Hyaluronidase Activity in Saliva of European Culicoides (Diptera: Ceratopogonidae).

Biting midges of the genus Culicoides transmit pathogens of veterinary importance such as bluetongue virus (Reoviridae: Orbivirus). The saliva of Culicoides is known to contain bioactive molecules including peptides and proteins with vasodilatory and immunomodulative properties. In this study, we detected activity of enzyme hyaluronidase in six Culicoides species that commonly occur in Europe and that are putative vectors of arboviruses. Hyaluronidase was present in all species studied, although its molecular size, sensitivity to SDS, and substrate specificity differed between species. Further studies on the potential effect of hyaluronidase activity on the vector competence of Culicoides species for arboviruses would be beneficial.

Description of Culicoides paradoxalis sp. nov. from France and Portugal (Diptera: Ceratopogonidae).

A new species, Culicoides paradoxalis Ramilo and Delécolle (Diptera: Ceratopogonidae), is described from specimens collected in France (Corsica and southeast region) and Portugal. This species resembles Culicoides lupicaris Downes and Kettle, and can be distinguished from this species and from Culicoides newsteadi Austen by its wing pattern, in addition to the absence of spines on the tarsomere 4 of female mid leg. In male, the presence of two appendices on the sternite 9 together with the absence of sensilla coeloconica on the flagellomere 11 is also useful to distinguish these three species. Separation from other members of the Culicoides subgenus is confirmed by the analysis of the Cytochrome Oxidase I (COI) mitochondrial marker. 

Modelling the monthly abundance of Culicoides biting midges in nine European countries using Random Forests machine learning.

BACKGROUND: Culicoides biting midges transmit viruses resulting in disease in ruminants and equids such as bluetongue, Schmallenberg disease and African horse sickness. In the past decades, these diseases have led to important economic losses for farmers in Europe. Vector abundance is a key factor in determining the risk of vector-borne disease spread and it is, therefore, important to predict the abundance of Culicoides species involved in the transmission of these pathogens. The objectives of this study were to model and map the monthly abundances of Culicoides in Europe. METHODS: We obtained entomological data from 904 farms in nine European countries (Spain, France, Germany, Switzerland, Austria, Poland, Denmark, Sweden and Norway) from 2007 to 2013. Using environmental and climatic predictors from satellite imagery and the machine learning technique Random Forests, we predicted the monthly average abundance at a 1 km2 resolution. We used independent test sets for validation and to assess model performance. RESULTS: The predictive power of the resulting models varied according to month and the Culicoides species/ensembles predicted. Model performance was lower for winter months. Performance was higher for the Obsoletus ensemble, followed by the Pulicaris ensemble, while the model for Culicoides imicola showed a poor performance. Distribution and abundance patterns corresponded well with the known distributions in Europe. The Random Forests model approach was able to distinguish differences in abundance between countries but was not able to predict vector abundance at individual farm level. CONCLUSIONS: The models and maps presented here represent an initial attempt to capture large scale geographical and temporal variations in Culicoides abundance. The models are a first step towards producing abundance inputs for R0 modelling of Culicoides-borne infections at a continental scale.

Spatial distribution modelling of Culicoides (Diptera: Ceratopogonidae) biting midges, potential vectors of African horse sickness and bluetongue viruses in Senegal.

BACKGROUND: In Senegal, the last epidemic of African horse sickness (AHS) occurred in 2007. The western part of the country (the Niayes area) concentrates modern farms with exotic horses of high value and was highly affected during the 2007 outbreak that has started in the area. Several studies were initiated in the Niayes area in order to better characterize Culicoides diversity, ecology and the impact of environmental and climatic data on dynamics of proven and suspected vectors. The aims of this study are to better understand the spatial distribution and diversity of Culicoides in Senegal and to map their abundance throughout the country. METHODS: Culicoides data were obtained through a nationwide trapping campaign organized in 2012. Two successive collection nights were carried out in 96 sites in 12 (of 14) regions of Senegal at the end of the rainy season (between September and October) using OVI (Onderstepoort Veterinary Institute) light traps. Three different modeling approaches were compared: the first consists in a spatial interpolation by ordinary kriging of Culicoides abundance data. The two others consist in analyzing the relation between Culicoides abundance and environmental and climatic data to model abundance and investigate the environmental suitability; and were carried out by implementing generalized linear models and random forest models. RESULTS: A total of 1,373,929 specimens of the genus Culicoides belonging to at least 32 different species were collected in 96 sites during the survey. According to the RF (random forest) models which provided better estimates of abundances than Generalized Linear Models (GLM) models, environmental and climatic variables that influence species abundance were identified. Culicoides imicola, C. enderleini and C. miombo were mostly driven by average rainfall and minimum and maximum normalized difference vegetation index. Abundance of C. oxystoma was mostly determined by average rainfall and day temperature. Culicoides bolitinos had a particular trend; the environmental and climatic variables above had a lesser impact on its abundance. RF model prediction maps for the first four species showed high abundance in southern Senegal and in the groundnut basin area, whereas C. bolitinos was present in southern Senegal, but in much lower abundance. CONCLUSIONS: Environmental and climatic variables of importance that influence the spatial distribution of species abundance were identified. It is now crucial to evaluate the vector competence of major species and then combine the vector densities with densities of horses to quantify the risk of transmission of AHS virus across the country.

