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This is the third in a planned series of data papers presenting modelled vector distributions produced during the ECDC and EFSA funded VectorNet project. The data package presented here includes those Culicoides vectors species first modelled in 2015 as part of the VectorNet gap analysis work namely C. imicola, C. obsoletus, C. scoticus, C. dewulfi, C. chiopterus, C. pulicaris, C. lupicaris, C. punctatus, and C. newsteadi. The known distributions of these species within the Project area (Europe, the Mediterranean Basin, North Africa, and Eurasia) are currently incomplete to a greater or lesser degree. The models are designed to fill the gaps with predicted distributions, to provide a) first indication of vector species distributions across the project geographical extent, and b) assistance in targeting surveys to collect distribution data for those areas with no field validated information. The models are based on input data from light trap surveillance of adult Culicoides across continental Europe and surrounding regions (71.8°N –33.5°S, – 11.2°W – 62°E), concentrated in Western countries, supplemented by transect samples in eastern and northern Europe. Data from central EU are relatively sparse.

Biting midges (Diptera, Ceratopogonidae) of the genus Culicoides are important vectors of pathogens causing diseases in free living and production animals and can lead to large economic losses in many European countries. In Europe, Culicoides imicola and the Obsoletus group are considered to be the main vectors of bluetongue virus that mostly affects ruminants such as cattle and sheep. Spatio-temporal modelling of vector distribution and abundance allows us to identify high risk areas for virus transmission and can aid in applying effective surveillance and control measures. We used presence-absence and monthly abundance data of Culicoides from 1005 sites across 9 countries (Spain, France, Denmark, Poland, Switzerland, Austria, Poland, Sweden, Norway) collected between the years 2007 and 2013. The dataset included information on the vector species abundance (number of specimens caught per night), GPS coordinates of each trap, start and end dates of trapping. We used 120 environmental predictor variables together with Random Forest machine learning algorithms to predict the overall species distribution (probability of occurrence) and monthly abundance in Europe. We generated maps for every month of the year, to visualize the abundance of C. imicola and Obsoletus group in Europe as well as distribution maps showing the probability of occurrence. We were able to create predictive maps of both Culicoides sp. occurrence and abundance using Random Forest models, and although the variance was large, the predicted abundance values for each site had a positive correlation with the observed abundance. We found relatively large spatial variations in probability of occurrence and abundance for both C. imicola and the Obsoletus group. For C. imicola probability of occurrence and abundance was higher in southern Spain, where as the Obsoletus group had higher probability of occurrence and abundance in central and northern Europe such as France and Germany. Temp

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

This study establishes the first faunistic inventory of livestock associated Culicoides (Diptera: Ceratopogonidae) species of Reunion Island (Indian Ocean), where bluetongue and epizootic hemorrhagic disease are regularly recorded. Single night-catches were performed at 41 sites using light suction traps at altitudes ranging from 0 to 1525. m, from March to April 2005. Five species were recorded: Culicoides imicola, Culicoides bolitinos, Culicoides enderleini, Culicoides grahamii, and Culicoides kibatiensis, among which at least the first three species are known to be involved in virus transmission to ruminants and equids. This is the first record of C. bolitinos, C. kibatiensis, and C. enderleini on the island. C. imicola was the most abundant species along the sea coast. C. bolitinos was more abundant inland and on two sites on the east coast. C. kibatiensis and C. grahamii were less abundant than the other three species and limited to two foci. Spatial distribution analysis of the different species showed that C. bolitinos, C. enderleini and C. imicola were collected at low altitudes, while the other two species were found at higher altitude. A morphological identification key for adult females and males is given, as well as cytochrome oxydase subunit I sequences. Phylogenetic reconstructions showed a clear divergence between C. bolitinos from Reunion Island and mainland Africa. This monograph will help to identify the Culicoides species in the poorly known entomological fauna of the south-western Indian Ocean region.

