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Abstract Background and methods The appearance of bluetongue virus (BTV) in 2006 within northern Europe exposed a lack of expertise and resources available across this region to enable the accurate morphological identification of species of Culicoides Latreille biting midges, some of which are the major vectors of this pathogen. This work aims to organise extant Culicoides taxonomic knowledge into a database and to produce an interactive identification key for females of Culicoides in the Western Palaearctic (IIKC: Interactive identification key for Culicoides). We then validated IIKC using a trial carried out by six entomologists based in this region with variable degrees of experience in identifying Culicoides. Results The current version of the key includes 98 Culicoides species with 10 morphological variants, 61 descriptors and 837 pictures and schemes. Validation was carried out by six entomologists as a blind trial with two users allocated to three classes of expertise (beginner, intermediate and advanced). Slides were identified using a median of seven steps and seven minutes and user confidence in the identification varied from 60% for failed identifications to a maximum of 80% for successful ones. By user class, the beginner group successfully identified 44.6% of slides, the intermediate 56.8% and the advanced 74.3%. Conclusions Structured as a multi-entry key, IIKC is a powerful database for the morphological identification of female Culicoides from the Western Palaearctic region. First developed for use as an interactive identification key, it was revealed to be a powerful back-up tool for training new taxonomists and to maintain expertise level. The development of tools for arthropod involvement in pathogen transmission will allow clearer insights into the ecology and dynamics of Culicoides and in turn assist in understanding arbovirus epidemiology.

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. [ABSTRACT FROM AUTHOR]

Background: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. Methods: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. Results: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. Conclusions: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region. [ABSTRACT FROM AUTHOR]

Background: Introduction of vector species into new areas represents a main driver for the emergence and worldwide spread of vector-borne diseases. This poses a substantial threat to livestock economies and public health. Culicoides imicola Kieffer, a major vector species of economically important animal viruses, is described with an apparent range expansion in Europe where it has been recorded in south-eastern continental France, its known northern distribution edge. This questioned on further C. imicola population extension and establishment into new territories. Studying the spatio-temporal genetic variation of expanding populations can provide valuable information for the design of reliable models of future spread. Methods: Entomological surveys and population genetic approaches were used to assess the spatio-temporal population dynamics of C. imicola in France. Entomological surveys (2-3 consecutive years) were used to evaluate population abundances and local spread in continental France (28 sites in the Var department) and in Corsica (4 sites). We also genotyped at nine microsatellite loci insects from 3 locations in the Var department over 3 years (2008, 2010 and 2012) and from 6 locations in Corsica over 4 years (2002, 2008, 2010 and 2012). Results: Entomological surveys confirmed the establishment of C. imicola populations in Var department, but indicated low abundances and no apparent expansion there within the studied period. Higher population abundances were recorded in Corsica. Our genetic data suggested the absence of spatio-temporal genetic changes within each region but a significant increase of the genetic differentiation between Corsican and Var populations through time. The lack of intra-region population structure may result from strong gene flow among populations. We discussed the observed temporal variation between Corsica and Var as being the result of genetic drift following introduction, and/or the genetic characteristics of populations at their range edge. Conclusions: Our results suggest that local range expansion of C. imicola in continental France may be slowed by the low population abundances and unsuitable climatic and environmental conditions. [ABSTRACT FROM AUTHOR]