An update of the Culicoides (Diptera: Ceratopogonidae) checklist for the Balkans.

BACKGROUND: The prime significance of species belonging to the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) is their ability to transmit viruses such as bluetongue virus (BTV) to wild and domestic ruminants. Prior to 1998, BTV was considered exotic in Europe, but according to recent history of its outbreaks, it has become endemic in southern and eastern European countries circulating beyond its expected historical limits, into the Balkan region. The wind-borne long-distance dispersal of Culicoides spp. over water bodies and local spreading between farms emphasize the necessity of filling in the information gaps regarding vector species distribution. In most Balkan countries, data on Culicoides fauna and species distribution are lacking, or information is old and scarce. RESULTS: During this study, 8586 specimens belonging to 41 species were collected. We present the first faunistic data on Culicoides species in the former Yugoslav Republic of Macedonia (FYROM), Kosovo, Montenegro and Serbia. For other countries (Bosnia and Herzegovina, Bulgaria and Croatia), all historical records were compiled for the first time and then expanded with our findings to various extents. In all countries, confirmed or suspected BTV vector species belonging to the subgenera Avaritia and Culicoides were collected. The total number of species sampled during our field collections was 20 in Bosnia and Herzegovina (15 new records), 10 in Bulgaria (2 new records), 10 in Croatia (5 new records), 13 in FYROM, 9 in Kosovo, 15 in Montenegro, and 28 in Serbia. Of these, 14 species were registered for the first time in this part of the Balkans. CONCLUSIONS: This paper provides the first data about Culicoides fauna in FYROM, Kosovo, Montenegro and Serbia, as well as new records and an update on the checklists for Bosnia and Herzegovina, Bulgaria and Croatia. These findings provide preliminary insights into the routes of BTV introduction and spreading within the Balkans, and present a valuable contribution to further research related to Culicoides-borne diseases in Europe.

Monthly variation in the probability of presence of adult Culicoides populations in nine European countries and the implications for targeted surveillance.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are small hematophagous insects responsible for the transmission of bluetongue virus, Schmallenberg virus and African horse sickness virus to wild and domestic ruminants and equids. Outbreaks of these viruses have caused economic damage within the European Union. The spatio-temporal distribution of biting midges is a key factor in identifying areas with the potential for disease spread. The aim of this study was to identify and map areas of neglectable adult activity for each month in an average year. Average monthly risk maps can be used as a tool when allocating resources for surveillance and control programs within Europe. METHODS: We modelled the occurrence of C. imicola and the Obsoletus and Pulicaris ensembles using existing entomological surveillance data from Spain, France, Germany, Switzerland, Austria, Denmark, Sweden, Norway and Poland. The monthly probability of each vector species and ensembles being present in Europe based on climatic and environmental input variables was estimated with the machine learning technique Random Forest. Subsequently, the monthly probability was classified into three classes: Absence, Presence and Uncertain status. These three classes are useful for mapping areas of no risk, areas of high-risk targeted for animal movement restrictions, and areas with an uncertain status that need active entomological surveillance to determine whether or not vectors are present. RESULTS: The distribution of Culicoides species ensembles were in agreement with their previously reported distribution in Europe. The Random Forest models were very accurate in predicting the probability of presence for C. imicola (mean AUC = 0.95), less accurate for the Obsoletus ensemble (mean AUC = 0.84), while the lowest accuracy was found for the Pulicaris ensemble (mean AUC = 0.71). The most important environmental variables in the models were related to temperature and precipitation for all three groups. CONCLUSIONS: The duration periods with low or null adult activity can be derived from the associated monthly distribution maps, and it was also possible to identify and map areas with uncertain predictions. In the absence of ongoing vector surveillance, these maps can be used by veterinary authorities to classify areas as likely vector-free or as likely risk areas from southern Spain to northern Sweden with acceptable precision. The maps can also focus costly entomological surveillance to seasons and areas where the predictions and vector-free status remain uncertain.

