The dying of the light: crepuscular activity in Culicoides and impact on light trap efficacy at temperate latitudes.

The light trap is the tool of choice for conducting large-scale Culicoides (Diptera: Ceratopogonidae) vector surveillance programmes. Its efficacy is in doubt, however. To assess this, hourly changes in Culicoides activity over the 24-h diel were determined comparatively by way of light trapping and aerial sweeping, and correlated against light intensity. In the Netherlands, sweeping around cattle at pasture revealed that, in early summer, Culicoides are active throughout the diel, and that their abundance peaks during the crepuscular period and falls to a low during the brightest hours of the day. By contrast, the light trap was able to accumulate Culicoides only at night (i.e. after illuminance levels had dropped to 0 lux and midge activity had begun to decline). Although Culicoides chiopterus and species of the Culicoides obsoletus complex were similarly abundant around livestock, they differed critically in their hours of peak activity, being largely diurnal and nocturnal, respectively. This polarity helps to explain why, routinely, the C. obsoletus complex dominates light trap collections and C. chiopterus does not. Inability to accumulate Culicoides at light intensity levels above 0 lux means that, at ever-higher latitudes, particularly beyond 45° N, the progressive northward lengthening of the twilight period will have an increasingly adverse impact upon the efficacy of the light trap as a vector surveillance tool.

Culicoides (Diptera: Ceratopogonidae) and livestock in the Netherlands: comparing host preference and attack rates on a Shetland pony, a dairy cow, and a sheep.

Culicoides (Diptera: Ceratopogonidae) host preferences and attack rates were quantified in early summer at a dairy farm in the Netherlands using livestock tethered at pasture. Midges were aspirated hourly over seven consecutive hours (17:00-23:00) from a dairy cow, a Shetland pony, and a sheep and correspondingly yielded seventeen, thirteen, and nine species. Of the 14,181 midges obtained, approximately 95% belonged to the C. obsoletus complex, C. dewulfi, C. chiopterus, and C. punctatus that together include all proven or potential vectors for arboviral diseases in livestock in northwestern Europe. On average, 7.6 and 3.5 times more Culicoides were collected, respectively, from the cow and the Shetland pony than from the sheep. In descending order of abundance, the C. obsoletus complex, C. dewulfi, and C. chiopterus dominated attacks on all three hosts, whereas C. punctatus and C. pulicaris favored only the two larger hosts. Irrespective of the host species involved, the three body regions attracted the same component species, C. chiopterus favoring the legs, C. punctatus and C. achrayi the belly, and the C. obsoletus complex, C. dewulfi, and C. pulicaris the head, back, and flanks. That known and potential vectors for animal diseases feed indiscriminately on a broad range of mammal hosts means that all major livestock species, including equines, are rendered susceptible to one or more Culicoides-borne pathogens.

Mosquitoes and Culicoides biting midges: vector range and the influence of climate change.

Vector-borne animal diseases pose a continuous and substantial threat to livestock economies around the globe. Increasing international travel, the globalisation of trade, and climate change are likely to play a progressively more important role in the introduction, establishment and spread of arthropod-borne pathogens worldwide. A review of the literature reveals that many climatic variables, functioning singly or in combination, exert varying effects on the distribution and range of Culicoides vector midges and mosquitoes. For example, higher temperatures may be associated with increased insect abundance--thereby amplifying the risk of disease transmission--but there are no indications yet of dramatic shifts occurring in the geographic range of Culicoides midges. However, the same cannot be said for mosquitoes: over the last few decades, multiple Asian species have established themselves in Europe, spread and are unlikely to ever be eradicated. Research on how insects respond to changes in climate is still in its infancy. The authors argue that we need to grasp how other annectant changes, such as extremes in precipitation (drought and flooding), may affect the dispersal capability of mosquitoes. Models are useful for assessing the interplay between mosquito vectors expanding their range and the native flora and fauna; however, ecological studies employing classical mark-release-recapture techniques remain essential for addressing fundamental questions about the survival and dispersal of mosquito species, with the resulting parameters fed directly into new-generation disease transmission models. Studies on the eventual impact of mosquitoes on animal and human health should be tackled through large-scale integrated research programmes. Such an approach calls for more collaborative efforts, along the lines of the One Health Initiative.

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.

Schmallenberg Virus in Culicoides Biting Midges in the Netherlands in 2012.

