Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Population genetics of the biting midge Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) from Thailand and its genetic relationships with global populations.

Culicoides oxystoma Kieffer is a vector of viruses, filarial nematodes and protozoa of the genus Leishmania transmitted to humans and other animals. Understanding genetic diversity, genetic structure and genetic relationships among geographically widespread populations will provide important information related to disease epidemiology. In this study, genetic diversity, genetic structure and genetic relationships between Thai C. oxystoma and those reported from other countries were inferred based on mitochondrial cytochrome oxidase I (COI) and nuclear internal transcribed spacer 1 (ITS-1) sequences. A high level of genetic diversity was found in C. oxystoma from Thailand. The maximum K2P intraspecific genetic divergence for COI gene and ITS-1 sequences were 4.29% and 6.55%, respectively. Despite high genetic diversity, no significant genetic differentiation was found within the 13 Thai populations. This could be a result of unspecialized habitat requirement of the larval habitat, abundance and continuous distribution of host blood sources, potential for long distance movement with host via trading. Mitochondrial genealogy analysis of the global population of C. oxystoma revealed three (A, B and C) genetically divergent lineages. Specimens from Thailand were included in the main lineage (A) with those from all other countries except those from Senegal that formed lineage B and those of Lineage C that was exclusively found in Bangladesh. The nuclear (ITS-1) genetic markers genealogy indicated that Thai C. oxystoma belong to the same genet.

Little-known virus is on the rise in South America.

Deforestation and climate change may be helping Oropouche virus spread far beyond the Amazon Basin.

Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Culicoides Midge Abundance across Years: Modeling Inter-Annual Variation for an Avian Feeder and a Candidate Vector of Hemorrhagic Diseases in Farmed Wildlife.

(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.

Transcriptomic Investigation of the Virus Spectrum Carried by Midges in Border Areas of Yunnan Province.

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.

Seasonal change and influence of environmental variables on host-seeking activity of the biting midge Culicoides sonorensis at a southern California dairy, USA.

BACKGROUND: As a primary vector of bluetongue virus (BTV) in the US, seasonal abundance and diel flight activity of Culicoides sonorensis has been documented, but few studies have examined how time of host-seeking activity is impacted by environmental factors. This knowledge is essential for interpreting surveillance data and modeling pathogen transmission risk. METHODS: The diel host-seeking activity of C. sonorensis was studied on a California dairy over 3 years using a time-segregated trap baited with CO2. The relationship between environmental variables and diel host-seeking activity (start, peak, and duration of activity) of C. sonorensis was evaluated using multiple linear regression. Fisher's exact test and paired-sample z-test were used to evaluate the seasonal difference and parity difference on diel host-seeking activity. RESULTS: Host-seeking by C. sonorensis began and reached an activity peak before sunset at a higher frequency during colder months relative to warmer months. The time that host-seeking activity occurred was associated low and high daily temperature as well as wind speed at sunset. Colder temperatures and a greater diurnal temperature range were associated with an earlier peak in host-seeking. Higher wind speeds at sunset were associated with a delayed peak in host-seeking and a shortened duration of host-seeking. Parous midges reached peak host-seeking activity slightly later than nulliparous midges, possibly because of the need for oviposition by gravid females before returning to host-seeking. CONCLUSIONS: This study demonstrates that during colder months C. sonorensis initiates host-seeking and reaches peak host-seeking activity earlier relative to sunset, often even before sunset, compared to warmer months. Therefore, the commonly used UV light-baited traps are ineffective for midge surveillance before sunset. Based on this study, surveillance methods that do not rely on light trapping would provide a more accurate estimate of host-biting risk across seasons. The association of environmental factors to host-seeking shown in this study can be used to improve modeling or prediction of host-seeking activity. This study identified diurnal temperature range as associated with host-seeking activity, suggesting that Culicoides may respond to a rapidly decreasing temperature by shifting to an earlier host-seeking time, though this association needs further study.

Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022.

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3-8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Species-level Microbiota of Biting Midges and Ticks from Poyang Lake.