Spatial and temporal variation in the abundance of Culicoides biting midges (Diptera: Ceratopogonidae) in nine European countries.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are vectors of bluetongue virus (BTV), African horse sickness virus and Schmallenberg virus (SBV). Outbreaks of both BTV and SBV have affected large parts of Europe. The spread of these diseases depends largely on vector distribution and abundance. The aim of this analysis was to identify and quantify major spatial patterns and temporal trends in the distribution and seasonal variation of observed Culicoides abundance in nine countries in Europe. METHODS: We gathered existing Culicoides data from Spain, France, Germany, Switzerland, Austria, Denmark, Sweden, Norway and Poland. In total, 31,429 Culicoides trap collections were available from 904 ruminant farms across these countries between 2007 and 2013. RESULTS: The Obsoletus ensemble was distributed widely in Europe and accounted for 83% of all 8,842,998 Culicoides specimens in the dataset, with the highest mean monthly abundance recorded in France, Germany and southern Norway. The Pulicaris ensemble accounted for only 12% of the specimens and had a relatively southerly and easterly spatial distribution compared to the Obsoletus ensemble. Culicoides imicola Kieffer was only found in Spain and the southernmost part of France. There was a clear spatial trend in the accumulated annual abundance from southern to northern Europe, with the Obsoletus ensemble steadily increasing from 4000 per year in southern Europe to 500,000 in Scandinavia. The Pulicaris ensemble showed a very different pattern, with an increase in the accumulated annual abundance from 1600 in Spain, peaking at 41,000 in northern Germany and then decreasing again toward northern latitudes. For the two species ensembles and C. imicola, the season began between January and April, with later start dates and increasingly shorter vector seasons at more northerly latitudes. CONCLUSION: We present the first maps of seasonal Culicoides abundance in large parts of Europe covering a gradient from southern Spain to northern Scandinavia. The identified temporal trends and spatial patterns are useful for planning the allocation of resources for international prevention and surveillance programmes in the European Union.

Exploring the diversity of blood-sucking Diptera in caves of Central Africa.

Caves house pathogenic microorganisms, some of which are transmitted by blood-sucking arthropods. In Africa, previous studies identified mosquitoes, sand flies and biting midges as the main potential vectors of cave-dwelling pathogens. However, to understand their involvement in pathogen spillover, it is crucial to characterize their diversity, community composition and dynamics. Using CDC light traps, we collected hematophagous Diptera in six caves of Gabon during one-shot or longitudinal sampling, and investigated their species diversity and dynamics in relation with external rainfall. Overall, we identified 68 species of mosquitoes, sand flies and biting midges, including 45 new records for Gabon. The dominant species were: Uranotaenia nigromaculata, Anopheles smithii s.l., Culex. rima group and Culex quasiguiarti for mosquitoes, Spelaeophlebotomus gigas and Spelaeomyia emilii for sand flies and the Culicoides trifasciellus group and Culicoides fulvithorax for biting midges. The survey revealed that species assemblages were cave-specific and included mainly troglophilous and trogloxenous species. Both diversity and abundance varied according to the cave and sampling time, and were significantly associated with rainfall. These associations were modulated by the cave specific environmental conditions. Moreover, the presence of trogloxenous and troglophilous species could be of high significance for pathogen transfers between cave and epigeous hosts, including humans.

Spatio-temporal genetic variation of the biting midge vector species Culicoides imicola (Ceratopogonidae) Kieffer in France.

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.

Modelling the Abundances of Two Major Culicoides (Diptera: Ceratopogonidae) Species in the Niayes Area of Senegal.

In Senegal, considerable mortality in the equine population and hence major economic losses were caused by the African horse sickness (AHS) epizootic in 2007. Culicoides oxystoma and Culicoides imicola, known or suspected of being vectors of bluetongue and AHS viruses are two predominant species in the vicinity of horses and are present all year-round in Niayes area, Senegal. The aim of this study was to better understand the environmental and climatic drivers of the dynamics of these two species. Culicoides collections were obtained using OVI (Onderstepoort Veterinary Institute) light traps at each of the 5 sites for three nights of consecutive collection per month over one year. Cross Correlation Map analysis was performed to determine the time-lags for which environmental variables and abundance data were the most correlated. C. oxystoma and C. imicola count data were highly variable and overdispersed. Despite modelling large Culicoides counts (over 220,000 Culicoides captured in 354 night-traps), using on-site climate measures, overdispersion persisted in Poisson, negative binomial, Poisson regression mixed-effect with random effect at the site of capture models. The only model able to take into account overdispersion was the Poisson regression mixed-effect model with nested random effects at the site and date of capture levels. According to this model, meteorological variables that contribute to explaining the dynamics of C. oxystoma and C. imicola abundances were: mean temperature and relative humidity of the capture day, mean humidity between 21 and 19 days prior a capture event, density of ruminants, percentage cover of water bodies within a 2 km radius and interaction between temperature and humidity for C. oxystoma; mean rainfall and NDVI of the capture day and percentage cover of water bodies for C. imicola. Other variables such as soil moisture, wind speed, degree days, land cover or landscape metrics could be tested to improve the models. Further work should also assess whether other trapping methods such as host-baited traps help reduce overdispersion.