A total of 130 pools of Culicoides biting midges collected between May and September 2012 in the Netherlands were assayed for Schmallenberg virus (SBV). The Culicoides midges were caught in the same area as where in 2011 a high proportion of Culicoides pools tested positive for SBV, in majority with a high viral load (Ct values between 20 and 30). Two of a total of 42 pools comprising 50 midges/pool of the Obsoletus complex from the 2012 collection tested weak positive (Ct values: 34.96 and 37.66), indicating a relatively low viral load. On an individual midge level, the proportion of SBV-infected Culicoides of the Obsoletus complex caught in the same area and in a comparable period of the year was significantly lower in 2012 (0.1% = 1 per 1050 tested) compared with 2011 (0.56% = 13 per 2300 tested).

Culicoides (Diptera: Ceratopogonidae) host preferences and biting rates in the Netherlands: comparing cattle, sheep and the black-light suction trap.

Host preference is an important determinant of feeding behaviour in biting insects and a critical component in the transmission of vector-borne diseases. The aim of the study was to quantify Culicoides (Diptera: Ceratopogonidae) host preferences and biting rates using tethered livestock at pasture (a dairy cow and a sheep) and to compare the numbers of biting midges aspirated off them to those captured simultaneously in a black-light suction trap acting as a surrogate host. Culicoides collections were made hourly over seven hours (from five hours before official sunset to two hours after) between 27 May and 19 June, 2013 at a dairy farm (eastern Netherlands). The study involved 13 replicates of a site × host randomised design. Culicoides collected by black-light suction trap and by direct aspiration were identified to species morphologically and age-graded. The C. obsoletus complex, C. dewulfi and C. pulicaris predominated on the back and flanks of the animals, C. punctatus on the belly, and C. chiopterus on the legs. Using comparable collection periods, 9.3 times (95% confidence interval: 8.6-10.0) more Culicoides were caught on the cow than on the sheep and 25.4 times (95% confidence interval: 18.4-35.1) less in the black-light suction trap compared to the sheep. Mean Culicoides biting rates on the cow across the 7-h collection period were 4.6, 3.5, 1.0, 1.0 and 0.5 min(-1) for C. dewulfi, the C. obsoletus complex, C. chiopterus, C. punctatus and C. pulicaris, respectively; for the sheep they were 0.6, 0.4 and 0.1 min(-1) for the C. obsoletus complex, C. dewulfi and C. punctatus, respectively. Though midges were aspirated off livestock during each of the seven hours, they only began to appear in the black-light suction trap 5h later, from sunset onwards. After sunset, its efficacy improved markedly, but occurred when midge activity overall had begun to decline. Though it was quite accurate in ranking Culicoides species abundance, the black-light suction trap proved to be of limited value for determining hours of peak biting activity, levels of abundance, and host preference, in Culicoides.

The Mondrian matrix: Culicoides biting midge abundance and seasonal incidence during the 2006-2008 epidemic of bluetongue in the Netherlands.

During the northern Europe epidemic of bluetongue (BT), Onderstepoort-type blacklight traps were used to capture Culicoides Latreille (Diptera: Ceratopogonidae) biting midges weekly between November 2006 and December 2008 on 21 livestock farms in the Netherlands. Proven and potential vectors for the bluetongue virus (BTV) comprised almost 80% of the midges collected: the Obsoletus complex, constituting C. obsoletus (Meigen) and C. scoticus Downes & Kettle (44.2%), C. dewulfi Goetghebuer (16.4%), C. chiopterus (Meigen) (16.3%) and C. pulicaris (Linnaeus) (0.1%). Half of the 24 commonest species of Culicoides captured completed only one (univoltine) or two (bivoltine) generations annually, whereas multivoltine species (including all BTV vectors) cycled through five to six generations (exceeding the one to four generations calculated in earlier decades). Whether this increment signals a change in the phenology of northern Europe Culicoides or simply is an adaptive response that manifests during warmer episodes, thus heightening periodically the incursive potential of midge-borne arboviruses, remains to be clarified. Culicoides duddingstoni Kettle & Lawson, C. grisescens Edwards, C. maritimus Kieffer, C. pallidicornis Kieffer and C. riethi Kieffer are new records for the biting midge fauna of the Netherlands. It is suggested that C. punctatus (Meigen) be added to the European list of vector Culicoides.

Principal climatic and edaphic determinants of Culicoides biting midge abundance during the 2007-2008 bluetongue epidemic in the Netherlands, based on OVI light trap data.