Culicoides biting midges (Diptera: Ceratopogonidae) are important vectors of arboviruses in Africa. Culicoides oxystoma has been recently recorded in the Niayes region of Senegal (West Africa) and its high abundance on horses suggests a potential implication in the transmission of the African horse sickness virus in this region. This species is also suspected to transmit bluetongue virus to imported breeds of sheep. Little information is available on the biology and ecology of Culicoides in Africa. Therefore, understanding the circadian host-seeking activity of this putative vector is of primary importance to assess the risk of the transmission of Culicoides-borne pathogens. To achieve this objective, midges were collected using a sheep-baited trap over two consecutive 24-h periods during four seasons in 2012. A total of 441 Culicoides, belonging to nine species including 418 (94.8 %) specimens of C. oxystoma, were collected. C. oxystoma presented a bimodal circadian host-seeking activity at sunrise and sunset in July and was active 3 h after sunrise in April. Daily activity appeared mainly related to time periods. Morning activity increased with the increasing temperature up to about 27 °C and then decreased with the decreasing humidity, suggesting thermal limits for C. oxystoma activity. Evening activity increased with the increasing humidity and the decreasing temperature, comprised between 20 and 27 °C according to seasons. Interestingly, males were more abundant in our sampling sessions, with similar activity periods than females, suggesting potential animal host implication in the facilitation of reproduction. Finally, the low number of C. oxystoma collected render practical vector-control recommendations difficult to provide and highlight the lack of knowledge on the bio-ecology of this species of veterinary interest. [ABSTRACT FROM AUTHOR]

Background: Culicoides biting midges are biological vectors of internationally important arboviruses of livestock and equines. Insecticides are often employed against Culicoides as a part of vector control measures, but systematic assessments of their efficacy have rarely been attempted. The objective of the present study is to determine baseline susceptibility of multiple Culicoides vector species and populations in Europe and Africa to the most commonly used insecticide active ingredients. Six active ingredients are tested: three that are based on synthetic pyrethroids (alpha-cypermethrin, deltamethrin and permethrin) and three on organophosphates (phoxim, diazinon and chlorpyrifos-methyl). Methods: Susceptibility tests were conducted on 29,064 field-collected individuals of Culicoides obsoletus Meigen, Culicoides imicola Kieffer and a laboratory-reared Culicoides nubeculosus Meigen strain using a modified World Health Organization assay. Populations of Culicoides were tested from seven locations in four different countries (France, Spain, Senegal and South Africa) and at least four concentrations of laboratory grade active ingredients were assessed for each population. Results: The study revealed that insecticide susceptibility varied at both a species and population level, but that broad conclusions could be drawn regarding the efficacy of active ingredients. Synthetic pyrethroid insecticides were found to inflict greater mortality than organophosphate active ingredients and the colony strain of C. nubeculosus was significantly more susceptible than field populations. Among the synthetic pyrethroids, deltamethrin was found to be the most toxic active ingredient for all species and populations. Conclusions: The data presented represent the first parallel and systematic assessment of Culicoides insecticide susceptibility across several countries. As such, they are an important baseline reference to monitor the susceptibility status of Culicoides to current insecticides and also to assess the toxicity of new active ingredients with practical implications for vector control strategies. [ABSTRACT FROM AUTHOR]

Background African horse sickness (AHS) is an equine disease endemic to Senegal. The African horse sickness virus (AHSV) is transmitted to the mammalian hosts by midges of the Culicoides Latreille genus. During the last epizootic outbreak of AHS in Senegal in 2007, 1,169 horses died from this disease entailing an estimated cost of 1.4 million euros. In spite of the serious animal health and economic implications of AHS, very little is known about determinants involved in transmission such as contact between horses and the Culicoides species suspected of being its vectors. Methods The monthly variation in host/vector contact was determined in the Niayes area, Senegal, an area which was severely affected by the 2007 outbreak of AHS. A horse-baited trap and two suction light traps (OVI type) were set up at each of five sites for three consecutive nights every month for one year. Results Of 254,338 Culicoides midges collected 209,543 (82.4%) were female and 44,795 (17.6%) male. Nineteen of the 41 species collected were new distribution records for Senegal. This increased the number of described Culicoides species found in Senegal to 53. Only 19 species, of the 41 species found in light trap, were collected in the horse-baited trap (23,669 specimens) largely dominated by Culicoides oxystoma (22,300 specimens, i.e. 94.2%) followed by Culicoides imicola (482 specimens, i.e. 2.0%) and Culicoides kingi (446 specimens, i.e. 1.9%). Conclusions Culicoides oxystoma should be considered as a potential vector of AHSV in the Niayes area of Senegal due to its abundance on horses and its role in the transmission of other Culicoides-borne viruses. [ABSTRACT FROM AUTHOR]