Objective: The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area, namely, Qunlu Practice Base, Peach Blossom Garden, and Huangtong Animal Husbandry, and whether vectors carry any bacterial pathogens that may cause diseases to humans, to provide scientific basis for prospective pathogen discovery and disease prevention and control. Methods: Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit (OPU) analysis, we characterized the species-level microbial community structure of two important vector species, biting midges and ticks, including 33 arthropod samples comprising 3,885 individuals, collected around Poyang Lake. Results: A total of 662 OPUs were classified in biting midges, including 195 known species and 373 potentially new species, and 618 OPUs were classified in ticks, including 217 known species and 326 potentially new species. Surprisingly, OPUs with potentially pathogenicity were detected in both arthropod vectors, with 66 known species of biting midges reported to carry potential pathogens, including Asaia lannensis and Rickettsia bellii, compared to 50 in ticks, such as Acinetobacter lwoffii and Staphylococcus sciuri. We found that Proteobacteria was the most dominant group in both midges and ticks. Furthermore, the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria. Pantoea sp7 was predominant in biting midges, while Coxiella sp1 was enriched in ticks. Meanwhile, Coxiella spp., which may be essential for the survival of Haemaphysalis longicornis Neumann, were detected in all tick samples. The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors. Conclusion: Biting midges and ticks carry large numbers of known and potentially novel bacteria, and carry a wide range of potentially pathogenic bacteria, which may pose a risk of infection to humans and animals. The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Decoding the genome of bloodsucking midge Forcipomyia taiwana (Diptera: Ceratopogonidae): Insights into odorant receptor expansion.

Biting midges, notably those within the Ceratopogonidae family, have long been recognized for their epidemiological significance, both as nuisances and vectors for disease transmission in vertebrates. Despite their impact, genomic insights into these insects, particularly beyond the Culicoides genus, remain limited. In this study, we assembled the Forcipomyia taiwana (Shiraki) genome, comprising 113 scaffolds covering 130.4 Mbps-with the longest scaffold reaching 7.6 Mbps and an N50 value of 2.6 Mbps-marking a pivotal advancement in understanding the genetic architecture of ceratopogonid biting midges. Phylogenomic analyses reveal a shared ancestry between F. taiwana and Culicoides sonorensis Wirth & Jones, dating back approximately 124 million years, and highlight a dynamic history of gene family expansions and contractions within the Ceratopogonidae family. Notably, a substantial expansion of the odorant receptor (OR) gene family was observed, which is crucial for the chemosensory capabilities that govern biting midges' interactions with their environment, including host seeking and oviposition behaviors. The distribution of OR genes across the F. taiwana genome displays notable clusters on scaffolds, indicating localized tandem gene duplication events. Additionally, several collinear regions were identified, hinting at segmental duplications, inversions, and translocations, contributing to the olfactory system's evolutionary complexity. Among the 156 ORs identified in F. taiwana, 134 are biting midge-specific ORs, distributed across three distinct clades, each exhibiting unique motif features that distinguish them from the others. Through weighted gene co-expression network analysis, we correlated distinct gene modules with sex and reproductive status, laying the groundwork for future investigations into the interplay between gene expression and adaptive behaviors in F. taiwana. In conclusion, our study not only highlights the unique olfactory repertoire of ceratopogonid biting midges but also sets the stage for future studies into the genetic underpinnings of their unique biological traits and ecological strategies.

Establishment of a mouse allergy model for Culicoides (Diptera: Ceratopogonidae).

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.

Comparative genomics of a novel Erwinia species associated with the Highland midge (Culicoides impunctatus).

Erwinia (Enterobacterales: Erwiniaceae) are a group of cosmopolitan bacteria best known as the causative agents of various plant diseases. However, other species in this genus have been found to play important roles as insect endosymbionts supplementing the diet of their hosts. Here, I describe Candidatus Erwinia impunctatus (Erwimp) associated with the Highland midge Culicoides impunctatus (Diptera: Ceratopogonidae), an abundant biting pest in the Scottish Highlands. The genome of this new Erwinia species was assembled using hybrid long and short read techniques, and a comparative analysis was undertaken with other members of the genus to understand its potential ecological niche and impact. Genome composition analysis revealed that Erwimp is similar to other endophytic and ectophytic species in the genus and is unlikely to be restricted to its insect host. Evidence for an additional plant host includes the presence of a carotenoid synthesis operon implicated as a virulence factor in plant-associated members in the sister genus Pantoea. Unique features of Erwimp include several copies of intimin-like proteins which, along with signs of genome pseudogenization and a loss of certain metabolic pathways, suggests an element of host restriction seen elsewhere in the genus. Furthermore, a screening of individuals over two field seasons revealed the absence of the bacteria in Culicoides impunctatus during the second year indicating this microbe-insect interaction is likely to be transient. These data suggest that Culicoides impunctatus may have an important role to play beyond a biting nuisance, as an insect vector transmitting Erwimp alongside any conferred impacts to surrounding biota.

Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis.

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.

Diversity of Culicoides in the middle belt of Ghana with Implications on the transmission of Mansonella perstans; a molecular approach.

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.

Three new species of Forcipomyia (Lepidohelea) (Diptera: Ceratopogonidae) from West Bengal, India.