Palaearctic Culicoides midges (Diptera: Ceratopogonidae) represent a vital link in the northward advance of certain arboviral pathogens of livestock such as that caused by bluetongue virus. The effects of relevant ecological factors on weekly Culicoides vector abundances during the bluetongue virus serotype 8 epidemics in the Netherlands in 2007 and 2008 were quantified within a hurdle modelling framework. The relative role of meteorological parameters showed a broadly consistent association across species, with larger catches linked to temperature-related variables and lower wind speed. Moreover, vector abundance was found to be influenced by edaphic factors, likely related to species-specific breeding habitat preferences that differed markedly amongst some species. This is the first study on Culicoides vector species in the Netherlands identified during an entomological surveillance programme, in which an attempt is made to pinpoint the factors that influence midge abundance levels. In addition to providing key inputs into risk-mitigating tools for midge-borne pathogens and disease transmission models, the adoption of methods that explicitly address certain features of abundance datasets (frequent zero-count observations and over-dispersion) helped enhance the robustness of the ecological analysis.

Seasonal dynamics of biting midges (Diptera: Ceratopogonidae: Culicoides), the potential vectors of bluetongue virus, in Sweden.

The outbreak of bluetongue (BT) in northern Europe 2006 initiated the monitoring of vectors, biting midges of the genus Culicoides in Sweden. In order to determine the diversity, distribution and seasonal dynamics of Culicoides, weekly collections were made during 2008 and during March-December 2009 using the Ondestepoort Veterinary Institute black light trap. Twenty sampling sites were selected in 12 provinces. In total of 30,704 Culicoides were collected in 2008 and 32,252 in 2009. The most abundant species were the potential vectors of BTV Culicoides obsoletus/C. scoticus that comprised of 77% of the total catches. Other biting midges collected were Culicoides impunctatus (9%), Culicoides grisescens (3%), Culicoides punctatus (2%), Culicoides chiopterus (2%) and Culicoides pulicaris (2%). Culicoides obsoletus/C. scoticus were most abundant during May-June and August-September. The majority of the species were active from March to November in 2008 and April to October in 2009. Species considered as potential vectors of bluetongue virus (BTV) occurred as far north as latitude 65°N (Kalix).

Mapping the basic reproduction number (R0) for vector-borne diseases: a case study on bluetongue virus.

Geographical maps indicating the value of the basic reproduction number, R0, can be used to identify areas of higher risk for an outbreak after an introduction. We develop a methodology to create R0 maps for vector-borne diseases, using bluetongue virus as a case study. This method provides a tool for gauging the extent of environmental effects on disease emergence. The method involves integrating vector-abundance data with statistical approaches to predict abundance from satellite imagery and with the biologically mechanistic modelling that underlies R0. We illustrate the method with three applications for bluetongue virus in the Netherlands: 1) a simple R0 map for the situation in September 2006, 2) species-specific R0 maps based on satellite-data derived predictions, and 3) monthly R0 maps throughout the year. These applications ought to be considered as a proof-of-principle and illustrations of the methods described, rather than as ready-to-use risk maps. Altogether, this is a first step towards an integrative method to predict risk of establishment of diseases based on mathematical modelling combined with a geographic information system that may comprise climatic variables, landscape features, land use, and other relevant factors determining the risk of establishment for bluetongue as well as of other emerging vector-borne diseases.

Endophily in Culicoides associated with BTV-infected cattle in the province of Limburg, south-eastern Netherlands, 2006.

