Culicoides Scoticus Research Articles

Emergence dynamics of adult Culicoides biting midges at two farms in south-east England

BackgroundCulicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of livestock arboviruses that cause diseases with significant economic, social and welfare impacts. Within temperate regions, livestock movement during arbovirus outbreaks can be facilitated by declaring a ‘seasonal vector-free period’ (SVFP) during winter when adult Culicoides are not active. In this study we carry out long-term monitoring of Culicoides adult emergence from larval development habitats at two farms in the UK to validate current definitions of the SVFP and to provide novel bionomic data for known vector species.MethodsStandard emergence traps were used to collect emerging adult Culicoides from dung heaps at two cattle farms in the south-east of England from June to November 2016 and March 2017 to May 2018. Culicoides were morphologically identified to species or complex level and count data were analysed using a simple population dynamic model for pre-adult Culicoides that included meteorological components.ResultsMore than 96,000 Culicoides were identified from 267 emergence trapping events across 2 years, revealing clear evidence of bivoltinism from peaks of male populations of Culicoidesobsoletus emerging from dung heaps. This pattern was also reflected in the emergence of adult female Obsoletus complex populations, which dominated the collections (64.4% of total catch) and emerged throughout the adult active period. Adult male C. obsoletus were observed emerging earlier than females (protandry) and emergence of both sexes occurred throughout the year. Culicoides chiopterus and Culicoides scoticus were also identified in spring emergence collections, providing the first evidence for the overwintering of larvae in dung heaps for these species.ConclusionsThis study demonstrates continual and highly variable rates of emergence of Culicoides throughout the year. A lack of evidence for mass emergence in spring along with the ability to observe male generations highlights the need for complementary surveillance techniques in addition to light-trap data when investigating seasonality and phenology. Evidence was found of other vector species, C. chiopterus and C. scoticus, utilising cattle dung heaps as an overwintering habitat, further highlighting the importance of these habitats on farms.Graphical

Culicoides scoticus

This datasheet on Culicoides scoticus covers Identity, Distribution, Hosts/Species Affected.

A study of the composition of the Obsoletus complex and genetic diversity of Culicoides obsoletus populations in Spain

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

Thermal limits for flight activity of field-collected Culicoides in the United Kingdom defined under laboratory conditions

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

Species composition and relative abundance of the genus Culicoides (Diptera: Ceratopogonidae) in Romania

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

Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain.

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host-feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood-engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.

Indoor and outdoor winter activity of Culicoides biting midges, vectors of bluetongue virus, in Italy.

Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black-light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C.obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C.obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors.

A Mini-review of Culicoides (Diptera: Ceratopogonidae) Fauna and its Vectorial Role in Tunisia

Following the bluetongue outbreaks in Tunisia from 1999 to 2006, bluetongue virus serotype 2 was isolated and several entomological investigations were performed. The present study reviewed the current status of Culicoides fauna in Tunisia and their role as proven and potential vectors of bluetongue virus based on the available and scattered reports. In total, about 35 species of Culicoides are known to occur in the country. Culicoides imicola is considered as the main and the proven vector of bluetongue virus. Other species of the genus Culicoides were suggested as potential vectors bluetongue virus. We cited mainly Culicoides obsoletus, Culicoides scoticus, Culicoides dewulfi and Culicoides pulicaris. Priority recommendations to prepare the country for future veterinary health challenges were suggested.

Molecular phylogeny of 42 species ofCulicoides(Diptera, Ceratopogonidae) from three continents

The genus Culicoides includes vectors of important animal diseases such as bluetongue and Schmallenberg virus (BTV and SBV). This genus includes 1300 species classified in 32 subgenera and 38 unclassified species. However, the phylogenetic relationships between different subgenera of Culicoides have never been studied. Phylogenetic analyses of 42 species belonging to 12 subgenera and 8 ungrouped species of genus Culicoides from Ecuador, France, Gabon, Madagascar and Tunisia were carried out using two molecular markers (28S rDNA D1 and D2 domains and COI mtDNA). Sequences were subjected to non-probabilistic (maximum parsimony) and probabilistic (Bayesian inference (BI)) approaches. The subgenera Monoculicoides, Culicoides, Haematomyidium, Hoffmania, Remmia and Avaritia (including the main vectors of bluetongue disease) were monophyletic, whereas the subgenus Oecacta was paraphyletic. Our study validates the subgenus Remmia (= Schultzei group) as a valid subgenus, outside of the subgenus Oecacta. In Europe, Culicoides obsoletus, Culicoides scoticus and Culicoides chiopterus should be part of the Obsoletus complex whereas Culicoides dewulfi should be excluded from this complex. Our study suggests that the current Culicoides classification needs to be revisited with modern tools.

