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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. Fishers 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.

Background: Clinical cases of leishmaniasis caused by Leishmania (Mundinia) parasites have been increasingly reported in Southeast Asia, particularly Thailand. Recent evidence has shown that Leishmania (Mundinia) parasites successfully developed into infective metacyclic promastigotes in Culicoides biting midges, strongly supporting their putative role in disease transmission. However, Culicoides diversity, host preference, and Leishmania prevalence in endemic areas remain largely unknown. Methods: We investigated the seasonal dynamics, infection prevalence, and blood meal identification of Culicoides collected from the emerging focus of visceral leishmaniasis in Lampang Province, Northern Thailand, during 2021–2023. Midge samples were molecularly screened for Leishmania using SSU rRNA-qPCR and ITS1-PCR, followed by Sanger plasmid sequencing, and parasite haplotype diversity was analyzed. Host blood meal origins were comparatively identified using host-specific Cytb-PCRs and a nanopore-based metabarcoding approach. Results: A total of 501 parous and gravid females and 46 blood-engorged ones belonging to at least 17 species of five subgenera (Remmia, Trithecoides, Avaritia, Hoffmania, and Meijerehelea) and two species groups (Shortti and Calvipalpis) were collected with temporal differences in abundance. Leishmania was detected by SSU rRNA-qPCR in 31 samples of at least 11 midge species, consisting of Culicoides oxystoma, C. guttifer, C. orientalis, C. mahasarakhamense, C (Trithecoides) spp., C. innoxius, C. shortti, C. arakawae, C. sumatrae, C. actoni, and C. fulvus, with the overall infection prevalence of 5.7%. The latter six species represent the new records as putative leishmaniasis vectors in Northern Thailand. The ITS1-PCR and plasmid sequencing revealed that Leishmania martiniquensis was predominantly identified in all qPCR-positive species, whereas L. orientalis was identified only in three C. oxystoma samples. The most dominant haplotype of L. martiniquensis in Thailand was genetically intermixed with those from other geographical regions, confirming its globalization. Neutrality test statistics were also significantly negative on regional and country-wide scales, suggesting rapid population expansion or selective sweeps. Nanopore-based blood meal analysis revealed that most Culicoides species are mammalophilic, with peridomestic and wild mammals (cow, pig, deer, and goat-like species) and humans as hosts, while C. guttifer and C. mahasarakhamense fed preferentially on chickens. Conclusions: This study revealed seasonal dynamics and sympatric circulation of L. martiniquensis and L. orientalis in different species of Culicoides. Evidence of human blood feeding was also demonstrated, implicating Culicoides as putative vectors of human leishmaniasis in endemic areas. Further research is therefore urgently needed to develop vector control strategies and assess the infection status of their reservoir hosts to effectively minimize disease transmission. [ABSTRACT FROM AUTHOR]

Abstract Background Culicoides biting midges exhibit a global spatial distribution and are the main vectors of several viruses of veterinary importance, including bluetongue (BT) and African horse sickness (AHS). Many environmental and anthropological factors contribute to their ability to live in a variety of habitats, which have the potential to change over the years as the climate changes. Therefore, as new habitats emerge, the risk for new introductions of these diseases of interest to occur increases. The aim of this study was to model distributions for two primary vectors for BT and AHS (Culicoides imicola and Culicoides bolitinos) using random forest (RF) machine learning and explore the relative importance of environmental and anthropological factors in a region of South Africa with frequent AHS and BT outbreaks. Methods Culicoides capture data were collected between 1996 and 2022 across 171 different capture locations in the Western Cape. Predictor variables included climate-related variables (temperature, precipitation, humidity), environment-related variables (normalised difference vegetation index—NDVI, soil moisture) and farm-related variables (livestock densities). Random forest (RF) models were developed to explore the spatial distributions of C. imicola, C. bolitinos and a merged species map, where both competent vectors were combined. The maps were then compared to interpolation maps using the same capture data as well as historical locations of BT and AHS outbreaks. Results Overall, the RF models performed well with 75.02%, 61.6% and 74.01% variance explained for C. imicola, C. bolitinos and merged species models respectively. Cattle density was the most important predictor for C. imicola and water vapour pressure the most important for C. bolitinos. Compared to interpolation maps, the RF models had higher predictive power throughout most of the year when species were modelled individually; however, when merged, the interpolation maps performed better in all seasons except winter. Finally, midge densities did not show any conclusive correlation with BT or AHS outbreaks. Conclusion This study yielded novel insight into the spatial abundance and drivers of abundance of competent vectors of BT and AHS. It also provided valuable data to inform mathematical models exploring disease outbreaks so that Culicoides-transmitted diseases in South Africa can be further analysed. Graphical Abstract

Background: Culicoides biting midges exhibit a global spatial distribution and are the main vectors of several viruses of veterinary importance, including bluetongue (BT) and African horse sickness (AHS). Many environmental and anthropological factors contribute to their ability to live in a variety of habitats, which have the potential to change over the years as the climate changes. Therefore, as new habitats emerge, the risk for new introductions of these diseases of interest to occur increases. The aim of this study was to model distributions for two primary vectors for BT and AHS (Culicoides imicola and Culicoides bolitinos) using random forest (RF) machine learning and explore the relative importance of environmental and anthropological factors in a region of South Africa with frequent AHS and BT outbreaks. Methods: Culicoides capture data were collected between 1996 and 2022 across 171 different capture locations in the Western Cape. Predictor variables included climate-related variables (temperature, precipitation, humidity), environment-related variables (normalised difference vegetation index—NDVI, soil moisture) and farm-related variables (livestock densities). Random forest (RF) models were developed to explore the spatial distributions of C. imicola, C. bolitinos and a merged species map, where both competent vectors were combined. The maps were then compared to interpolation maps using the same capture data as well as historical locations of BT and AHS outbreaks. Results: Overall, the RF models performed well with 75.02%, 61.6% and 74.01% variance explained for C. imicola, C. bolitinos and merged species models respectively. Cattle density was the most important predictor for C. imicola and water vapour pressure the most important for C. bolitinos. Compared to interpolation maps, the RF models had higher predictive power throughout most of the year when species were modelled individually; however, when merged, the interpolation maps performed better in all seasons except winter. Finally, midge densities did not show any conclusive correlation with BT or AHS outbreaks. Conclusion: This study yielded novel insight into the spatial abundance and drivers of abundance of competent vectors of BT and AHS. It also provided valuable data to inform mathematical models exploring disease outbreaks so that Culicoides-transmitted diseases in South Africa can be further analysed. [ABSTRACT FROM AUTHOR]

Background: Culicoides midges have been well-studied in Spain, particularly over the last 20 years, mainly because of their role as vectors of arboviral diseases that affect livestock. Most studies on Culicoides are conducted using suction light traps in farmed environments, but studies employing alternative trapping techniques or focusing on natural habitats are scarce. Methods: In the present study, we analyze Culicoides captured in 2023 at 476 sites in western Andalusia (southern Spain) using carbon dioxide-baited Biogents (BG)-sentinel traps across different ecosystems. Results: We collected 3,084 Culicoides midges (3060 females and 24 males) belonging to 23 species, including the new species Culicoides grandifovea sp. nov. and the first record of Culicoides pseudolangeroni for Europe. Both species were described with morphological and molecular methods and detailed data on spatial distribution was also recorded. The new species showed close phylogenetic relations with sequences from an unidentified Culicoides from Morocco (92.6% similarity) and with Culicoides kurensis. Culicoides imicola was the most abundant species (17.4%), followed by Culicoides grandifovea sp. nov. (14.6%) and Culicoides kurensis (11.9%). Interestingly, Culicoides montanus was the only species of the obsoletus and pulicaris species complexes captured, representing the first record of this species in southern Spain. A total of 53 valid Culicoides species have been reported in the area, with 48 already reported in literature records and 5 more added in the present study. Information on the flight period for the most common Culicoides species is also provided. Conclusions: To the best of our knowledge, our study represents the most comprehensive effort ever done on nonfarmland habitats using carbon-dioxide baited suction traps for collecting Culicoides. Our data suggests that using carbon dioxide traps offers a completely different perspective on Culicoides communities compared with routinely used light traps, including the discovery of previously unrecorded species. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical Abstract