Culicoides were captured at a BTV-infected dairy near Gulpen in the province of Limburg (south-east Netherlands) between 14 September and 4 October 2006. Onderstepoort-type blacklight traps were used to sample Culicoides both inside and outside a partially open shed housing 11 cattle. A total of 28 light trap collections were made at the shed and yielded: 9371 Culicoides representing 11 species; >90% comprised five potential vectors of BTV and in order of abundance were Culicoides obsoletus and Culicoides scoticus (of the Obsoletus Complex), Culicoides dewulfi, Culicoides pulicaris and Culicoides chiopterus; Culicoides imicola, the principal Mediterranean (and African) vector of BTV, was absent. 2339 Culicoides representing seven species were captured inside (endophily) the cattle shed; >95% comprised the Obsoletus Complex and C. dewulfi. Conversely, the Pulicaris Complex, represented by five species and including C. pulicaris, showed strong exophily with >97% captured outside the shed. 7032 Culicoides were captured outside the shed, approximately threefold more than inside. This trend was reversed on an overcast day, when eightfold more Culicoides were captured inside; this indicates that when the light intensity outdoors is low Culicoides will attack (i) earlier in the day while cattle are still at pasture, and (ii) might follow cattle into the sheds in the late afternoon leading to elevated numbers of biting midges being trapped inside the shed during the subsequent hours of darkness. Culicoides were captured inside the shed on all 14 sampling nights. On occasion up to 33% were freshly blood fed indicating they had avidly attacked the cattle inside (endophagy); because half the cattle had seroconverted to BTV, and because no cattle were left outdoors at night, the data indicate that (i) the housing of animals in partially open buildings does not interrupt the transmission of BTV, and/or (ii) BTV is being transmitted while cattle are grazing outdoors during the day. The capture of partially engorged midges inside the shed shows they are being disturbed while feeding; this may lead to cattle being attacked repeatedly, and if these attacks include older parous BTV-infected Culicoides, may enhance virus dissemination (particularly in sheds where cattle stand close together). Endo- and exophagy by potential vector Culicoides--coupled to increased adult longevity and multiple feeding events in single (potentially) infected midges--would ensure an R0 of >1, resulting in the continued maintenance and spread of BTV within local vertebrate populations. Four light trap collections made additionally in a mature deciduous forest 70 m from the shed yielded a high proportion (48%) of gravid females amongst which 10% had incompletely digested blackened blood meals in their abdomens; the absence of this age category in Culicoides captured at the sheds indicates that all Culicoides, after engorgement, exit the buildings to undergo oogenesis elsewhere. In Europe, the blacklight trap is used widely for the nocturnal monitoring of Culicoides; a drawback to this approach is that this trap cannot be used to sample midges that are active during the day. Because diurnal biting in vector Culicoides may constitute a significant and underestimated component of BTV transmission a novel capture methodology will be required in future and is discussed briefly.

Indoor activity of Culicoides associated with livestock in the bluetongue virus (BTV) affected region of northern France during autumn 2006.

In August 2006, bluetongue virus (BTV) was detected in the Netherlands, Belgium, western Germany, Luxembourg and northern France for the first time. Consequently, a longitudinal entomological study was conducted in the affected region of northern France (Ardennes) throughout the autumn of 2006. Data on the spatio-temporal distribution of Culicoides (Diptera: Ceratopogonidae) associated with livestock were collected and an attempt was made to identify the vector(s) involved in BTV transmission by means of virus detection in wild-caught biting midges. Weekly sampling using standardized Onderstepoort-type blacklight traps were performed simultaneously both outdoors and indoors in one BTV-free and three BTV-affected farms between September and December 2006. Culicoides were sorted according to farm, location (outdoors vs. indoors), time point (in weeks), species and physiological stage. BTV detection was conducted by RT-PCR on monospecific pools of non-bloodfed parous female Culicoides. The principal results showed: (i) the absence of the Mediterranean vector, C. imicola, (ii) the relatively low abundance of C. dewulfi and C. pulicaris, (iii) the widespread occurrence and abundance of C. obsoletus/C. scoticus with longevity and behaviour compatible with BTV transmission, and (iv) all Culicoides pools tested for BTV were negative. In France, the very low levels of BTV-8 circulation were probably due to the limited introduction of the virus from affected neighbouring countries, and not due to the absence of local vector populations. A key finding has been the substantiation, for the first time, that Culicoides, and particularly the potential vectors C. obsoletus/C. scoticus and C. dewulfi, can be active at night inside livestock buildings and not only outside, as originally believed. The endophagic tendencies of members of the Obsoletus group are discussed in light of the prolonged period of BTV transmission during the autumn of 2006 and the risk of BTV overwintering and resurgence in the spring of 2007. Overall, there is an urgent need to improve our knowledge on the ecology of local Culicoides species before any clear, effective and reliable recommendations can be provided to the veterinary authorities in terms of prevention and control.

The Culicoides 'snapshot': a novel approach used to assess vector densities widely and rapidly during the 2006 outbreak of bluetongue (BT) in The Netherlands.