Eco-climatic indicators for three Culicoides species of the Obsoletus complex in Italy.

Bluetongue, Schmallenberg and African horse sickness viruses are transmitted by vectors belonging to the genus Culicoides (Diptera: Ceratopogonidae). Among this genus, species of the Obsoletus complex play a crucial role in Europe. In Italy the complex includes three species: Culicoides obsoletus sensu strictu, Culicoides scoticus and Culicoides montanus. These three sibling species were identified in 80 collection sites across Italy through a multiplex PCR test. Sixty‑four sites were classified with a predominant species through a Bayesian approach. Environmental, topographic and climatic factors together with landscape metrics were investigated in each species group using the non‑parametric Mann‑Whitney test. Culicoides obsoletus s.s. resulted the most widely distributed species across the peninsula (51 sites). This species was collected in many eco‑climatic conditions, at a wide range of temperature and altitudes, mainly in anthropogenic landscape. Conversely C. scoticus collection sites were dominated by natural vegetated areas or forest, at medium higher altitudes, preferably in a wilder and more pristine environment (predominant species in 8 sites). Culicoides montanus was a rarer species, statistically predominant in only 3 sites located in Southern Italy, characterized by temperatures higher than the other species. These results, together with other on‑going researches on the vector competence of the species belonging to the Obsoletus complex, and with investigation of larval and breeding habitats, could greatly improve epidemiological knowledge of possible spreading Culicoides‑borne viruses.

An unrecognized species of the Culicoides obsoletus complex feeding on livestock in the Netherlands

In studies on Culicoides attacking livestock in the Netherlands, we chanced upon a species of the Obsoletus complex that we do not recognize, but whose dark wing pattern is distinctive. Nine cytochrome c oxidase (CO1) sequences of our so-called ‘dark obsoletus’ support its status as a separate species, the sequences differing significantly from those representing Culicoides obsoletus (Meigen) (90–91% homology) and Culicoides scoticus Downes & Kettle (87–88% homology). In the last decade, several research groups in Europe have encountered ‘mystery species’ related to C. obsoletus and in some instances have made their sequences for various genetic loci available in GenBank. These include a CO1 series submitted from Sweden in 2012 (annotated as ‘obsoletus 01, 02, or 03 MA-2012′) and of which some share a 99% identity with our sequences for ‘dark obsoletus’. Without doubt, the series from the Netherlands, along with a portion of the Swedish submissions, together represent a single species (‘dark obsoletus’). Whether this species is referable to the Russian Culicoides gornostaevae Mirzaeva recorded recently from Norway, Sweden and Poland, and based solely upon the external morphology of the male, is not clear. The presence in Western Europe of multiple undescribed species related to C. obsoletus means that the taxonomy of this important vector complex is not fully resolved; consequently, we know little about these cryptic species with regard to seasonality, geographic range and host preference. This is undesirable given that Culicoides-borne arboviruses causing disease in livestock are moving more regularly out of the tropics and spreading north into temperate latitudes.

Environmental drivers of Culicoides phenology: how important is species-specific variation when determining disease policy?