Three new species of the subgenus Lepidohelea Kieffer, 1917 of the genus Forcipomyia were described from the northern region of West Bengal based on morphological data. Forcipomyia (L.) inflatistyla sp. n. was collected from the Dooars region (Jayanti, near the Core area of Buxa Tiger Reserve), while two new species F. (L.) ranula and F. (L.) sulcoa were procured from Kalimpong in the Eastern Himalaya. Discovery of the three new species raises the total number of species of the genus Forcipomyia to 86 in India. A key to the Indian species of Lepidohelea is also provided.

Taxonomy and feeding of Forcipomyia (Microhelea) fuliginosa Meigen and Forcipomyia (Microhelea) esakiana Tokunaga (Diptera: Ceratopogonidae) on Antheraea mylitta Drury (Lepidoptera: Saturniidae).

Taxonomic equivocality and complexity exist in the two species of Ceratopogonids, Forcipomyia (Microhelea) fuliginosa Meigen and Forcipomyia (Microhelea) esakiana Tokunaga. Incongruencies and inaccuracies in species identification restrict further biological and ecological studies on the host-ectoparasite association. Preferential landing and hemolymphophagy of F. fuliginosa and F. esakiana on Antheraea mylitta Drury larva were studied under field conditions. The silkworm A. mylitta is reared in the tasar sericulture industry, contributing 1466 metric tons (202122) of indigenous raw silk in India. Ectoparasitic behavior of the biting midges, F. fuliginosa, and F. esakiana is an increasing threat to the silkworm, necessitating proper identification. Intra and inter-variations of these two closely related species have been stated. Morphological-based identification of these species has been substantiated with COX1 molecular data. A Bayesian-modeled approach to reconstruct the dendrogram of the two species based on the COX1 sequences generated has been presented along with the referred sequences of F. fuliginosa from Genebank. The species F. esakiana is being reported for the first time from India, along with its ectoparasitic hemolymphophagous nature. The role of these insectivorous species in transmitting pathogens to the larvae of tasar silk needs further investigation.

Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021-2022.

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.

Large-Scale Cytochrome C Oxidase Subunit I Gene Data Analysis for the Development of a Multiplex Polymerase Chain Reaction Test Capable of Identifying Biting Midge Vector Species and Haplotypes (Diptera: Ceratopogonidae) of the Culicoides Subgenus Avaritia Fox, 1955.

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%).

Isolation and whole-genome sequence analysis of Balagodu virus in Japan.

Whole-genome sequencing of a virus isolated from Culicoides biting midges in southern Japan in 2020 revealed that it is a strain of Balagodu virus (BLGV; genus Orthobunyavirus; family Peribunyaviridae; order Bunyavirales). A solitary instance of BLGV isolation occurred in India in 1963. All assembled segments comprise complete protein-coding sequences that are similar to those of other orthobunyaviruses. The consensus 3'- and 5'-terminal sequences of orthobunyaviruses' genomic RNAs are also conserved in the Japanese BLGV strain. Here, we update the geographic distribution of BLGV and provide its complete sequence, contributing to the clarification of orthobunyavirus phylogeny.

Variable gut pH as a potential mechanism of tolerance to Bacillus thuringiensis subsp. israelensis toxins in the biting midge Culicoides sonorensis.

BACKGROUND: Toxins of Bacillus thuringiensis subsp. israelensis (Bti) are safer alternatives for controlling dipteran pests such as black flies and mosquitoes. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is an important pest of livestock in much of the United States and larval midges utilize semi-aquatic habitats which are permissive for Bti product application. Reports suggest that Bti products are ineffective at killing biting midges despite their taxonomic relation to black flies and mosquitoes. Here, we investigate the toxicity of a Bti-based commercial insecticide and its active ingredient in larval Culicoides sonorensis. A suspected mechanism of Bti tolerance is an acidic larval gut, and we used a pH indicator dye to examine larval Culicoides sonorensis gut pH after exposure to Bti. RESULTS: The lethal concentration to kill 90% (LC90) of larvae of the commercial product (386 mg/L) was determined to be almost 10 000 times more than that of some mosquito species, and no concentration of active ingredient tested achieved 50% larval mortality. The larval gut was found to be more acidic after exposure to Bti which inhibits Bti toxin activity. By comparison, 100% mortality was achieved in larval Aedes aegypti at the product's label rate for this species and mosquito larvae had alkaline guts regardless of treatment. Altering the larval rearing water to alkaline conditions enhanced Bti efficacy when using the active ingredient. CONCLUSION: We conclude that Bti is not practical for larval Culicoides sonorensis control at the same rates as mosquitos but show that alterations or additives to the environment could make the products more effective. © 2024 Society of Chemical Industry.