Over the past three decades, a range of mosquito-borne viruses that threaten public and veterinary health have emerged or re-emerged in Europe. Mosquito surveillance activities have highlighted the Culex pipiens species complex as being critical for the maintenance of a number of these viruses. This species complex contains morphologically similar forms that exhibit variation in phenotypes that can influence the probability of virus transmission. Critical amongst these is the choice of host on which to feed, with different forms showing different feeding preferences. This influences the ability of the mosquito to vector viruses and facilitate transmission of viruses to humans and domestic animals. Biases towards blood-feeding on avian or mammalian hosts have been demonstrated for different Cx. pipiens ecoforms and emerging evidence of hybrid populations across Europe adds another level of complexity to virus transmission. A range of molecular methods based on DNA have been developed to enable discrimination between morphologically indistinguishable forms, although this remains an active area of research. This review provides a comprehensive overview of developments in the understanding of the ecology, behaviour and genetics of Cx. pipiens in Europe, and how this influences arbovirus transmission.

Correct mosquito species identification is essential for mosquito and disease control programs. However, this is complicated by the difficulties in morphologically identifying some mosquito species. In this study, variation of a partial sequence of the cytochromecoxidase unit I (COI) gene was used for the molecular identification of British mosquito species and to facilitate the discovery of cryptic diversity, and monitoring invasive species. Three DNA extraction methods were compared to obtain DNA barcodes from adult specimens. In total, we analyzed 42 species belonging to the generaAedesMeigen, 1818 (21 species),AnophelesMeigen, 1818 (7 species),CoquillettidiaTheobald, 1904 (1 species),CulexLinnaeus, 1758 (6 species),CulisetaFelt, 1904 (7 species), andOrthopodomyiaTheobald, 1904 (1 species). Intraspecific genetic divergence ranged from 0% to 5.4%, while higher interspecific divergences were identified betweenAedesgeminusPeus, 1971/Culisetalitorea(Shute, 1928) (24.6%) andAe.geminus/An.plumbeusStephens, 1828 (22.5%). Taxonomic discrepancy was shown betweenAn.daciaeLinton, Nicolescu & Harbach, 2004 andAn.messeaeFalleroni, 1828 indicating the poor resolution of theCOIDNA barcoding region in separating these taxa. Other species such asAe.cantans(Meigen, 1818)/Ae.annulipes(Meigen, 1830) showed similar discrepancies indicating some limitation of this genetic marker to identify certain mosquito species. The combination of morphology and DNA barcoding is an effective approach for the identification of British mosquitoes, for invasive mosquitoes posing a threat to the UK, and for the detection of hidden diversity within species groups.

The range of vertebrate hosts on which species of mosquito blood-feed is an important parameter for identifying potential vectors and in assessing the risk of incursion and establishment of vector-borne pathogens. In the United Kingdom, studies of mosquito host range have collected relatively few specimens and used techniques that could only broadly identify host species. This study conducted intensive collection and analysis of mosquitoes from a grazing marsh environment in southeast England. This site provides extensive wetland habitat for resident and migratory birds and has abundant human nuisance biting mosquitoes. The aim was to identify the blood-feeding patterns of mosquito species present at the site which could contribute to the transmission of pathogens. Twice-weekly collections of mosquitoes were made from Elmley Nature Reserve, Kent, between June and October 2014. Mosquitoes were collected using resting boxes, by aspiration from man-made structures and using a Mosquito Magnet Pro baited with 1-octen-3-ol. Blood-fed specimens were classified according to the degree of blood meal digestion using the Sella scale and vertebrate origin determined using sequencing of a fragment of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes that were morphologically cryptic were identified to species level using multiplex PCR and sequencing methods. A total of 20,666 mosquitoes of 11 species were collected, and 2,159 (10.4%) were blood-fed (Sella scale II-VI); of these 1,341 blood-fed specimens were selected for blood meal analysis. Vertebrate origin was successfully identified in 964 specimens (72%). Collections of blood-fed individuals were dominated by Anopheles maculipennis complex (73.5%), Culiseta annulata (21.2%) and Culex pipiens form pipiens (10.4%). Nineteen vertebrate hosts comprising five mammals and 14 birds were identified as hosts for mosquitoes, including two migratory bird species. Feeding on birds by Culex modestus and Anopheles atroparvus populations in England was demonstrated. This study expands the vertebrate host range of mosquitoes in the Thames estuary region of the UK. Feeding on both resident and migratory bird species by potential arbovirus vectors including Cx. pipiens f. pipiens and Cx. modestus indicates the potential for enzootic transmission of an introduced arbovirus between migratory and local bird species by native mosquito species.