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]

Abstract Background Bluetongue is a non-contagious viral disease that affects 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 first 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-qPCR-positive pools of the studied Culicoides species exhibited high levels of BTV positivity in each body part (head, thorax, and abdomen), confirming 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 significant risk of Culicoides transmitting the BTV in Morocco. Conclusions The detection of BTV positivity in Culicoides spp. (lacking wing spots that allow their differentiation according to morphological identification keys) suggested that other Culicoides species are competent for BTV transmission in Morocco. The study results indicated, for the first 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. Graphical Abstract

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]

Abstract 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. Graphical Abstract

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]

Background: Culicoides Latreille (Diptera: Ceratopogonidae) is a genus of hematophagous midges feeding on various vertebrate hosts and serving as a vector for numerous pathogens important to livestock and wildlife health. North American pathogens include bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses. Little is known about Culicoides spp. distribution and abundance and species composition in Ontario, Canada, despite bordering numerous U.S. states with documented Culicoides spp. and BT and EHD virus activity. We sought to characterize Culicoides spp. distribution and abundance and to investigate whether select meteorological and ecological risk factors influenced the abundance of Culicoides biguttatus, C. stellifer, and the subgenus Avaritia trapped throughout southern Ontario. Methods: From June to October of 2017 to 2018, CDC-type LED light suction traps were placed on twelve livestock-associated sites across southern Ontario. Culicoides spp. collected were morphologically identified to the species level when possible. Associations were examined using negative binomial regression among C. biguttatus, C. stellifer, and subgenus Avaritia abundance, and select factors: ambient temperature, rainfall, primary livestock species, latitude, and habitat type. Results: In total, 33,905 Culicoides spp. midges were collected, encompassing 14 species from seven subgenera and one species group. Culicoides sonorensis was collected from three sites during both years. Within Ontario, the northern trapping locations had a pattern of seasonal peak abundance in August (2017) and July (2018), and the southern locations had abundance peaks in June for both years. Culicoides biguttatus, C. stellifer, and subgenus Avaritia were significantly more abundant if ovine was the primary livestock species at trapping sites (compared to bovine). Culicoides stellifer and subgenus Avaritia were significantly more abundant at mid- to high-temperature ranges on trap days (i.e., 17.3–20.2 and 20.3–31.0 °C compared to 9.5–17.2 °C). Additionally, subgenus Avaritia were significantly more abundant if rainfall 4 weeks prior was between 2.7 and 20.1 mm compared to 0.0 mm and if rainfall 8 weeks prior was between 0.1 and 2.1 mm compared to 0.0 mm. Conclusions: Results from our study describe Culicoides spp. distribution in southern Ontario, the potential for spread and maintenance of EHD and BT viruses, and concurrent health risks to livestock and wildlife in southern Ontario in reference to certain meteorological and ecological risk factors. We identified that Culicoides spp. are diverse in this province, and appear to be distinctly distributed spatially and temporally. The livestock species present, temperature, and rainfall appear to have an impact on the abundance of C. biguttatus, C. stellifer, and subgenus Avaritia trapped. These findings could help inform targeted surveillance, control measures, and the development of management guides for Culicoides spp. and EHD and BT viruses in southern Ontario, Canada. [ABSTRACT FROM AUTHOR]

Abstract Background Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. Method We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a − 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. Results We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. Conclusions These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera. Graphical Abstract

Background: Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. Method: We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a − 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. Results: We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. Conclusions: These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera. [ABSTRACT FROM AUTHOR]

Background: Orbivirus-induced hemorrhagic diseases cause high mortality in wild and captive white-tailed deer in North America. The role of different Culicoides species in Orbivirus transmission outside of areas of intensive animal production has not been established. At our study location, bluetongue virus (BTV) RNA-positive female Culicoides debilipalpis pools have been detected annually since 2012 when BTV transmission was noted in a captive deer herd. Identifying specific larval habitats of suspected vectors at active transmission sites is crucial both for identifying the source of the vectors and for subsequently planning intervention actions. Since C. debilipalpis larvae are known to develop in tree holes, this study was designed to use DNA barcoding to identify larvae collected from tree holes. Methods: Adult female Culicoides were collected using light or emergence traps and morphologically identified to 11 species. Culicoides sonorensis were also obtained from a laboratory colony. Substrate was collected from tree holes and flooded with water to harvest floating larvae. Total DNA from three to seven adult females per species and 19 larvae was extracted. Two loci of the nuclear 18S ribosomal RNA (rRNA) gene, one locus each of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear 28S rRNA gene were amplified using loci-specific primers. Results: All 61 adults were sequenced at each of the four loci under study. Since no single locus delineated all putative species and the COI locus yielded unreliable pseudogenes for two individuals of C. arboricola, sequences of all four loci were concatenated to maximize species separation and allow for larval association with identified adults. Sixteen larvae were clearly assigned to species based on DNA barcoding and phylogenetic results. Multiple larvae were assigned to each of the C. debilipalpis clade, the C. villosipennis clade, the C. arboricola clade and the C. nanus clade. Conclusions: Of the approximately 62 species described in the southeast USA, 21 have now been barcoded and sequences are publicly available. In this study, we constructed a database composed of species-specific sequences of adult Culicoides and then identified larvae to species by matching their corresponding sequences with adults. Since Culicoides larvae are difficult to identify, using DNA barcoding to facilitate larval habitat surveys can be a valuable tool. [ABSTRACT FROM AUTHOR]

Abstract Background Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that can cause fatal vector-borne diseases in white-tailed deer (Odocoileus virginianus). Trapping methods for collecting potential Culicoides vectors of orbiviruses were compared to optimize surveillance studies. Methods The number of captured midges and the virus infection rates of midge pools were compared for dry ice-baited Centers for Disease Control and Prevention (CDC) traps with or without black light. The number of individual midges of different Culicoides species captured at different crepuscular and nocturnal periods using rotator traps also was determined. The number of species/specimens of Culicoides was measured using five different trap methods including three animal-baited methods, a CDC trap with black light, and a CDC trap with no light. Results In trial one, there was no significant difference (P = 0.37) in the proportion of BTV-infected flies caught in traps with light compared to traps without light. However, there was a significant difference (P = 0.026) for EHDV-infected flies, and 89% were captured in traps with light. In trial two, more specimens of C. debilipalpis were captured in the morning hours (06:00–08:00) than in the evening hours (18:00–20:00). For trial three, the animal-baited traps did not capture any species of Culicoides that were not captured in the CDC light traps. There was no significant difference (P = 0.22) in total specimens captured among all five trap types. Conclusions Specimens of Culicoides infected with BTV were not repelled by light traps in the first trial, while the majority of the specimens positive for EHDV were caught in traps with light. For the second trial, specimens of C. debilipalpis were most abundant during early morning hours, and thus spray applications of insecticides for control of that species may be more effective at sunrise rather than sunset. For objective three, no animal-baited trapping method collected different species of midges when compared to the CDC traps with light, which is unlike certain studies conducted in other geographical regions. Graphical abstract