We modelled the ecoclimatic niche of Culicoides imicola, a major arthropod vector of midge-borne viral pathogens affecting ruminants and equids, at fine scale and on a global extent, so as to provide insight into current and future risks of disease epizootics, and increase current knowledge of the species' ecology. Based on the known distribution and ecology of C. imicola, the species' response to monthly climatic conditions was characterised using CLIMEX with 10′ spatial resolution climatic datasets. The species' climatic niche was projected worldwide and under future climatic scenarios. The validated model highlights the role of irrigation in supporting the occurrence of C. imicola in arid regions. In Europe, the modelled potential distribution of C. imicola extended further West than its reported distribution, raising questions regarding ongoing process of colonization and non-climatic habitat factors. The CLIMEX model highlighted similar ecological niches for C. imicola and the Australasian C. brevitarsis raising questions on biogeography and biosecurity. Under the climate change scenarios considered, its' modelled potential distribution could expand northward in the Northern hemisphere, whereas in Africa its range may contract in the future. The biosecurity risks from bluetongue and African horse sickness viruses need to be re-evaluated in regions where the vector's niche is suitable. Under a warmer climate, the risk of vector-borne epizootic pathogens such as bluetongue and African horse sickness viruses are likely to increase as the climate suitability for C. imicola shifts poleward, especially in Western Europe. [ABSTRACT FROM AUTHOR]

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex ( C. obsoletus and C. scoticus ) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola , only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) is distributed throughout Africa to northern Asia and Australasia and includes several potential vector species of livestock pathogens. The taxonomy of the species belonging to this species group is confounded by the wide geographical distribution and morphological variation exhibited by many species. In this work, morphological and molecular approaches were combined to assess the taxonomic validity of the species and morphological variants of the Schultzei group found in Senegal by comparing their genetic diversity with that of specimens from other geographical regions. The species list for Senegal was updated with four species: Culicoides kingi, C. oxystoma, C. enderleini and C. nevilli being recorded. This is the first record of C. oxystoma from Africa south of Sahara, and its genetic relationship with samples from Israel, Japan and Australia is presented. This work provides a basis for ecological studies of the seasonal and spatial dynamics of species of this species group that will contribute to better understanding of the epidemiology of the viruses they transmit. [ABSTRACT FROM AUTHOR]

Bluetongue ( BT) is a commonly cited example of a disease with a distribution believed to have recently expanded in response to global warming. The BT virus is transmitted to ruminants by biting midges of the genus Culicoides, and it has been hypothesized that the emergence of BT in Mediterranean Europe during the last two decades is a consequence of the recent colonization of the region by Culicoides imicola and linked to climate change. To better understand the mechanism responsible for the northward spread of BT, we tested the hypothesis of a recent colonization of Italy by C. imicola, by obtaining samples from more than 60 localities across Italy, Corsica, Southern France, and Northern Africa (the hypothesized source point for the recent invasion of C. imicola), and by genotyping them with 10 newly identified microsatellite loci. The patterns of genetic variation within and among the sampled populations were characterized and used in a rigorous approximate Bayesian computation framework to compare three competing historical hypotheses related to the arrival and establishment of C. imicola in Italy. The hypothesis of an ancient presence of the insect vector was strongly favoured by this analysis, with an associated P ≥ 99%, suggesting that causes other than the northward range expansion of C. imicola may have supported the emergence of BT in southern Europe. Overall, this study illustrates the potential of molecular genetic markers for exploring the assumed link between climate change and the spread of diseases. [ABSTRACT FROM AUTHOR]

