaicum were trapped from different ecotopes of five villages in Khoda-Afarin County, Iran. By using a sensitive nested PCR assay and targeting the nuclear 18S rDNA-ITS1 marker, filarial infections were found in 38 (1.89%) of 2005 black flies. Homology exploration of 360 bp of the sequences indicated that the filarial worms are members of the family Onchocercidae, with maximum alignment scores of 93–95%. Phylogenetic analysis showed that two Iranian Onchocerca isolates were clustered in the O. fasciata–O. volvulus lineage and were well separated from other filarial nematodes. Both the entomological evidence (empty abdomen of the specimens) and climatologic data (adequate accumulated degree days for development) suggest that the filarial DNA was probably that of infective larvae of vertebrates. This is the first report of an infection by Onchocerca species in S. turgaicum and the first record of onchocercids in black flies in Iran; however, more research is required to demonstrate transmission of these filarial worms by black flies in nature. [ABSTRACT FROM AUTHOR]

Mosquitoes (Diptera: Culicidae) and biting midges (Diptera: Ceratopogonidae) are among the most important vectors of human and veterinary pathogens. For modelling the distribution of these pathogens, entomological aspects are essential, which in turn are highly dependent on environmental factors, such as temperature. In this study, mosquitoes and biting midges were sampled in multiple microclimates at two low (360, 480 meters above sea level, m.a.s.l.) and two high (1250, 1530 m.a.s.l.) altitude locations in Switzerland. Sets of various traps (CO2‐baited CDC, LED‐UV, resting boxes, oviposition cups) equipped with dataloggers were placed in transects at five sites with similar vegetation at each location. Only the CDC and the LED‐UV traps collected enough insects for analyses. Taxonomic diversity was greater for mosquitoes but lower for biting midges at lower altitudes. Both mosquitoes and biting midges had a thermal preference. Culicoides preferred the traps with warmer microclimate, especially at lower altitudes, whereas mosquito preferences depended on the species, but not on altitude. Relative humidity had a significant positive impact on catches of biting midges but not mosquitoes. To obtain better data on thermal preferences of resting and ovipositing vectors in addition to host seeking individuals, new and improved collecting methods are needed. [ABSTRACT FROM AUTHOR]

Bluetongue is a non‐contagious viral disease causing significant economic losses throughout the world. The bluetongue vectors Culicoides oxystoma and Culicoides actoni, which play a significant role in the transmission of various pathogens, are distributed across different geographical realms. Adults are minute in size with wide phenotypic variation, so morphology‐based species identification is severely constrained by preparatory time and shortage of taxonomic expertise. To make the identification process rapid and effective, a specific primer was designed for the identification of C. actoni based on the multiple sequence alignment of ITS1 sequences of 11 Culicoides species. Along with this, a refined version of existing C. oxystoma specific primer was proposed. The primer sets distinguished C. oxystoma and C. actoni from a pooled sample consisting of other Culicoides species as well as closely related genera such as Forcipomyia and Alluaudomyia. Our findings suggest that the primers were species specific, sensitive and have potential to discriminate vector species C. oxystoma and C. actoni from pooled samples. To the best of our knowledge, these are the first ITS1 sequences generated and submitted in GenBank for Culicoides innoxius, Culicoides shortti, Culicoides palpifer and Culicoides anophelis and the first for Culicoides peregrinus, Culicoides fulvus and C. actoni from India. [ABSTRACT FROM AUTHOR]

Culicoides are important vectors for livestock and human pathogens. Wild animals act as reservoirs for important orbiviruses such as bluetongue and African horse sickness viruses. There are only limited studies on the distribution of Culicoides species in forest habitats. In this study, we collected Culicoides from different wildlife sanctuaries and national parks of Karnataka. We collected and morphologically identified 8597 Culicoides. We found 18 species of Culicoides in different sites, with C. oxystoma and C. imicola being the predominant species across the sites. The sequence alignment and phylogenetic analysis of the Cox1 gene of Cuilicoides species revealed a huge level of sequence similarity and their wide distribution around the world. Most of the isolates from our study were closely related to Chinese isolates. The abundance of the species was analyzed using the Bayesian ordination method. We used a hierarchical joint distribution negative binomial regression model to detect the correlation between species owing to environmental covariates and residual correlation. The presence of potential vectors for important livestock pathogens in wild habitats in our study warrants further research on the detection of pathogens in Culicoides collected from forest habitats and adopt surveillance in wild animal habitats to prevent disease spread from wild animals to livestock and vice versa. [ABSTRACT FROM AUTHOR]