Abstract Background Bluetongue is a serious disease of ruminants caused by the bluetongue virus (BTV). BTV is transmitted by biting midges (Culicoides spp.). Serological evidence from livestock and the presence of at least one competent vector species of Culicoides suggests that transmission of BTV is possible and may have occurred in Kazakhstan. Methods We estimated the risk of transmission using a mathematical model of the reproduction number R 0 for bluetongue. This model depends on livestock density and climatic factors which affect vector density. Data on climate and livestock numbers from the 2466 local communities were used. This, together with previously published model parameters, was used to estimate R 0 for each month of the year. We plotted the results on isopleth maps of Kazakhstan using interpolation to smooth the irregular data. We also mapped the estimated proportion of the population requiring vaccination to prevent outbreaks of bluetongue. Results The results suggest that transmission of bluetongue in Kazakhstan is not possible in the winter from October to March. Assuming there are vector-competent species of Culicoides endemic in Kazakhstan, then low levels of risk first appear in the south of Kazakhstan in April before spreading north and intensifying, reaching maximum levels in northern Kazakhstan in July. The risk declined in September and had disappeared by October. Conclusion These results should aid in surveillance efforts for the detection and control of bluetongue in Kazakhstan by indicating where and when outbreaks of bluetongue are most likely to occur. The results also indicate where vaccination efforts should be focussed to prevent outbreaks of disease. Graphical abstract

Background: Aquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019. Methods: Culicoides were captured by light suction traps baited with CO2 and by sweep netting vegetation. Blood meals and species identification of blood-fed specimens were determined using cytochrome c oxidase I subunit (COI) DNA barcoding. Multivariate generalized linear models were used to evaluate the associations between the abundance of Culicoides, the species richness and other parameters. Results: The 4973 identified specimens comprised 28 species of Culicoides. These included two species reported for the first time in northern Spain, thus raising to 54 the number of Culicoides species described in the region. Specimens of all 28 species and 99.6% of the total specimens collected were caught in suction traps, while sweep netting vegetation revealed just 11 species and 0.4% of the total specimens. Midge abundance peaked in June/early July, with five species comprising > 80% of the captures: Culicoides alazanicus (24.9%), Culicoides griseidorsum (20.3%), Culicoides poperinghensis (16.2%), Culicoides kibunensis (10.7%) and Culicoides clastrieri (9.6%). DNA barcode analysis of blood meals from eight Culicoides species revealed that they fed on 17 vertebrate species (3 mammals and 14 birds). Species in the subgenus Avaritia were primarily ornithophilic, except for C. griseidorsum and C. poperinghensis. Host DNA from blood meals was successfully amplified from 75% of blood-fed females. A pictorial blood meal digestion scale is provided to accurately assess the blood-fed status of female Culicoides. Conclusions: The large number of different blood meal sources identified in the midges captured in this study signals the likely importance of wild birds and mammals (e.g. red deer and wild boar) as reservoir/amplifying hosts for pathogens. Available hosts are more exposed to being bitten by biting midge populations in aquatic ecosystems in late spring and early summer. [ABSTRACT FROM AUTHOR]

Abstract Background Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. Methods Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. Results Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoides jacobsoni was identified as TIBOV, while the other two viruses from C. orientalis and C. tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. Conclusions The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known. Graphical abstract

Abstract Background Reunion Island regularly faces outbreaks of bluetongue and epizootic hemorrhagic diseases, two insect-borne orbiviral diseases of ruminants. Hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae) are the vectors of bluetongue (BTV) and epizootic hemorrhagic disease (EHDV) viruses. In a previous study, statistical models based on environmental and meteorological data were developed for the five Culicoides species present in the island to provide a better understanding of their ecology and predict their presence and abundance. The purpose of this study was to couple these statistical models with a Geographic Information System (GIS) to produce dynamic maps of the distribution of Culicoides throughout the island. Methods Based on meteorological data from ground weather stations and satellite-derived environmental data, the abundance of each of the five Culicoides species was estimated for the 2214 husbandry locations on the island for the period ranging from February 2016 to June 2018. A large-scale Culicoides sampling campaign including 100 farms was carried out in March 2018 to validate the model. Results According to the model predictions, no husbandry location was free of Culicoides throughout the study period. The five Culicoides species were present on average in 57.0% of the husbandry locations for C. bolitinos Meiswinkel, 40.7% for C. enderleini Cornet & Brunhes, 26.5% for C. grahamii Austen, 87.1% for C. imicola Kieffer and 91.8% for C. kibatiensis Goetghebuer. The models also showed high seasonal variations in their distribution. During the validation process, predictions were acceptable for C. bolitinos, C. enderleini and C. kibatiensis, with normalized root mean square errors (NRMSE) of 15.4%, 13.6% and 16.5%, respectively. The NRMSE was 27.4% for C. grahamii. For C. imicola, the NRMSE was acceptable (11.9%) considering all husbandry locations except in two specific areas, the Cirque de Salazie—an inner mountainous part of the island—and the sea edge, where the model overestimated its abundance. Conclusions Our model provides, for the first time to our knowledge, an operational tool to better understand and predict the distribution of Culicoides in Reunion Island. As it predicts a wide spatial distribution of the five Culicoides species throughout the year and taking into consideration their vector competence, our results suggest that BTV and EHDV can circulate continuously on the island. As further actions, our model could be coupled with an epidemiological model of BTV and EHDV transmission to improve risk assessment of Culicoides-borne diseases on the island. Graphic Abstract

Background: Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides Latreille is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis Wirth & Jones is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, single-nucleotide polymorphism (SNP) analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance. Methods: The aim of the current study was to develop a reliable and affordable way of distinguishing between the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers, which were also tested for species differentiation. Results: A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recovered the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately distinguishing between the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested. Conclusions: These microsatellites provide an affordable way to distinguish between the sibling species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America. [ABSTRACT FROM AUTHOR]

Abstract Background Culicoides imicola (Diptera: Ceratopogonidae) is an important Afrotropical and Palearctic vector of disease, transmitting viruses of animal health and economic significance including African horse sickness and bluetongue viruses. Maternally inherited symbiotic bacteria (endosymbionts) of arthropods can alter the frequency of COI (cytochrome c oxidase subunit I) mitochondrial haplotypes (mitotypes) in a population, masking the true patterns of host movement and gene flow. Thus, this study aimed to assess the mtDNA structure of C. imicola in relation to infection with Candidatus Cardinum hertigii (Bacteroides), a common endosymbiont of Culicoides spp. Methods Using haplotype network analysis, COI Sanger sequences from Cardinium-infected and -uninfected C. imicola individuals were first compared in a population from South Africa. The network was then extended to include mitotypes from a geographic range where Cardinium infection has previously been investigated. Results The mitotype network of the South African population demonstrated the presence of two broad mitotype groups. All Cardinium-infected specimens fell into one group (Fisher’s exact test, P = 0.00071) demonstrating a linkage disequilibrium between endosymbiont and mitochondria. Furthermore, by extending this haplotype network to include other C. imicola populations from the Mediterranean basin, we revealed mitotype variation between the Eastern and Western Mediterranean basins (EMB and WMB) mirrored Cardinium-infection heterogeneity. Conclusions These observations suggest that the linkage disequilibrium of Cardinium and mitochondria reflects endosymbiont gene flow within the Mediterranean basin but may not assist in elucidating host gene flow. Subsequently, we urge caution on the single usage of the COI marker to determine population structure and movement in C. imicola and instead suggest the complementary utilisation of additional molecular markers.