Background: African horse sickness (AHS) is a major, Culicoides-borne viral disease in equines whose introduction into Europe could have dramatic consequences. The disease is considered to be endemic in sub-Saharan Africa. Recent introductions of other Culicoides-borne viruses (bluetongue and Schmallenberg) into northern Europe have highlighted the risk that AHS may arrive in Europe as well. The aim of our study was to provide a spatiotemporal quantitative risk model of AHS introduction into France. The study focused on two pathways of introduction: the arrival of an infectious host (PW-host) and the arrival of an infectious Culicoides midge via the livestock trade (PW-vector). The risk of introduction was calculated by determining the probability of an infectious animal or vector entering the country and the probability of the virus then becoming established: i.e., the virus's arrival in France resulting in at least one local equine host being infected by one local vector. This risk was assessed using data from three consecutive years (2010 to 2012) for 22 regions in France. Results: The results of the model indicate that the annual risk of AHS being introduced to France is very low but that major spatiotemporal differences exist. For both introduction pathways, risk is higher from July to October and peaks in July. In general, regions with warmer climates are more at risk, as are colder regions with larger equine populations; however, regional variation in animal importation patterns (number and species) also play a major role in determining risk. Despite the low probability that AHSV is present in the EU, intra-EU trade of equines contributes most to the risk of AHSV introduction to France because it involves a large number of horse movements. Conclusion: It is important to address spatiotemporal differences when assessing the risk of ASH introduction and thus also when implementing efficient surveillance efforts. The methods and results of this study may help de

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. METHODS: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. RESULTS: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. CONCLUSIONS: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

Background: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. Methods: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. Results: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. Conclusions: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Feeding success depends on host availability, host defensive reactions and host preferences. Host choice is a critical determinant of the intensity at which pathogens are transmitted. The aim of the current study was to describe host preferences of Palaea

The emergence and massive spread of bluetongue in Western Europe during 2006-2008 had disastrous consequences for sheep and cattle production and confirmed the ability of Palaearctic Culicoides (Diptera: Ceratopogonidae) to transmit the virus. Some aspects of Culicoides ecology, especially host-seeking and feeding behaviors, remain insufficiently described due to the difficulty of collecting them directly on a bait animal, the most reliable method to evaluate biting rates. Our aim was to compare typical animal-baited traps (drop trap and direct aspiration) to both a new sticky cover trap and a UV-light/ suction trap (the most commonly used method to collect Culicoides).

Culicoides are small biting midges involved worldwide in the transmission of bluetongue and African horse sickness viruses. Feeding behaviours of Palaearctic biting midge species and their spatio-temporal dynamics remain unclear at the specific level. Three multiplex species-specific PCR-based assays were developed and used to identify blood meal source of engorged females of Palaearctic midge species of veterinary interest. Species-specific primers of potential hosts from livestock, domestic animals and wildlife (cattle, goat, sheep, red deer, roe deer, chamois, dog, pig, cat, horse) were designed and multiplexed from the mitochondrial cytochrome b gene. The assays also make possible to identify whether multiple blood meals have been taken. The first results from several Culicoides populations sampled in France highlight the utility of this valuable diagnostic tool combined with species identification assays, and suggest that most of the Culicoides species may have an opportunistic feeding behaviour regarding the host distribution and density. Noteworthy is the peculiar trophic behaviour of Culicoides chiopterus showing clear trends to cattle. Information on host preference and feeding behaviours are crucial for a better understanding of vector-host interactions and disease epidemiology.

One of the aims of the DynVect project was to set up a network of European entomologists working on Culicoides, the vectors of bluetongue virus, to create a platform for discussion, data sharing and data analysis. The first task consisted in describing the surveillance systems in place in each country. For this, a questionnaire was sent to 9 countries: Belgium, Denmark, England, France, Germany, Italy, Spain, Sweden and The Netherlands. Between 1 and 9 institutes per country were involved in the surveillance. The first country to set up a surveillance system was Spain (in 2000), followed by other southern countries (Italy and France). A second wave of countries set up surveillance systems in 2006, with the arrival of bluetongue in northern Europe. All countries currently continue the surveillance except Germany (which stopped the surveillance in mid 2008). All countries shared common aims: defining "vector-free" periods and describing the diversity and dynamics of species. Except in Italy, all surveillance protocols evolved over the years, for example from covering specific regions to the entire country, and/or with an increasing number of traps. Surveillance systems vary between countries in terms of i) number of traps per country (5 - 300), ii) types of traps used (OVI, CDC, BG, Rieb, Rothamsted suction trap), although currently all countries use OVI traps except for Spain, iii) trapping frequency (day and night, weekly, fortnightly or monthly), iv) number of collection nights (mainly 1 night trapping, except Belgium with 2 consecutive nights and Germany with 7 consecutive days), v) trap location within farm (inside or outside buildings or both according to the season), vi) the level to which species are identified (Group, Complex or species) and vii) the definition of the vector-free period. Following this descriptive phase, participants discussed the data which could be shared and the associated aims as well as the database structure which should be used. This w