Background: After several years without bluetongue disease, a ruminant illness caused by Culicoides-borne bluetongue virus (BTV), two new autochthonous cases were reported in 2018 in Germany. By contrast, Schmallenberg virus (SBV), another Culicoides-borne virus pathogenic to ruminants, has continuously circulated in Germany since its first emergence in 2011. The disease outbreaks have triggered numerous studies on the biology of the Culicoides vectors, but many ecological details are still obscure. Methods: Culicoides biting midge species were collected with UV-light traps on 10 farms in Germany, with one trap inside and one trap outside barns on each of the farms. Traps were run once a week for 24 h from January to December 2019. Collected biting midges were morphologically identified, counted and statistically evaluated, with a focus on the Obsoletus Group and the Pulicaris Complex of the ceratopogonid genus Culicoides, which are believed to contain the major virus vectors. Temperature and relative humidity recorded at each trap were linked to the quantity of caught Culicoides. Correlations between relative Culicoides abundance and presence of livestock or type of husbandry were also investigated. Results: A total of 38,886 Culicoides biting midges were trapped, with most of them belonging to the Obsoletus Group (51.0%) and the Pulicaris Complex (38.8%). The majority of captured specimens were collected in traps inside the barns. Obsoletus Group individuals were caught from late January to the last week of December while Pulicaris Complex individuals were captured from the end of March to early December. The lowest average temperatures at which members of the two groups were collected were 10.7 °C and 12.8 °C, respectively. While temperature had a statistically significant effect on the activity of both the Obsoletus Group and the Pulicaris Complex, relative humidity only significantly affected the activity of the latter. The presence of livestock significantly influenced the number of captured Obsoletus Group, but not of Pulicaris Complex specimens. Inside the barns, no statistical difference was found between numbers of caught Obsoletus Group and Pulicaris Complex specimens in livestock holdings with deep litter and manure scraper or slatted floor husbandry systems. Conclusions: The almost year-round presence of Obsoletus Group biting midges and the demonstrated high relative abundance of other potential Culicoides vector species inside barns suggest a high risk of indoor virus transmission to ruminants should BTV or SBV circulate locally. Appropriate structural, organisational and vector control measures to reduce biting midge exposure should be implemented. [ABSTRACT FROM AUTHOR]

Background: Culicoides biting midges (Diptera: Ceratopogonidae) are economically important blood-feeding pests closely associated with livestock production. They are the principal vectors of two hemorrhagic disease viruses affecting both wild and domestic ruminants within the US: bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). BTV impacts the US agriculture sector through direct commodity loss and strict international livestock trade restrictions. Yet, despite posing a considerable threat to US livestock, Culicoides are understudied, and management strategies are lacking. Current control tools for Culicoides are limited to synthetic chemicals, predominantly pyrethroids. With limited products available for livestock producers, proper pesticide rotation is difficult. The present study investigates the efficacy of fluralaner, an isoxazoline insecticide, beyond its current labeled use as an ectoparasiticide in anticipation of adding a new class of pesticides into rotation for use against biting midges. Methods: The efficacy of fluralaner was evaluated by conducting contact, topical, and oral toxicity bioassays on adult female Culicoides sonorensis. Contact toxicity was assessed by using a modified WHO cone assay, which simulates exposure through landing on an insecticide-treated surface. A modified WHO topical toxicity assay, in which fluralaner dilutions were administered to the lateral thorax, was used to assess topical toxicity. For evaluation of oral toxicity, females were offered a blood meal spiked with fluralaner in an artificial membrane feeding system to simulate a systemic insecticide. Results: Contact exposure of fluralaner did not cause extensive or consistent mortality. Even the highest concentration tested (100 mg/ml) resulted in an average of only 24.3% mortality at 24 h, and mortality did not significantly differ between exposed and control midges at any concentration. One hundred percent mortality was consistently achieved at concentrations of 1 mg/ml when fluralaner was applied topically. The LC50 for topical exposure to fluralaner at 24 h was estimated to be 0.011 mg/ml. Oral exposure to fluralaner through ingestion of a spiked blood meal proved to be the most effective exposure method, significantly increasing mortality in a dose-dependent manner at 1 h post-exposure. The LC50 at 24 h following ingestion was 14.42 ng/ml. Conclusion: Our results suggest that fluralaner is a viable candidate for use as an insecticide against adult biting midges if exposed orally, such as in a systemic given to livestock. As withdrawal period requirements for meat animals present unique yet definitive challenges, pharmacokinetic studies of isoxazoline drugs need to be pursued and finalized for livestock before fluralaner may be used as a management strategy in this manner. Alternatively, livestock not raised for consumption, such as hair sheep, would directly benefit from administering oral fluralaner as a component of a BTV disease management program. [ABSTRACT FROM AUTHOR]