A novel method was developed and implemented during the recent outbreak of bluetongue (BT) in sheep and cattle in The Netherlands to obtain rapidly a 'snapshot' of Culicoides vector densities at the national level. The country was divided into 110 raster cells, each measuring 20 km x 20 km; within 106 of these cells, a farm was selected with a minimum of 10 cattle and sampled for Culicoides for one night only using the Onderstepoort-type blacklight trap. Prior to deployment of the light traps in the field, local veterinarians were trained in their use and in the preservation of captured Culicoides. The collections were dispatched daily by courier to a field laboratory where the Culicoides were counted and identified. The 'snapshot' commenced on 12 September 2006 and was completed on 28 September coinciding with the 5-7 weeks of BT virus (BTV) activity in The Netherlands and when the number of weekly cases of disease was on the rise. Analysis of the 106 collections was completed on 5 October. The number of grid cells in which a taxon occurred is represented by the index 20(2) gFR (=20 km x 20 km grid Frequency Rate); this index essentially reflects the percentage of examined raster cells found to contain the potential vector in question. The 'snapshot' results can be summarised as follows: A total of >35,000 Culicoides were captured (mu=333 midges/light trap) representing 16 species; Not a single specimen of C. imicola, the principal Afro-Asiatic vector of BTV in southern Europe, was captured; The three European species of Culicoides implicated previously as potential vectors in the Mediterranean region and the Balkans also occur in The Netherlands; The Obsoletus Complex (represented by both potential vectors Culicoides obsoletus and Culicoides scoticus) was the most prevalent taxon (20(2) gFR: 93.4%) followed by the Pulicaris Complex (76.4%); The Pulicaris Complex comprised at least six species. One of these, Culicoides pulicaris sensu stricto (ss), a potential vector in southern Europe, had a low 20(2) gFR of 17.9% indicating it played no role in the transmission of BTV; The next most prevalent taxa were Culicoides dewulfi (70.8%) and Culicoides chiopterus (67.0%) both of which breed in cattle dung. This close association with livestock raises their potential as vectors of BTV; Culicoides achrayi, Culicoides circumscriptus, C. dewulfi, Culicoides halophilus, Culicoides lupicaris and Culicoides stigma are new species records for The Netherlands and demonstrates that the extant knowledge on the Culicoides fauna of northern Europe is incomplete; Cooling trends in the weather induced a >50-fold decrease in overall Culicoides numbers but with subsequent warming these would rebound rapidly indicating that more intense cold is required before heightened mortalities are induced amongst adult biting midges. The northward advance of BT in Europe compels the competent authorities in affected and in neighbouring territories to acquire rapidly baseline information around which to plan sound vector surveillance and livestock movement strategies. The Culicoides 'snapshot' is a tool well suited to this purpose. It is stressed that a vector surveillance program must be built upon a firm taxonomic base because misidentifications will flaw the mapped seasonal and geographic distribution patterns upon which veterinary authorities depend.

The 2006 outbreak of bluetongue in northern Europe--the entomological perspective.

After bluetongue (BT) appeared in northern Europe in August 2006 entomological studies were implemented in all five affected Member States (MSs) to establish which species of Culicoides had acted as vectors. The findings can be summarised as follows: (i) C. imicola the principal southern European/African vector of BTV has not penetrated into northern Europe, (ii) three pools of C. obsoletus/C. scoticus and one of C. dewulfi assayed RT-PCR-positive to BTV-8, (iii) in support of these results it was found that both potential vectors had also high parity rates (approximately 40%) indicating increased longevity favouring BTV virogenesis and transmission, (iv) furthermore, C. obsoletus/C. scoticus and C. dewulfi occurred also widely and abundantly on sheep and cattle holdings across the entire affected region, (v) and during the latter part of the season showed strong endophily readily entering livestock buildings in significant numbers to bite the animals inside (endophagy), (vi) which demonstrates that housing at best offers only limited protection to livestock from Culicoides attacks, (vii) in contrast the potential vector C. pulicaris sensu stricto was restricted geographically, was captured rarely, had a low parity rate (10%) and was exophilic indicating it played no role in the outbreak of BT, (viii) the incrimination of C. dewulfi as a novel vector is significant because it breeds in cattle and horse dung this close association raising its vectorial potential, but (ix) problems with its taxonomy (and that of the Obsoletus and Pulicaris species complexes) illustrates the need for morphological and molecular techniques to become more fully integrated to ensure progress in the accurate identification of vector Culicoides, (x) midge densities (as adjudged by light traps) were generally low indicating northern European Culicoides to have a high vector potential and/or that significant numbers of midges are going undetected because they are biting (and transmitting BTV) during the day when light traps are not effective, and (xi) the sporadic capture of Culicoides in the winter of 2007 invites re-examination of the current definition of a vector-free period. The re-emergence of BT over a wide front in 2007 raises anew questions as to precisely how the virus overwinters and asks also that we scrutinise our monitoring systems in terms of their sensitivity and early warning capability.

Influence of biotic and abiotic factors on the distribution and abundance of Culicoides imicola and the Obsoletus Complex in Italy.

