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Summary: Following the emergence of the Schmallenberg virus (SBV) in 2011 in Germany and its rapid spread in Europe, Culicoides (Diptera: Ceratopogonidae) collected through the French surveillance network were analysed in order to record the presence of virus genome into species diversity collected, to assess the minimum infectious rates (MIR) and the virus circulation dynamics in Culicoides populations. Two vector activity periods were selected (2011, August to October, 53 sites and 2012, June to October, 35 sites) corresponding to 704 night collections. A total of 29,285 individual midges covering at least 50 species were tested either in pools of maximum 50 females or individually (for Culicoides obsoletus/Culicoides scoticus) using real‐time RT‐PCR. Nine species were found SBV positive (C. obsoletus, C. scoticus, Culicoides chiopterus, Culicoides dewulfi, Culicoides imicola, Culicoides pulicaris, Culicoides newsteadi, Culicoides lupicaris and Culicoides nubeculosus) with overall MIR ranging from 0.2% to 4.2%. While the Culicoides nubeculosus laboratory strain is generally considered to have only low vector competence for viruses, interestingly, field‐caught C. nubeculosus specimens were found positive twice for SBV. The first SBV‐positive pool was recorded in August 2011 in north‐eastern France, dating the virus circulation in France 5 months earlier than the first recorded congenital malformations and 2 months earlier than the former recorded date based on retrospective serological data. The MIR were maximum in October 2011, and in July 2012 according to dates of virus arrival in the studied areas. Moreover, our study also showed that virus circulation could be locally intense with infection rate (IR) reaching up to 16% for C. obsoletus/C. scoticus in July 2012 in one site of western France. This retrospective study demonstrates the importance of large‐scale analysis to describe the spatio‐temporal dynamics of virus circulation. [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.

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.

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.

Background: Investigations of Culicoides fauna, including inventories, were carried out in Morocco at different periods after the country had faced major bluetongue and African horse sickness outbreaks. However, no comprehensive reference publication has provided a clear overview of the Culicoides species diversity. This study reviewed available data on Culicoides biting midge species in Morocco from 1968 to 2015 (published and grey literature in French and English) in order to revise the current checklist, in light of state of the art taxonomic and systematic knowledge, and confirmed the checklist with morphological and molecular identifications of specimens collected from the region of Rabat., Methods: Literature related to Culicoides collections in Morocco was collated. Authors were contacted to obtain raw data and additional information for the collections. Fresh Culicoides material was collected and examined from two sites around Rabat, the capital of Morocco. Each collected individual was examined and morphologically identified, if possible, to the species level. In addition, molecular identification was performed to separate closely related species, to confirm difficult morphological identifications and to confirm new species records., Results: A total of 6121 individuals of Culicoides spp. were collected and analyzed and at least 17 species were identified: C. cataneii/C. gejgelensis, C. circumscriptus, C. fagineus, C. festivipennis, C. imicola, C. jumineri, C. kingi, C. longipennis, C. montanus, C. newsteadi, C. obsoletus, C. paolae, C. parotti, C. puncticollis, C. sahariensis, C. scoticus and C. subfagineus. Seven species were confirmed using phylogenetic analyses. Two new species records for Morocco are reported: C. paolae and C. subfagineus., Conclusions: The Moroccan fauna of Culicoides now includes 54 valid species. Further work would certainly increase this total, as one of the clades we identified was not affiliated to any described and valid species.

Background: Biting midge species of the genus Culicoides Latreille (Diptera: Ceratopogonidae) comprise more than 1300 species distributed worldwide. Several species of Culicoides are vectors of various viruses that can affect animals, like the African horse sickness virus (AHSV), known to be endemic in sub-Saharan Africa. The ecological and veterinary interest of Culicoides emphasizes the need for rapid and reliable identification of vector species. However, morphology-based identification has limitations and warrants integration of molecular data. DNA barcoding based on the mitochondrial gene cytochrome c oxidase subunit 1 (cox1) is used as a rapid and authentic tool for species identification in a wide variety of animal taxa across the globe. In this study, our objectives were as follows: (i) establish a reference DNA barcode for Afrotropical Culicoides species; (ii) assess the accuracy of cox1 in identifying Afrotropical Culicoides species; and (iii) test the applicability of DNA barcoding for species identification on a large number of samples of Culicoides larvae from the Niayes area of Senegal, West Africa., Results: A database of 230 cox1 sequences belonging to 42 Afrotropical Culicoides species was found to be reliable for species-level assignments, which enabled us to identify cox1 sequences of Culicoides larvae from the Niayes area of Senegal. Of the 933 cox1 sequences of Culicoides larvae analyzed, 906 were correctly identified by their barcode sequences corresponding to eight species of Culicoides. A total of 1131 cox1 sequences of adult and larval Culicoides were analyzed, and a hierarchical increase in mean divergence was observed according to two taxonomic levels: within species (mean = 1.92%, SE = 0.00), and within genus (mean = 17.82%, SE = 0.00)., Conclusions: Our study proves the efficiency of DNA barcoding for studying Culicoides larval diversity in field samples. Such a diagnostic tool offers great opportunities for investigating Culicoides immature stages ecology and biology, a prerequisite for the implementation of eco-epidemiological studies to better control AHSV in the Niayes region of Senegal, and more generally in sub-Saharan Africa.

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.

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.

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.

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.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.