Determining the host feeding patterns of mosquitoes by identifying the origin of their blood-meals is an important part of understanding the role of vector species in current and future disease transmission cycles. Collecting large numbers of blood-fed mosquitoes from the field is difficult, therefore it is important to maximise the information obtained from each specimen. This study aimed to use mosquito genome sequence to identify the species within Anopheles maculipennis sensu lato (An. maculipennis s.l.), identify the vertebrate hosts of field-caught blood-fed An. maculipennis s.l. , and to test for the presence of myxoma virus (Poxviridae, genus Leporipoxvirus) in specimens found to have fed on the European rabbit (Oryctolagus cuniculus). Blood-fed An. maculipennis s.l. were collected from resting sites at Elmley Nature Reserve, Kent, between June and September 2013. Hosts that An. maculipennis s.l. had fed on were determined by a PCR-sequencing approach based on the partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes were then identified to species by sequencing a region of the internal transcribed spacer-2. DNA extracts from all mosquitoes identified as having fed on rabbits were subsequently screened using PCR for the presence of myxoma virus. A total of 94 blood-fed Anopheles maculipennis s.l. were collected, of which 43 (46 %) provided positive blood-meal identification results. Thirty-six of these specimens were identified as Anopheles atroparvus, which had fed on rabbit (n = 33, 92 %) and cattle (n = 3, 8 %). Seven mosquitoes were identified as Anopheles messeae, which had fed on cattle (n = 6, 86 %) and dog (n = 1, 14 %). Of the 33 An. atroparvus that contained rabbit blood, nine (27 %) were positive for myxoma virus. Results demonstrate that a single DNA extract from a blood-fed mosquito can be successfully used for molecular identification of members of the An. maculipennis complex, blood-meal identification, and for the targeted detection of a myxoma virus. This study shows that An. atroparvus has a strong feeding preference for both healthy and myxoma-infected rabbits, providing evidence that this species may play a significant role in the transmission of myxomatosis among wild rabbit populations in the United Kingdom (UK).

Thirty-four species of Culicidae are present in the UK, of which 15 have been implicated as potential vectors of arthropod-borne viruses such as West Nile virus. Identification of mosquito feeding preferences is paramount to the understanding of vector-host-pathogen interactions which, in turn, would assist in the control of disease outbreaks. Results are presented on the application of DNA barcoding for vertebrate species identification in blood-fed female mosquitoes in rural locations. Blood-fed females (n = 134) were collected in southern England from rural sites and identified based on morphological criteria. Blood meals from 59 specimens (44%) were identified as feeding on eight hosts: European rabbit, cow, human, barn swallow, dog, great tit, magpie and blackbird. Analysis of the cytochrome c oxidase subunit I mtDNA barcoding region and the internal transcribed spacer 2 rDNA region of the specimens morphologically identified as Anopheles maculipennis s.l. revealed the presence of An. atroparvus and An. messeae. A similar analysis of specimens morphologically identified as Culex pipiens/Cx. torrentium showed all specimens to be Cx. pipiens (typical form). This study demonstrates the importance of using molecular techniques to support species-level identification in blood-fed mosquitoes to maximize the information obtained in studies investigating host feeding patterns.

Summary: Culicoides biting midges (Diptera, Ceratopogonidae) are vectors of arboviruses that cause significant economic and welfare impact. Local‐scale spread of Culicoides‐borne arboviruses is largely determined by the between‐farm movement of infected Culicoides. Study of the dispersal behaviour of Culicoides by capture–mark–recapture (CMR) is problematic due to the likelihood of mortality and changes in behaviour upon capture caused by the small size and fragility of these insects, evidenced by low recapture rates. To counter the problem of using CMR with Culicoides, this study utilised an ovalbumin immunomarking technique to quantify the within‐ and between‐farm dispersal of Culicoides in southern England. Both within‐ and between‐farm dispersal of Culicoides was observed. Of the 9058 Culicoides collected over 22 nights of trapping, 600 ovalbumin‐positive Culicoides, of 12 species including those implicated as arbovirus vectors, were collected with a maximum dispersal distance of 3125 m. This study provides the first species‐level data on the between‐farm dispersal of potential bluetongue, Schmallenberg and African horse sickness virus vectors in northern Europe. High‐resolution meteorological data determined upwind and downwind flight by Culicoides had occurred. Cumulative collection and meteorological data suggest 15·6% of flights over 1 km were upwind of the treatment area and 84·4% downwind. Synthesis and applications. The use of immunomarking eliminates the potential adverse effects on survival and behaviour of insect collection prior to marking, substantially improving the resolution and accuracy of estimates of the dispersal potential of small and delicate vector species such as Culicoides. Using this technique, quantification of the range of Culicoides dispersal with regard to meteorological conditions including wind direction will enable improved, data‐driven modelling of the spread of Culicoides‐borne arboviruses and will inform policy response to incursions and outbreaks.