How do species, population and active ingredient influence insecticide susceptibility in Culicoides biting midges (Diptera: Ceratopogonidae) of veterinary importance?

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.

Culicoides (Diptera: Ceratopogonidae) midges, the vectors of African horse sickness virus--a host/vector contact study in the Niayes area of Senegal.

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.

Feeding behaviour of Culicoides spp. (Diptera: Ceratopogonidae) on cattle and sheep in northeast Germany.

BACKGROUND: Culicoides spp. play an important role in the transmission of several vector-borne pathogens such as Bluetongue and Schmallenberg virus in Europe. To better understand the biology of local Culicoides species, a study divided into three parts was performed in northeast Germany to elucidate the feeding activity patterns (study A), preferential landing and feeding sites (study B) and host feeding preferences (study C) of Culicoides spp. using cattle and sheep as baits. METHODS: In study A, the activity of Culicoides spp. was monitored over a 72 h period by collecting insects at regular intervals from the interior of drop traps with cattle or sheep standing inside. In study B, Culicoides spp. were directly aspirated from the coat and fleece of cattle and sheep during the peak activity period of Culicoides. In study C, Culicoides spp. were collected using drop traps with either cattle or sheep standing inside and located 10 m apart. RESULTS: In study A, 3,545 Culicoides midges belonging to 13 species were collected, peak activity was observed at sunset. In study B, 2,024 Culicoides midges were collected. A significantly higher number of midges was collected from the belly and flank of cattle in comparison to their head region. In study C, 3,710 Culicoides midges were collected; 3,077 (83%) originated from cattle and 633 (17%) from sheep. Nearly half (46.7%) of the midges collected from cattle were engorged, significantly more than the number of engorged midges collected from sheep (7.5%). Culicoides from the Obsoletus complex (C. obsoletus and C. scoticus) were the most common Culicoides species encountered, followed by C. punctatus. Other species identified were C. dewulfi, C. chiopterus, C. pulicaris, C. lupicaris, C. pallidicornis, C. subfascipennis, C. achrayi, C. stigma, C. griseidorsum and C. subfagineus, the last two species are reported for the first time in Germany. Engorged C. chiopterus were collected in relatively high numbers from sheep, suggesting that this species may have a preference for sheep. CONCLUSIONS: An insight into the feeding behaviour of local Culicoides species under field conditions in northeast Germany was obtained, with implications for the implementation of control measures and midge-borne disease risk analysis.

Seasonal dynamics of Culicoides (Diptera: Ceratopogonidae) biting midges, potential vectors of African horse sickness and bluetongue viruses in the Niayes area of Senegal.

BACKGROUND: The African horse sickness epizootic in Senegal in 2007 caused considerable mortality in the equine population and hence major economic losses. The vectors involved in the transmission of this arbovirus have never been studied specifically in Senegal. This first study of the spatial and temporal dynamics of the Culicoides (Diptera: Ceratopogonidae) species, potential vectors of African horse sickness in Senegal, was conducted at five sites (Mbao, Parc Hann, Niague, Pout and Thies) in the Niayes area, which was affected by the outbreak. METHODS: Two Onderstepoort light traps were used at each site for three nights of consecutive collection per month over one year to measure the apparent abundance of the Culicoides midges. RESULTS: In total, 224,665 specimens belonging to at least 24 different species (distributed among 11 groups of species) of the Culicoides genus were captured in 354 individual collections. Culicoides oxystoma, Culicoides kingi, Culicoides imicola, Culicoides enderleini and Culicoides nivosus were the most abundant and most frequent species at the collection sites. Peaks of abundance coincide with the rainy season in September and October. CONCLUSIONS: In addition to C. imicola, considered a major vector for the African horse sickness virus, C. oxystoma may also be involved in the transmission of this virus in Senegal given its abundance in the vicinity of horses and its suspected competence for other arboviruses including bluetongue virus. This study depicted a site-dependent spatial variability in the dynamics of the populations of the five major species in relation to the eco-climatic conditions at each site.

First record of Culicoides oxystoma Kieffer and diversity of species within the Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) biting midges in Senegal.

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.

Host-seeking activity of bluetongue virus vectors: endo/exophagy and circadian rhythm of Culicoides in Western Europe.

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.

Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on Palaearctic biting midge species, vectors of Orbiviruses.

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.

Assessment of vector/host contact: comparison of animal-baited traps and UV-light/suction trap for collecting Culicoides biting midges (Diptera: Ceratopogonidae), vectors of Orbiviruses.

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.