Since 2006, arboviruses transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) have caused significant disruption to ruminant production in northern Europe. The most serious incursions involved strains of bluetongue virus (BTV), which cause bluetongue (BT) disease. To control spread of BTV, movement of susceptible livestock is restricted with economic and animal welfare impacts. The timing of BTV transmission in temperate regions is partly determined by the seasonal presence of adult Culicoides females. Legislative measures therefore allow for the relaxation of ruminant movement restrictions during winter, when nightly light-suction trap catches of Culicoides fall below a threshold (the ‘seasonally vector free period’: SVFP). We analysed five years of time-series surveillance data from light-suction trapping in the UK to investigate whether significant inter-specific and yearly variation in adult phenology exists, and whether the SVFP is predictable from environmental factors. Because female vector Culicoides are not easily morphologically separated, inter-specific comparisons in phenology were drawn from male populations. We demonstrate significant inter-specific differences in Culicoides adult phenology with the season of Culicoides scoticus approximately eight weeks shorter than Culicoides obsoletus. Species-specific differences in the length of the SVFP were related to host density and local variation in landscape habitat. When the Avaritia Culicoides females were modelled as a group (as utilised in the SFVP), we were unable to detect links between environmental drivers and phenological metrics. We conclude that the current treatment of Avaritia Culicoides as a single group inhibits understanding of environmentally-driven spatial variation in species phenology and hinders the development of models for predicting the SVFP from environmental factors. Culicoides surveillance methods should be adapted to focus on concentrated assessments of species-specific abundance during the start and end of seasonal activity in temperate regions to facilitate refinement of ruminant movement restrictions thereby reducing the impact of Culicoides-borne arboviruses.

Impact of mechanical disturbance on the emergence of Culicoides from cowpats

The outbreaks of Bluetongue virus and Schmallenberg virus between 2006 and 2012 highlighted the need for control methods for north-western Palaearctic biting midge species. There is especially a demand for alternative control methods without application of insecticides. Therefore, the objective of this paper was to evaluate the impact of mechanical disturbance as a control method for Culicoides biting midges in cowpats. We used a trash rake to destroy the compact structure of cowpats and spread the dung over the ground. We expected the disturbance to cause a faster alteration of physical characteristics and drying of the dung, resulting in lower emergence from disturbed cowpats. Emerging biting midges were collected with emergence traps. The number of emerged Culicoides was compared between treated and control cowpats. A total of 12,979 biting midges emerged, all belonging to the Obsoletus group. The majority of the 6,758 male individuals were identified as Culicoides chiopterus (Meigen, 1830) (91.4%), followed by Culicoides dewulfi (Goetghebuer, 1936) (6.5%), and a small number of Culicoides scoticus (Downes & Kettle, 1952) (1.2%). Our results showed no significant differences between the emergence of Culicoides (males and females) from disturbed and control cowpats. The lack of differences is discussed in light of the time period chosen for the experiment and the climatic conditions (especially low temperatures) during the study period. The climatic conditions during the study did not favour desiccation effects and therefore did not cause a severe alteration of the dung and the biotic parameters. In conclusion, immature Culicoides showed a high tolerance against mechanical disturbance.

Does covering of farm-associated Culicoides larval habitat reduce adult populations in the United Kingdom?

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of a range of internationally important arboviruses of livestock, including bluetongue virus (BTV) and the recently emerging Schmallenberg virus (SBV). Culicoides species in the subgenus Avaritia (in the UK: Culicoides obsoletus Meigen, Culicoides scoticus Downes & Kettle, Culicoides dewulfi Goetghebuer and Culicoides chiopterus Meigen) have been implicated in BTV transmission in northern Europe and to a varying degree utilise cattle dung as a larval development substrate. The collection of cattle dung into heaps on farms provides a localised source of Culicoides emergence in close proximity to livestock. This study assesses the impact of covering dung heaps prior to the onset of adult Culicoides activity with the aim of reducing recruitment to the local adult populations at four livestock farms in England. Light suction trap catches of adult Culicoides from these farms were compared with those from four untreated control farms from a wide geographic range across the UK. It was demonstrated that implementing control of emergence from dung heaps did not have a significant impact upon the local adult subgenus Avaritia abundance at the treated farm holdings and that the onset of Culicoides activity was similarly unaffected. Use of this method in isolation is unlikely to have an effect in reducing the risk of BTV and SBV transmission. The implications of these results for control of farm-associated Culicoides in Europe are discussed.