Abstract Background Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, in both replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides. This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the UK under laboratory conditions. Methods Over 18,000 Culicoides adults were collected from the field using 4 W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the UK during the summer months and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-h period. Results The proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4 °C) compared with populations collected in the summer (10 °C). Within the subgenus Avaritia, Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup. Populations of Culicoides impunctatus collected in the North East of England were only active once temperatures reached 14 °C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders. Conclusions These findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. These data, alongside that defining thresholds for virus replication within Culicoides, provide a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance.

Abstract Background Culicoides (Diptera: Ceratopogonidae) are vectors for many arboviruses. At least 20 species are considered as vectors or potential vectors of bluetongue virus (BTV) which cause bluetongue disease in ruminants. A BTV prevalence of 30–50% among cattle and goats in tropical southern Yunnan Province, China, prompted an investigation of the potential BTV vectors in this area. Methods Culicoides were collected by light trapping at three sites in the tropical region of Yunnan Province. Species were identified based on morphology and DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in these specimens. Phylogenetic analysis was used to analyze the cox1 sequences of Culicoides specimens infected with BTV. Results Approximately 67,000 specimens of Culicoides were collected, of which 748 were tested for the presence of BTV. Five specimens, including two of Culicoides jacobsoni, one of C. tainanus and two of C. imicola, were identified as infected with BTV. No specimens of C. (subgenus Trithecoides) or C. oxystoma tested were positive for BTV infection. Conclusions To our knowledge this is the first report of C. jacobsoni as a potential BTV vector and the fourth report of an association between C. tainanus and BTV, as well as the first direct evidence of an association between BTV and C. imicola in Asia. A fourth potential cryptic species within C. tainanus was identified in this study. Further analysis is required to confirm the importance of C. jacobsoni and C. tainanus in BTV epidemiology in Asia.

BackgroundPathogen manipulation of host behavior can greatly impact vector-borne disease transmission, but almost no attention has been paid to how it affects disease surveillance. Bluetongue virus (BTV), transmitted by Culicoides biting midges, is a serious disease of ruminant livestock that can cause high morbidity and mortality and significant economic losses. Worldwide, the majority of surveillance for Culicoides to assess BTV transmission risk is done using UV-light traps. Here we show that field infection rates of BTV are significantly lower in midge vectors collected using traps baited with UV light versus a host cue (CO2).MethodsWe collected Culicoides sonorensis midges in suction traps baited with CO2, UV-light, or CO2 + UV on three dairies in southern California to assess differences in the resulting estimated infection rates from these collections. Pools of midges were tested for BTV by qRT-PCR, and maximum likelihood estimates of infection rate were calculated by trap. Infection rate estimates were also calculated by trapping site within a dairy. Colonized C. sonorensis were orally infected with BTV, and infection of the structures of the compound eye was examined using structured illumination microscopy.ResultsUV traps failed entirely to detect virus both early and late in the transmission season, and underestimated virus prevalence by as much as 8.5-fold. CO2 + UV traps also had significantly lower infection rates than CO2-only traps, suggesting that light may repel infected vectors. We found very high virus levels in the eyes of infected midges, possibly causing altered vision or light perception. Collecting location also greatly impacts our perception of virus activity.ConclusionsBecause the majority of global vector surveillance for bluetongue uses only light-trapping, transmission risk estimates based on these collections are likely severely understated. Where national surveillance programs exist, alternatives to light-trapping should be considered. More broadly, disseminated infections of many arboviruses include infections in vectors' eyes and nervous tissues, and this may be causing unanticipated behavioral effects. Field demonstrations of pathogen-induced changes in vector behavior are quite rare, but should be studied in more systems to accurately predict vector-borne disease transmission.

Background: Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges. These two species are vectors of disease in livestock of economic importance. As described in the literature, morphological identification for discrimination between them is still unclear. However, species-specific identification is necessary to solve taxonomic challenges between species and to understand their roles in disease transmission and epidemiology. This study aims to develop accurate tools to discriminate C. oxystoma from C. kingi using traditional morphometry and polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) assays for use in developing countries. Methods: Specimens were collected from the region of Kairouan in central Tunisia. A total of 446 C. oxystoma/C. kingi individuals were identified using traditional morphometric analyses combined with PCR–RFLP of the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were performed from the head, wings, and abdomen of slide-mounted specimens, and six ratios were calculated between these measurements. Multivariate analyses of the morphometric measurements were explored to identify which variables could lead to accurate species identification. Results: Four variables, namely antennae, wings, spermathecae, and palpus length, were suitable morphometric characteristics to differentiate between the species. Digestion with the SspI restriction enzyme of the PCR product led to good discriminative ability. Molecular procedures and phylogenetic analysis confirmed the efficiency of this simple and rapid PCR–RFLP method. Conclusions: This study highlights for the first time in Tunisia the presence of C. oxystoma and its discrimination from C. kingi using abdominal measurements and the PCR–RFLP method. This approach could be applied in future epidemiological studies at the national and international levels. [ABSTRACT FROM AUTHOR]

Abstract Background Bovine ephemeral fever virus (Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined. Methods Colony lines of biting midges (Culicoides sonorensis) and mosquitoes (Aedes aegypti, Culex pipiens and Culex quinquefasciatus) were infected with a strain of BEFV originating from Israel by feeding on blood–virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves. Results There was no evidence of BEFV replication within mosquitoes fed on blood/virus suspensions for mosquitoes of any species tested for each of the three colony lines. In 170 C. sonorensis fed on the blood/virus suspension, BEFV RNA was detected in the bodies of 13 individuals and in the heads of two individuals, indicative of fully disseminated infections and an oral susceptibility rate of 1.2%. BEFV RNA replication was further demonstrated in all C. sonorensis that were inoculated by the intrathoracic route with virus after 5, 6 or 7 days post-infection. Despite this, transmission of BEFV could not be demonstrated when infected C. sonorensis were allowed to feed on calves. Conclusions No evidence for infection or dissemination of BEFV (bovine/Israel/2005-6) in mosquitoes of three different species was found. Evidence was found for infection of C. sonorensis by the oral route. However, attempts to transmit BEFV to calves from infected C. sonorensis failed. These results highlight the challenge of defining the natural vector of BEFV and of establishing an in vivo transmission model. The results are discussed with reference to the translation of laboratory-based studies to inference of vector competence in the field. Graphic abstract

Abstract Background We assessed the impact of two sand fly insecticide interventions (insecticide spraying and insecticide-impregnated dog collars) on the peridomestic abundance and distribution of mosquitoes (Culicidae) and biting midges (Ceratopogonidae) in western São Paulo (Brazil) in a long-term (42-month) evaluation. Both of these dipteran groups are vectors of diseases of medical and veterinary relevance to humans and domestic animals in Brazil. Methods The interventions in the 3-arm stratified randomised control trial were: pheromone + insecticide (PI) (chicken roosts were sprayed with microencapsulated lambda-cyhalothrin; pheromone lure has no effect on the Diptera pests studied here); dog-collars (DC) (dogs fitted with deltamethrin-impregnated collars); and control (C) (unexposed to pyrethroids) were extended by 12 months. During that time, adult mosquitoes and midges were sampled along 280 households at three household locations (inside human dwellings, dog sleeping sites and chicken roosts). Results We collected 3145 culicids (9 genera, 87.6% Culex spp.) distributed relatively uniformly across all 3 arms: 41.9% at chicken roosts; 37.7% inside houses; and 20.3% at dog sleeping sites. We collected 11,464 Culicoides (15 species) found mostly at chicken roosting sites (84.7%) compared with dog sleeping sites (12.9%) or houses (2.4%). Mosquitoes and Culicoides were most abundant during the hot and rainy season. Increased daytime temperature was marginally associated with increased mosquito abundance (Z = 1.97, P = 0.049) and Culicoides abundance (Z = 1.71, P = 0.087). There was no significant association with daily average rainfall for either group. Household-level mosquito and midge numbers were both significantly reduced by the PI intervention 56% [incidence rate ratio, IRR = 0.54 (95% CI: 0.30–0.97), P ≤ 0.05] and 53% [IRR = 0.47 (95% CI: 0.26–0.85), P ≤ 0.05], respectively, compared to the control intervention. The abundance of both dipteran groups at dog sleeping sites was largely unaffected by the PI and DC interventions. The PI intervention significantly reduced abundance of mosquitoes inside houses (41%) and at chicken roosting sites (48%) and reduced midge abundance by 51% in chicken roosting sites. Conclusions Sprayed insecticide at chicken roosting sites reduced the abundance of mosquitoes and midges at the peridomestic level while dog collars had no effect on numbers for any group.

