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Culicoides biting midges (Diptera: Ceratopogonidae) are a highly successful group of small (1-3 mm) hematophagous flies, infamous for the role they play as biological vectors for numerous pathogens of veterinary significance. The principal aim of the national animal disease surveillance program of Israel is to be able to rapidly sort and identify live field-captured insects including Culicoides for arbovirus screening. In this exploratory study, three identification methods-classical morphology, DNA barcoding, and MALDI-TOF MS-were applied simultaneously to individuals of 10 Culicoides species that commonly attack livestock in Israel. The strengths and limitations of the three methods are compared and evaluated. In essence, the CO1 barcoding and MALDI-TOF MS results closely matched those of classical morphology. Furthermore, at a higher level and in strong accordance with recognized subgenera, the 10 species, in the reconstructed phylogenies, coalesced into multiple deeper-branched monophyletic clades. However, some discrepancies between the molecular and protein profiling results did occur and proved difficult to assess in terms of taxonomic significance. This difficulty underscores how tricky it is to establish clear species limits when methods involving borderline cutoff values and similarity indices are used as a taxonomic aid. An added shortcoming of the pluralistic triple-method approach is that a significant percentage of the species-level depositions in the GenBank and BOLD databases are misidentified, hindering structured comparison and interpretation of the morphological and molecular results obtained. Aspects of the unresolved taxonomy of various biting midge assemblages within the Mediterranean basin, including minor changes to the Israeli Culicoides checklist, are discussed in light of the methods applied. It is observed that the direct access that classical morphology provides to the external environment (or species niche) is indispensable to the full and correct interpretation (and application) of concomitant molecular and protein profiling results. The Culicoides taxonomy of the future ought to be fully integrative, during which the assimilation of modern methodological advances should strengthen-rather than undermine-the morphological foundations laid down during the 260-year Linnaean epoch., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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

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.

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.