Wing geometry as a tool for discrimination of Obsoletus group (Diptera: Ceratopogonidae: Culicoides) in France

In Europe, Culicoides chiopterus, Culicoides dewulfi, Culicoides obsoletus and Culicoides scoticus, which belongs to the subgenus Avaritia and Obsoletus group are the most proficient Bluetongue and Schmallenberg vectors. Within this group, correct identification based on morphological traits is difficult but essential to assess disease transmission risk. The development of new tools has revolutionized taxonomy (i.e. geometric morphometrics and molecular biology). Wing morphology is of primary importance to entomologists interested in systematics. Here, we report phenotypic differentiation patterns among the species above mentioned using a landmark-based geometric morphometric approach that efficiently identified C. chiopterus and C. dewulfi. Wing shape of the C. scoticus sample exhibited large specific variability. Based on landmarks and phylogenetic analyses (Maximum Parsimony), we suggest that Obsoletus group in Europe includes only C. obsoletus and C. scoticus. C. dewulfi and C. chiopterus are clearly excluded. Their shape seems closer to C. obsoletus that is why we suggest that only these two species should be grouped in the Obsoletus group. In addition, the concordance between phenetic clusters and phylogenies inferred from molecular data based on a fragment of the mtDNA COI gene and rDNA 28S suggests the existence of a strong signal in wing shape. These findings encourage us to use this powerful tool in taxonomic studies.

Host preferences of PalaearcticCulicoidesbiting midges: implications for transmission of orbiviruses

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 Palaearctic Culicoides species (Diptera: Ceratopogonidae) Latreille using traps baited with the five different host species of poultry, horse, cattle, sheep and goat. Collections were carried out nightly in July and August 2009 in western France with three replicates of a 5 × 5 randomized Latin square (five sites, five hosts). Moreover, an ultraviolet (UV) light/suction trap was operated during host-baited collections to correlate Culicoides biting rates and UV light/suction trap catches. A total of 660 Culicoides belonging to 12 species, but comprised mainly of Culicoides scoticus Downes and Kettle, Culicoides dewulfi Goetghebuer and Culicoides obsoletus Meigen, were collected on animal baits. Abundance was highest for the horse, which accounted for 95% of all Culicoides caught, representing 10 species. The horse, the largest bait, was the most attractive host, even when abundance data were corrected by weight, body surface or Kleiber's scaling factor. Culicoides obsoletus was the only dominant species attracted by birds. Both C. scoticus and C. dewulfi were collected mainly from the upper body of the horse. Finally, the quantification of host preferences allows for discussion of implications for the transmission of Culicoides-borne pathogens such as bluetongue virus.

A survey of Culicoides developmental sites on a farm in northern Spain, with a brief review of immature habitats of European species

Culicoides species (Diptera: Ceratopogonidae) belonging to the Obsoletus and Pulicaris groups are considered to be the main vectors of bluetongue virus (BTV) in non Mediterranean Europe. Selected terrestrial microhabitats (n=17) on a farm in northern Spain were sampled repeatedly over a year-long period and characterized for use by Culicoides species for immature development. Concurrent use of CDC light traps showed the presence of 37 species and 66,575 specimens of adult Culicoides. A total of 28 species and 11,396 individuals emerged from laboratory-maintained soil samples. Culicoides obsoletus and Culicoides scoticus (pooled as Obsoletus complex) were particularly abundant (comprising 58.6% and 74.5% of the total collections in light traps and emergence traps respectively). Potential key vectors of animal viruses (such as BTV) were found in two main terrestrial types of microhabitats. In the case of C. obsoletus, different types of manure (old and composted manure, manure mixed with organic matter, and fresh manure) produced most of the specimens. In contrast, larvae of C. scoticus and Culicoides lupicaris were associated with soil substantially comprised of rotting leaf litter that included the parasitic plant Lathraea clandestina. Several species, Culicoides festivipennis, Culicoides punctatus and Culicoides brunnicans, were very common in mud at pond margins. Indeed, pond microhabitats and runoff below barn rooflines supported the greatest species richness. In the pond habitat, 49.4% of Culicoides specimens emerged from mud at the water edge, as opposed to 50cm above (32.4%) and 1 meter above waterline (18%). Similar species richness, but statistically significant differences in abundance, were observed among the four pond microhabitats. Overall, the majority of the specimens were found in the upper layer (0–3cm), except in manure, where they preferred deeper layers (>6cm). Maximum peaks of abundance occurred in both light traps and soil samples in summer months, whereas increased captures in autumn were noticed only in light traps. Both trapping systems failed to collect adult Culicoides midges in the coldest months of December, January and February. The literature on immature habitats of species suspected in BTV transmission in Europe, the Pulicaris group and particularly the Obsoletus group, is briefly reviewed.