Abstract Background The Culicoides fauna of Algeria has been historically investigated, leading to the description of many new species by Kieffer in the 1920s, Clastrier in the 1950s or Callot in the 1960s and to a comprehensive inventory by Szadziewski in the 1980s. The emergence of bluetongue in the late 1990s enhanced Culicoides collections made in the country over the last two decades, but information remained mostly unpublished. The aim of this study is therefore to provide a comprehensive and updated checklist of Culicoides biting midge species in Algeria. Methods The literature (published and grey, in French and in English) from 1920 to date on Culicoides collections in Algeria was collected and analyzed in the light of the current taxonomic and systematic knowledge and methods. Fresh Culicoides material was also analyzed using light/suction trap collections carried out from November 2015 to September 2018 in nine localities of the ‘wilayah’ of Tiaret (northwestern Algeria). Slide mounted specimens were identified morphologically using the interactive identification key IIKC and original descriptions. Specimens were then compared with non-type material originating from different countries and partly with type material. Results A total of 13,709 Culicoides, belonging to at least 36 species within 10 subgenera, were examined leading to 10 new records in Algeria, including C. chiopterus, C. dewulfi, C. navaiae, C. grisescens, C. paradoxalis, C. shaklawensis, C. simulator, C. univittatus, C. achrayi and C. picturatus. These new records and all previous records provided by the literature review were discussed. Conclusions We propose a Culicoides checklist for the Algerian fauna of 59 valid species, including species mainly with a large Palaearctic distribution and a specific Mediterranean distribution, and only a few species from the Afrotropical region. Among them, several species, mainly of the subgenera Avaritia and Culicoides, are confirmed or probable vectors of arboviruses important in animal health.

Abstract Background Culicoides biting midges are vectors involved in the biological transmission cycle of important animal diseases such as bluetongue and African horse sickness. In Romania, the first outbreaks of bluetongue were reported in 2014, leading to increased activities within the existing entomological surveillance network. The main goals of the surveillance activities were the establishment of the vector free period in relation to animal trade and the identification of Culicoides species involved in the transmission of the pathogen. This study was conducted on the composition and relative abundance of the species belonging to the genus Culicoides (Diptera: Ceratopogonidae) in certain regions of Romania and provided the opportunity to update the existing checklist of Culicoides species of this country. Methods The study was conducted in 33 of the 42 administrative units (counties), including a total of 659 catches, in 102 locations. The collections were carried out with UV blacklight suction traps (OVI type). The collected insects were preserved in 70% ethanol. Morphological insect identification was carried out using a stereomicroscope, according to established identification keys. In ten localities the relative abundance of the cryptic species of the Obsoletus complex was determined by multiplex PCR assay based on the ITS2 segment. The identification of the Culicoides chiopterus (Meigen) species by morphological examination was confirmed by PCR assay based on the ITS1 segment. Results Eleven species were identified using morphological and PCR tools. The rest of the individuals were separated into five taxa. The species of the Obsoletus complex (grouping Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle) were the most abundant, accounting for 59% of the total number of captured Culicoides spp. Three of the identified species are mentioned, according to our knowledge, for the first time in Romania: Culicoides newsteadi Austen, Culicoides flavipulicaris Dzhafarov and Culicoides bysta Sarvašová, Kočisová, Candolfi & Mathieu. Conclusions Our study demonstrates that the Culicoides species most commonly cited as being involved in the transmission of arboviruses in Europe (i.e. bluetongue and Schmallenberg viruses) make up a high proportion of adult Culicoides trapped in Romania.

Abstract Background Within the genus Culicoides (Diptera: Ceratopogonidae), the subgenus Avaritia is of particular interest as it contains a significant number of economically important vector species. Disagreements about the systematic classification of species within this subgenus have resulted in a taxonomic imbroglio. Methods A molecular phylogeny of the subgenus Avaritia was conducted to test the existing systematic classification, which is based on phenetic assessment of morphological characters. Three nuclear ribosomal markers, internal transcribed spacer 1 and 2 (ITS1, ITS2), 5.8S, and three mitochondrial markers, cytochrome c oxidase subunit 1 and 2, and cytochrome b (cox1, cox2 and cytb), were obtained for 37 species of the subgenus Avaritia from all six biogeographical regions. Phylogenetic reconstructions using these genes independently and in combination were implemented using Bayesian inference analysis and maximum likelihood methods. Results Phylogenetic reconstructions gave strong support to several monophyletic groups within the subgenus Avaritia. Both C. actoni and C. pusillus formed a single clade with C. grahamii so their respective groups, the Actoni and Pusillus groups, have been merged with the Grahamii group. Some support was provided for the Boophagus and Jacobsoni groups. A group of species currently placed into the Orientalis group clustered in a clade with poor support. The Obsoletus group was defined as a sister clade to all other Avaritia groups. The clade including the Imicola group was well supported based on phylogenetic criteria. Conclusions This phylogenetic study combining five distinct molecular markers has provided meaningful insights into the systematic relationships of Culicoides (Avaritia) and highlighted future directions to continue the study of this subgenus. While the cox2 marker appeared to be useful to investigate closely related species, the 5.8S marker was highly conserved and uninformative. Further investigations including species absent from this work are needed to confirm the proposed systematic scheme. However, this systematic scheme can now serve as a foundation to investigate cryptic species affiliation within the subgenus. We advocate that future studies employ a combination of morphological and molecular analyses.

Abstract Background Culicoides biting midges are biological vectors of arboviruses including bluetongue virus (BTV), Schmallenberg virus (SBV) and African horse sickness virus (AHSV). Zoos are home to a wide range of ‘at risk’ exotic and native species of animals. These animals have a high value both in monetary terms, conservation significance and breeding potential. To understand the risk these viruses pose to zoo animals, it is necessary to characterise the Culicoides fauna at zoos and determine which potential vector species are feeding on which hosts. Methods Light-suction traps were used at two UK zoos: the Zoological Society of London (ZSL) London Zoo (LZ) and ZSL Whipsnade Zoo (WZ). Traps were run one night each week from June 2014 to June 2015. Culicoides were morphologically identified to the species level and any blood-fed Culicoides were processed for blood-meal analysis. DNA from blood meals was extracted and amplified using previously published primers. Sequencing was then carried out to determine the host species. Results A total of 11,648 Culicoides were trapped and identified (n = 5880 from ZSL WZ; n = 5768 from ZSL LZ), constituting 25 different species. The six putative vectors of BTV, SBV and AHSV in northern Europe were found at both zoos and made up the majority of the total catch (n = 10,701). A total of 31 host sequences were obtained from blood-fed Culicoides. Culicoides obsoletus/C. scoticus, Culicoides dewulfi, Culicoides parroti and Culicoides punctatus were found to be biting a wide range of mammals including Bactrian camels, Indian rhinoceros, Asian elephants and humans, with Culicoides obsoletus/C. scoticus also biting Darwin’s rhea. The bird-biting species, Culicoides achrayi, was found to be feeding on blackbirds, blue tits, magpies and carrion crows. Conclusions To our knowledge, this is the first study to directly confirm blood-feeding of Culicoides on exotic zoo animals in the UK and shows that they are able to utilise a wide range of exotic as well as native host species. Due to the susceptibility of some zoo animals to Culicoides-borne arboviruses, this study demonstrates that in the event of an outbreak of one of these viruses in the UK, preventative and mitigating measures would need to be taken.