Culicoides imicola Kieffer (Culicoides, Diptera: Ceratopogonidae) is the principal vector of bluetongue virus (BTV) to ruminant livestock in southern Europe. The secondary potential vectors are Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle of the Obsoletus Complex, Culicoides pulicaris (Linnaeus) of the Pulicaris Complex and Culicoides dewulfi Goetghebuer of the subgenus Avaritia Fox. Between 2000 and 2004 >38,000 light-trap collections were made for Culicoides across Italy including the islands of Sardinia and Sicily. Mapping of the 100 largest collections of C. imicola and of the Obsoletus Complex showed them to be disjunct overlapping in only 2% of the 200 municipalities selected. For each municipality the average values were calculated for minimum temperature, aridity index, altitude, terrain slope, normalised difference vegetation index (NDVI) and percentage forest cover. A factor analysis identified two principal factors ('biotic' and 'abiotic') and explained 84% of the total variability; a discriminant analysis classified correctly 87.5% of the observations. The results indicate adult populations of C. imicola to occur in more sparsely vegetated habitats that are exposed to full sunlight, whereas species of the Obsoletus Complex favour a more shaded habitat, with increased green leaf density. Heliophily and umbrophily, by shortening or lengthening the respective adult life cycles of these two vectors, will likely impact on the ability of each to transmit BTV and is discussed in the light of the current outbreak of BTV across the Mediterranean Basin.

Comparative descriptions of the pupae of five species of the Culicoides imicola complex (Diptera, Ceratopogonidae) from South Africa.

The viruses causing the economically important livestock diseases of African horse sickness (AHS) and bluetongue (BT) are transmitted by biting midges of the genus Culicoides (Diptera, Ceratopogonidae). In the Old World the most important vectors of these diseases are Culicoides imicola Kieffer, 1913, Culicoides brevitarsis Kieffer, 1917 and Culicoides bolitinos Meiswinkel, 1989. All three of these vectors belong to the Imicola complex of the subgenus Avaritia Fox, 1955. This species complex now comprises 12 sibling species; ten occur in sub-Saharan Africa and are difficult to identify (based mostly on subtle variations in the wing latterns) and so additional methods of reliable identification are needed. The pupal exuviae of the five commonest sibling species (C. imicola, C. bolitinos, Culicoides loxodontis Meiswinkel, 1992, Culicoides tuttifrutti Meiswinkel, Cornet & Dyce, 2003 and Culicoides sp. # 107) harvested from a variety of large herbivore dung types and from decaying fruits, are described and illustrated in detail. It is shown that they can be differentiated clearly on a number of morphological characters and, furthermore, are separable into two distinct groups based (principally) on the shape of the respiratory organ. A key for identifying and differentiating these five pupae is provided. Also, the pupa of the Oriental-Australasian C. brevitarsis was compared with its allopatric sister taxon, C. bolitinos. Because they share a common larval habitat (cattle and buffalo dung) and are almost inseparable in the adult phenotype, the question of their possible synonymy is raised. However, their respective pupae could not be differentiated on gross morphology and so it is argued that this unresolved problem requires a molecular solution.

Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequences.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages - the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world.

Description and implementation of a surveillance network for bluetongue in the Balkans and in adjoining areas of south-eastern Europe.

During the recent severe outbreaks of bluetongue (BT) in the Mediterranean Basin, the BT virus (BTV) spread beyond its historical limits into the Balkan region. One of the primary impacts of BT is the cessation in livestock trade which can have severe economic and social consequences. The authors briefly describe the development of the collaborative East-BTnet programme which aims to assist all affected and at-risk Balkan states and adjoining countries in the management of BT, and in the development of individual national surveillance systems. The beneficiary countries involved, and led by the World organisation for animal health (Office International des Epizooties) Collaborating Centre for veterinary training, epidemiology, food safety and animal welfare of the Istituto Zooprofilattico dell'Abruzzo e del Molise 'G. Caporale' in collaboration with the Institute for the Protection and the Security of the Citizen, the European Commission Joint Research Centre (IPSC-JRC), were Albania, Bosnia-Herzegovina, Bulgaria, Croatia, Cyprus, the Former Yugoslavia Republic of Macedonia, Kosovo, Malta, Romania, Serbia and Montenegro, Slovenia and Turkey. A regional web-based surveillance network is a valuable tool for controlling and managing transboundary animal diseases such as BT. Its implementation in the Balkan region and in adjoining areas of south-eastern Europe is described and discussed.