Chemical composition of silage residues sustaining the larval development of the Culicoides obsoletus/Culicoides scoticus species (Diptera: Ceratopogonidae)

Culicoides (Diptera: Ceratopogonidae) are biological vectors of bluetongue virus (BTV). Bluetongue is a viral disease that affects domestic and wild ruminants. Since its recent emergence in northern Europe, this disease has caused considerable economic losses to the sheep and cattle industry. The biotopes, and more particularly the chemical characteristics which are suitable for larval development of the main vector species, are still relatively unknown. This study shows that the larvae of biting midges belonging to the species Culicoides obsoletus and Culicoides scoticus are able to breed in different types of silage residue (maize, grass, sugar beet pulp and their combinations). The chemical composition of substrates strongly influences the presence of the immature stages of these biting midges. Higher lignin and insoluble fibre contents seem to favour their presence and could play the role of a physical support for semi-aquatic larvae. In contrast, higher concentrations of magnesium and calcium are negatively correlated with the presence of these two species. These data will help to locate and monitor the breeding sites of these species and could contribute to the control of these insects on farms.

Investigation of Diel Activity ofCulicoidesBiting Midges (Diptera: Ceratopogonidae) in the United Kingdom by Using a Vehicle-Mounted Trap

Truck trap collections of Culicoides biting midges (Diptera: Ceratopogonidae) were made during 2 yr of sampling from 2008 to 2009 at a farm site in southern England. Samples were collected from 810 sample runs carried out over 52 d and contained 7,095 Culicoides of which more than half (50.3%) were identified as Culicoides obsoletus Meigen by using a multiplex polymerase chain reaction assay. Other commonly encountered species included Culicoides scoticus Downes & Kettle (14.7% of total Culicoides caught), Culicoides dewulfi Goetghebuer (3.7%), and Culicoides chiopterus Meigen (4.2%). The activity rates of these species were examined with regard to both meteorological factors (light intensity, humidity, temperature, and wind speed and direction) and other potentially contributing variables (lunar phase and brightness, sunset time, and year) by using generalized linear models. All the species examined were collected in greater abundance at sunset, although the relationship between underlying light intensity and numbers was less pronounced in C. dewulfi and C. chiopterus. Collections of Culicoides were reduced at temperatures above 21 degrees C and were inversely related to wind speed. Variation between species was recorded, however, in response to wind direction: C. dewulfi and C. chiopterus were associated with prevailing winds passing through fields containing livestock, whereas C. obsoletus and C. scoticus demonstrated no such relationship. A male:female ratio of 1:3.56 was observed in catches, and male populations were protandrous. These results are discussed with reference both to the ecology of these species and methods currently used to predict adult Culicoides movement and abundance in Europe.

Simultaneous quantification of the relative abundance of species complex members: Application to Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae), potential vectors of bluetongue virus

The two sympatric sibling species Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle (Diptera: Ceratopogonidae), are known to be competent vectors for bluetongue virus in the Palaearctic region. However, morphological identification of constituent species is only readily applicable to adult males and these two species distinguishing traits have overlapping character states. As their vector competence may differ in space and time, the correct identification and quantification of specimens of both species are essential for understanding bluetongue epidemiology. However, no molecular tools are available for high-throughput identification of the two species. We therefore developed a quantitative duplex real-time PCR assay to determine the relative abundance of each sibling species in a sample using TaqMan probes. For each species, standard curves were constructed from serial dilutions of purified plasmid DNA containing ITS1-5.8S-ITS2 (rDNA) in the range of 10−1 to 10−5ng/μL. Standard curves were used to quantify samples of mixed C. obsoletus/C. scoticus specimens. Specificity was evaluated with 5156 specimens representing 62 species. Based on the DNA quantities detected according to the standard curves, a quadratic model developed on 1100 males and validated on 555 females was able to predict the relative abundance of each species simultaneously in a one-shot reaction (Pearson coefficient of 0.999). Our assay showed a requirement of two specimens or less for 95% of the predictions, making it highly applicable to field collections. Extensive use of this real-time PCR assay will provide a better understanding of geographical distribution, dynamics, and bionomics on a species level, which is essential for risk assessment. This approach is an important contribution to medical entomology for investigating the vector role of arthropod sibling species.