Abstract Background Culicoides biting midges are potential vectors of different pathogens. However, especially for eastern Europe, there is a lack of knowledge on the host-feeding patterns of this vector group. Therefore, this study aimed to identify Culicoides spp. and their vertebrate hosts collected in a wetland ecosystem. Methods Culicoides spp. were collected weekly from May to August 2017, using Biogents traps with UV light at four sites in the Danube Delta Biosphere Reserve, Romania. Vectors and hosts were identified with a DNA barcoding approach. The mitochondrial cytochrome c oxidase subunit 1 was used to identify Culicoides spp., while vertebrate hosts were determined targeting cytochrome b or 16S rRNA gene fragments. A maximum likelihood phylogenetic tree was constructed to verify the biting midge identity against other conspecific Palaearctic Culicoides species. A set of unfed midges was used for morphological confirmation of species identification using slide-mounted wings. Results Barcoding allowed the species identification and detection of corresponding hosts for 1040 (82.3%) of the 1264 analysed specimens. Eight Culicoides spp. were identified with Culicoides griseidorsum, Culicoides puncticollis and Culicoides submaritimus as new species records for Romania. For 39 specimens no similar sequences were found in GenBank. This group of unknown Culicoides showed a divergence of 15.6–16.3% from the closest identified species and clustered in a monophyletic clade, i.e. a novel species or a species without reference sequences in molecular libraries. For all Culicoides spp., nine mammalian and 24 avian species were detected as hosts. With the exception of C. riethi (n = 12), at least one avian host was detected for all Culicoides spp., but this host group only dominated for Culicoides kibunensis and the unknown Culicoides sp.. The most common host group were mammals (n = 993, 87.6% of all identified blood sources) dominated by cattle (n = 817, 70.6%). Conclusions Most Culicoides spp. showed a broad host-feeding pattern making them potential bridge vectors. At the same time, new records of biting midge species for Romania, as well as a potentially unknown Culicoides species, highlight the lack of knowledge regarding the biting midge species and their genetic diversity in eastern Europe.

Background: Reunion Island regularly faces outbreaks of bluetongue and epizootic hemorrhagic diseases, two insect-borne orbiviral diseases of ruminants. Hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae) are the vectors of bluetongue (BTV) and epizootic hemorrhagic disease (EHDV) viruses. In a previous study, statistical models based on environmental and meteorological data were developed for the five Culicoides species present in the island to provide a better understanding of their ecology and predict their presence and abundance. The purpose of this study was to couple these statistical models with a Geographic Information System (GIS) to produce dynamic maps of the distribution of Culicoides throughout the island. Methods: Based on meteorological data from ground weather stations and satellite-derived environmental data, the abundance of each of the five Culicoides species was estimated for the 2214 husbandry locations on the island for the period ranging from February 2016 to June 2018. A large-scale Culicoides sampling campaign including 100 farms was carried out in March 2018 to validate the model. Results: According to the model predictions, no husbandry location was free of Culicoides throughout the study period. The five Culicoides species were present on average in 57.0% of the husbandry locations for C. bolitinos Meiswinkel, 40.7% for C. enderleini Cornet & Brunhes, 26.5% for C. grahamii Austen, 87.1% for C. imicola Kieffer and 91.8% for C. kibatiensis Goetghebuer. The models also showed high seasonal variations in their distribution. During the validation process, predictions were acceptable for C. bolitinos, C. enderleini and C. kibatiensis, with normalized root mean square errors (NRMSE) of 15.4%, 13.6% and 16.5%, respectively. The NRMSE was 27.4% for C. grahamii. For C. imicola, the NRMSE was acceptable (11.9%) considering all husbandry locations except in two specific areas, the Cirque de Salazie—an inner mountainous part of the island—and the sea edge, where the model overestimated its abundance. Conclusions: Our model provides, for the first time to our knowledge, an operational tool to better understand and predict the distribution of Culicoides in Reunion Island. As it predicts a wide spatial distribution of the five Culicoides species throughout the year and taking into consideration their vector competence, our results suggest that BTV and EHDV can circulate continuously on the island. As further actions, our model could be coupled with an epidemiological model of BTV and EHDV transmission to improve risk assessment of Culicoides-borne diseases on the island. [ABSTRACT FROM AUTHOR]

Background: Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that can cause fatal vector-borne diseases in white-tailed deer (Odocoileus virginianus). Trapping methods for collecting potential Culicoides vectors of orbiviruses were compared to optimize surveillance studies. Methods: The number of captured midges and the virus infection rates of midge pools were compared for dry ice-baited Centers for Disease Control and Prevention (CDC) traps with or without black light. The number of individual midges of different Culicoides species captured at different crepuscular and nocturnal periods using rotator traps also was determined. The number of species/specimens of Culicoides was measured using five different trap methods including three animal-baited methods, a CDC trap with black light, and a CDC trap with no light. Results: In trial one, there was no significant difference (P = 0.37) in the proportion of BTV-infected flies caught in traps with light compared to traps without light. However, there was a significant difference (P = 0.026) for EHDV-infected flies, and 89% were captured in traps with light. In trial two, more specimens of C. debilipalpis were captured in the morning hours (06:00–08:00) than in the evening hours (18:00–20:00). For trial three, the animal-baited traps did not capture any species of Culicoides that were not captured in the CDC light traps. There was no significant difference (P = 0.22) in total specimens captured among all five trap types. Conclusions: Specimens of Culicoides infected with BTV were not repelled by light traps in the first trial, while the majority of the specimens positive for EHDV were caught in traps with light. For the second trial, specimens of C. debilipalpis were most abundant during early morning hours, and thus spray applications of insecticides for control of that species may be more effective at sunrise rather than sunset. For objective three, no animal-baited trapping method collected different species of midges when compared to the CDC traps with light, which is unlike certain studies conducted in other geographical regions. [ABSTRACT FROM AUTHOR]

Background: Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. Methods: The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO2-CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO2-CDC traps were identified by morphological and molecular analysis. Results: From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus (n = 273), Sergentomyia minuta (n = 5), P. mascittii (n = 2) and P. perfiliewi (n = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from −2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. Conclusions: Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta, the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic. [ABSTRACT FROM AUTHOR]

Background: Culicoides imicola (Diptera: Ceratopogonidae) is an important Afrotropical and Palearctic vector of disease, transmitting viruses of animal health and economic significance including African horse sickness and bluetongue viruses. Maternally inherited symbiotic bacteria (endosymbionts) of arthropods can alter the frequency of COI (cytochrome c oxidase subunit I) mitochondrial haplotypes (mitotypes) in a population, masking the true patterns of host movement and gene flow. Thus, this study aimed to assess the mtDNA structure of C. imicola in relation to infection with Candidatus Cardinum hertigii (Bacteroides), a common endosymbiont of Culicoides spp. Methods: Using haplotype network analysis, COI Sanger sequences from Cardinium-infected and -uninfected C. imicola individuals were first compared in a population from South Africa. The network was then extended to include mitotypes from a geographic range where Cardinium infection has previously been investigated. Results: The mitotype network of the South African population demonstrated the presence of two broad mitotype groups. All Cardinium-infected specimens fell into one group (Fisher's exact test, P = 0.00071) demonstrating a linkage disequilibrium between endosymbiont and mitochondria. Furthermore, by extending this haplotype network to include other C. imicola populations from the Mediterranean basin, we revealed mitotype variation between the Eastern and Western Mediterranean basins (EMB and WMB) mirrored Cardinium-infection heterogeneity. Conclusions: These observations suggest that the linkage disequilibrium of Cardinium and mitochondria reflects endosymbiont gene flow within the Mediterranean basin but may not assist in elucidating host gene flow. Subsequently, we urge caution on the single usage of the COI marker to determine population structure and movement in C. imicola and instead suggest the complementary utilisation of additional molecular markers. [ABSTRACT FROM AUTHOR]