Feeding success of free-living hematophagous insects depends on their ability to be active when hosts are available and to reach places where hosts are accessible. When the hematophagous insect is a vector of pathogens, determining the components of host-seeking behavior is of primary interest for the assessment of transmission risk. Our aim was to describe endo/exophagy and circadian host-seeking activity of Palaearctic Culicoides species, which are major biting pests and arbovirus vectors, using drop traps and suction traps baited with four sheep, as bluetongue virus hosts. Collections were carried out in the field, a largely-open stable and an enclosed stable during six collection periods of 24 hours in April/May, in late June and in September/October 2010 in western France. A total of 986 Culicoides belonging to 13 species, mainly C. brunnicans and C. obsoletus, was collected on animal baits. Culicoides brunnicans was clearly exophagic, whereas C. obsoletus was able to enter stables. Culicoides brunnicans exhibited a bimodal pattern of host-seeking activity with peaks just after sunrise and sunset. Culicoides obsoletus was active before sunset in spring and autumn and after sunset in summer, thus illustrating influence of other parameters than light, especially temperature. Description of host-seeking behaviors allowed us to discuss control strategies for transmission of Culicoides-borne pathogens, such as bluetongue virus. However, practical vector-control recommendations are difficult to provide because of the variation in the degree of endophagy and time of host-seeking activity. [ABSTRACT FROM AUTHOR]

Background: 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). Methods/results: Collections were made from 1.45 hours before sunset to 1.45 hours after sunset in June/July 2009 at an experimental sheep farm (INRA, Nouzilly, Western France), with 3 replicates of a 4 sites × 4 traps randomized Latin square using one sheep per site. Collected Culicoides individuals were sorted morphologically to species, sex and physiological stages for females. Sibling species were identified using a molecular assay. A total of 534 Culicoides belonging to 17 species was collected. Abundance was maximal in the drop trap (232 females and 4 males from 10 species) whereas the diversity was the highest in the UV-light/suction trap (136 females and 5 males from 15 species). Significant between-trap differences abundance and parity rates were observed. Conclusions: Only the direct aspiration collected exclusively host-seeking females, despite a concern that human manipulation may influence estimation of the biting rate. The sticky cover trap assessed accurately the biting rate of abundant species even if it might act as an interception trap. The drop trap collected the highest abundance of Culicoides and may have caught individuals not attracted by sheep but by its structure. Finally, abundances obtained using the UV-light/suction trap did not estimate accurately Culicoides biting rate. [ABSTRACT FROM AUTHOR]

Species recognition and identification are crucial in any biological studies, especially when dealing with insect species involved in pathogen transmission. In recent years, molecular approaches have helped the clarification of systematic schemes and taxonomic status. Kiehl et al. (Parasitol Res 105:331–336, ) used molecular data to discuss the taxonomic status of biting midge species in the Palaearctic region. In the present work, the statements that “[Thus] there is no molecular support for the existence of a separate species C. montanus” and “[Therefore] probably C. scoticus should be considered only as a race of C. obsoletus” are discussed. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) is a Culicoides-borne pathogen infecting both domestic and wild ruminants. In Europe, the Red Deer (Cervus elaphus) (RD) is considered a potential BTV reservoir, but persistent sylvatic cycle has not yet been demonstrated. In this paper, we explored the dynamics of BTV1 and BTV8 serotypes in the RD in France, and the potential role of that species in the re-emergence of BTV8 in livestock by 2015 (i.e., 5 years after the former last domestic cases). We performed 8 years of longitudinal monitoring (2008–2015) among 15 RD populations and 3065 individuals. We compared Culicoides communities and feeding habits within domestic and wild animal environments (51,380 samples). Culicoides diversity (>30 species) varied between them, but bridge-species able to feed on both wild and domestic hosts were abundant in both situations. Despite the presence of competent vectors in natural environments, BTV1 and BTV8 strains never spread in RD along the green corridors out of the domestic outbreak range. Decreasing antibody trends with no PCR results two years after the last domestic outbreak suggests that seropositive young RD were not recently infected but carried maternal antibodies. We conclude that RD did not play a role in spreading or maintaining BTV in France. [ABSTRACT FROM AUTHOR]