Background: Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, in both replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides. This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the UK under laboratory conditions. Methods: Over 18,000 Culicoides adults were collected from the field using 4 W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the UK during the summer months and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-h period. Results: The proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4 °C) compared with populations collected in the summer (10 °C). Within the subgenus Avaritia, Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup. Populations of Culicoides impunctatus collected in the North East of England were only active once temperatures reached 14 °C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders. Conclusions: These findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. These data, alongside that defining thresholds for virus replication within Culicoides, provide a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance. [ABSTRACT FROM AUTHOR]

Background: Culicoides insignis is a confirmed vector of bluetongue virus (BTV) throughout the American tropics and a possible vector of epizootic hemorrhagic disease virus (EHDV) in Florida. Despite its importance, fundamental information on the biology and ecology of this vector species is lacking. In this study, we examined the oviposition of C. insignis under laboratory conditions, monitored the development of immature stages and attempted colonization of this species. Methods: Live C. insignis females were collected from the field using CDC-UV-LED traps, allowed to blood-feed on live chicken and given various natural substrates for oviposition in two-choice assays. The eggs deposited were transferred to 0.3% agar slants, and the hatched larvae were provided a diet of Panagrellus redivivus Linnaeus nematodes and the development of all immature stages was monitored. Results: Culicoides insignis females exhibited an overall oviposition preference for dishes containing mud from their larval habitat as gravid females deposited a significantly higher number of eggs on these dishes (35.3 eggs/female) than on controls (17.7 eggs/female). The ovipositing females also deposited a higher percentage of eggs on substrates with habitat mud and other organically enriched muds (≥ 75.2%) compared to controls (31.0%). The larvae developed successfully to adulthood on the nematode diet, exhibiting high overall larval survival rates (85.0%). Sex ratios of the F1 generation were male biased, approximately 3:1 (male:female). Captive mating could not be induced in the F1 adults. Conclusions: Mud from the larval habitat and other organically enriched muds provide strong oviposition cues to C. insignis under laboratory conditions. Further studies will be needed to identify the key biotic/abiotic factors influencing midge oviposition in the field. The agar/nematode method is effective for the rearing of C. insignis larvae. However, further studies will be needed to address the issue of male-biased sex ratios in the progeny and to examine the mating habits/cues of C. insignis in nature, which may provide clues towards inducing captive mating in the F1 adults. [ABSTRACT FROM AUTHOR]

Background: Bluetongue is a serious disease of ruminants caused by the bluetongue virus (BTV). BTV is transmitted by biting midges (Culicoides spp.). Serological evidence from livestock and the presence of at least one competent vector species of Culicoides suggests that transmission of BTV is possible and may have occurred in Kazakhstan. Methods: We estimated the risk of transmission using a mathematical model of the reproduction number R0 for bluetongue. This model depends on livestock density and climatic factors which affect vector density. Data on climate and livestock numbers from the 2466 local communities were used. This, together with previously published model parameters, was used to estimate R0 for each month of the year. We plotted the results on isopleth maps of Kazakhstan using interpolation to smooth the irregular data. We also mapped the estimated proportion of the population requiring vaccination to prevent outbreaks of bluetongue. Results: The results suggest that transmission of bluetongue in Kazakhstan is not possible in the winter from October to March. Assuming there are vector-competent species of Culicoides endemic in Kazakhstan, then low levels of risk first appear in the south of Kazakhstan in April before spreading north and intensifying, reaching maximum levels in northern Kazakhstan in July. The risk declined in September and had disappeared by October. Conclusion: These results should aid in surveillance efforts for the detection and control of bluetongue in Kazakhstan by indicating where and when outbreaks of bluetongue are most likely to occur. The results also indicate where vaccination efforts should be focussed to prevent outbreaks of disease. [ABSTRACT FROM AUTHOR]

Background: The Culicoides obsoletus species complex (henceforth 'Obsoletus complex') is implicated in the transmission of several arboviruses that can cause severe disease in livestock, such as bluetongue, African horse sickness, epizootic hemorrhagic disease and Schmallenberg disease. Thus, this study aimed to increase our knowledge of the composition and genetic diversity of the Obsoletus complex by partial sequencing of the cytochrome c oxidase I (cox1) gene in poorly studied areas of Spain. Methods: A study of C. obsoletus populations was carried out using a single-tube multiplex polymerase chain reaction (PCR) assay that was designed to differentiate the Obsoletus complex sibling species Culicoides obsoletus and Culicoides scoticus, based on the partial amplification of the cox1 gene, as well as cox1 georeferenced sequences from Spain available at GenBank. We sampled 117 insects of the Obsoletus complex from six locations and used a total of 238 sequences of C. obsoletus (ss) individuals (sampled here, and from GenBank) from 14 sites in mainland Spain, the Balearic Islands and the Canary Islands for genetic diversity and phylogenetic analyses. Results: We identified 90 C. obsoletus (ss), 19 Culicoides scoticus and five Culicoides montanus midges from the six collection sites sampled, and found that the genetic diversity of C. obsoletus (ss) were higher in mainland Spain than in the Canary Islands. The multiplex PCR had limitations in terms of specificity, and no cryptic species within the Obsoletus complex were identified. Conclusions: Within the Obsoletus complex, C. obsoletus (ss) was the predominant species in the analyzed sites of mainland Spain. Information about the species composition of the Obsoletus complex could be of relevance for future epidemiological studies when specific aspects of the vector competence and capacity of each species have been identified. Our results indicate that the intraspecific divergence is higher in C. obsoletus (ss) northern populations, and demonstrate the isolation of C. obsoletus (ss) populations of the Canary Islands. [ABSTRACT FROM AUTHOR]

Background: In the last two decades, recurrent epizootics of bluetongue virus and Schmallenberg virus have been reported in the western Palearctic region. These viruses affect domestic cattle, sheep, goats and wild ruminants and are transmitted by native hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae). Culicoides dispersal is known to be stratified, i.e. due to a combination of dispersal processes occurring actively at short distances and passively or semi-actively at long distances, allowing individuals to jump hundreds of kilometers. Methods: Here, we aim to identify the environmental factors that promote or limit gene flow of Culicoides obsoletus, an abundant and widespread vector species in Europe, using an innovative framework integrating spatial, population genetics and statistical approaches. A total of 348 individuals were sampled in 46 sites in France and were genotyped using 13 newly designed microsatellite markers. Results: We found low genetic differentiation and a weak population structure for C. obsoletus across the country. Using three complementary inter-individual genetic distances, we did not detect any significant isolation by distance, but did detect significant anisotropic isolation by distance on a north-south axis. We employed a multiple regression on distance matrices approach to investigate the correlation between genetic and environmental distances. Among all the environmental factors that were tested, only cattle density seems to have an impact on C. obsoletus gene flow. Conclusions: The high dispersal capacity of C. obsoletus over land found in the present study calls for a re-evaluation of the impact of Culicoides on virus dispersal, and highlights the urgent need to better integrate molecular, spatial and statistical information to guide vector-borne disease control. [ABSTRACT FROM AUTHOR]

Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches. [ABSTRACT FROM AUTHOR]

Light trapping is an important tool for monitoring insect populations. This is especially true for biting Diptera, where light traps play a crucial role in disease surveillance by tracking the presence and abundance of vector species. Physiological and behavioural data have been instrumental in identifying factors that influence dipteran phototaxis and have spurred the development of more effective light traps. However, the development of less attractive domestic lights has received comparatively little interest but could be important for reducing interactions between humans and vector insects, with consequences for reducing disease transmission. Here, we discuss how dipteran eyes respond to light and the factors influencing positive phototaxis, and conclude by identifying key areas for further research. In addition, we include a synthesis of attractive and unattractive wavelengths for a number of vector species. A more comprehensive understanding of how Diptera perceive and respond to light would allow for more efficient vector sampling as well as potentially limiting the risk posed by domestic lighting. [ABSTRACT FROM AUTHOR]

Background: Culicoides (Diptera: Ceratopogonidae) are vectors for many arboviruses. At least 20 species are considered as vectors or potential vectors of bluetongue virus (BTV) which cause bluetongue disease in ruminants. A BTV prevalence of 30–50% among cattle and goats in tropical southern Yunnan Province, China, prompted an investigation of the potential BTV vectors in this area. Methods: Culicoides were collected by light trapping at three sites in the tropical region of Yunnan Province. Species were identified based on morphology and DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in these specimens. Phylogenetic analysis was used to analyze the cox1 sequences of Culicoides specimens infected with BTV. Results: Approximately 67,000 specimens of Culicoides were collected, of which 748 were tested for the presence of BTV. Five specimens, including two of Culicoides jacobsoni, one of C. tainanus and two of C. imicola, were identified as infected with BTV. No specimens of C. (subgenus Trithecoides) or C. oxystoma tested were positive for BTV infection. Conclusions: To our knowledge this is the first report of C. jacobsoni as a potential BTV vector and the fourth report of an association between C. tainanus and BTV, as well as the first direct evidence of an association between BTV and C. imicola in Asia. A fourth potential cryptic species within C. tainanus was identified in this study. Further analysis is required to confirm the importance of C. jacobsoni and C. tainanus in BTV epidemiology in Asia. [ABSTRACT FROM AUTHOR]

Background: Aedes vigilax is one of the most significant arbovirus vector and pest species in Australia's coastal regions. Occurring in multiple countries, this mosquito species occurs as a species complex which has been separated into three clades with two detected in Australia. Until recently, Ae. vigilax has largely been absent from Victoria, only occasionally caught over the years, with no reported detections from 2010 to 2016. Complicating the detection of Ae. vigilax is the shared sympatric distribution to the morphologically similar Ae. camptorhynchus, which can exceed 10,000 mosquitoes in a single trap night in Victoria. Currently, there are no molecular assays available for the detection of Ae. vigilax. We aim to develop a quantitative PCR (qPCR) for the detection of Ae. vigilax, with the specificity and sensitivity of this assay assessed as well as a method to process whole mosquito traps. Methods: Trapping was performed during the 2017–2020 mosquito season in Victoria in two coastal areas across these 3 consecutive years. A qPCR assay was designed to allow rapid identification of Ae. vigilax as well as a whole mosquito trap homogenizing and processing methodology. Phylogenetic analysis was performed to determine which clade Ae. vigilax from Victoria was closest to. Results: Aedes vigilax was successfully detected each year across two coastal areas of Victoria, confirming the presence of this species. The qPCR assay was proven to be sensitive and specific to Ae. vigilax, with trap sizes up to 1000 mosquitoes showing no inhibition in detection sensitivity. Phylogenetic analysis revealed that Ae. vigilax from Victoria is associated with clade III, showing high sequence similarity to those previously collected in New South Wales, Queensland and Western Australia. Conclusions: Aedes vigilax is a significant vector species that shares an overlapping distribution to the morphologically similar Ae. camptorhynchus, making detection difficult. Here, we have outlined the implementation of a specific and sensitive molecular screening assay coupled with a method to process samples for detection of Ae. vigilax in collections with large numbers of non-target species. [ABSTRACT FROM AUTHOR]

Background: Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. Methods: Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. Results: Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoidesjacobsoni was identified as TIBOV, while the other two viruses from C.orientalis and C.tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. Conclusions: The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known. [ABSTRACT FROM AUTHOR]

Background: Wolbachia pipientis are endosymbiotic bacteria present in a large proportion of terrestrial arthropods. The species is known to sometimes affect the ability of its host to transmit vector-borne pathogens. Central Sweden is endemic for Sindbis virus (SINV), where it is mainly transmitted by the vector species Culex pipiens and Culex torrentium, with the latter established as the main vector. In this study we investigated the Wolbachia prevalence in these two vector species in a region highly endemic for SINV. Methods: Culex mosquitoes were collected using CDC light traps baited with carbon dioxide over 9 years at 50 collection sites across the River Dalälven floodplains in central Sweden. Mosquito genus was determined morphologically, while a molecular method was used for reliable species determination. The presence of Wolbachia was determined through PCR using general primers targeting the wsp gene and sequencing of selected samples. Results: In total, 676 Cx. pipiens and 293 Cx. torrentium were tested for Wolbachia. The prevalence of Wolbachia in Cx. pipiens was 97% (95% CI 94.8–97.6%), while only 0.7% (95% CI 0.19–2.45%) in Cx. torrentium. The two Cx. torrentium mosquitoes that were infected with Wolbachia carried different types of the bacteria. Conclusions: The main vector of SINV in the investigated endemic region, Cx. torrentium, was seldom infected with Wolbachia, while it was highly prevalent in the secondary vector, Cx. pipiens. The presence of Wolbachia could potentially have an impact on the vector competence of these two species. Furthermore, the detection of Wolbachia in Cx. torrentium could indicate horizontal transmission of the endosymbiont between arthropods of different species. [ABSTRACT FROM AUTHOR]

Background: Culicoides biting midges (Diptera, Ceratopogonidae) are known vectors of avian Haemoproteus parasites. These parasites cause diseases, pathology and even mortality in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in the transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites. Methods: Biting midges were collected using a UV trap as well as sticky traps installed in bird nest boxes. Individual parous females were diagnosed for the presence of haemoproteids using both PCR-based and microscopic methods. Results: We collected and dissected 232 parous Culicoides females from 9 species using a UV trap and 293 females from 11 species from bird nest boxes. Culicoides obsoletus was the dominant species collected using a UV trap, and Culicoides kibunensis dominated among midges collected in nest boxes. PCR-based screening showed that 5.2% of parous biting midges collected using a UV trap and 4.4% of midges collected from nest boxes were infected with avian haemosporidian parasites. Haemoproteid DNA was detected in C. kibunensis, Culicoides pictipennis, Culicoides punctatus, Culicoides segnis and Culicoides impunctatus females. The sporozoites of Haemoproteus minutus (genetic lineages hTURDUS2 and hTUPHI01) were detected in the salivary glands of two C. kibunensis females using microscopy, and this finding was confirmed by PCR. Conclusions: Culicoides kibunensis was detected as a new natural vector of Haemoproteus minutus (hTURDUS2 and hTUPHI01). Haemoproteid DNA was detected in females from five Culicoides species. This study contributes to the epizootiology of avian Haemoproteus infections by specifying Culicoides species as vectors and species that are likely to be responsible for the transmission of haemoproteids in Europe. [ABSTRACT FROM AUTHOR]