Comparison of surveillance trapping methods to monitor Culicoides biting midge activity in Trinidad, West Indies.

Culicoides biting midges (Diptera: Ceratopogonidae) are biting nuisances and arbovirus vectors of both public health and veterinary significance in Trinidad. We compared sampling methods to define the behaviour and bionomics of adult Culicoides populations at a commercial dairy goat farm. Three static trap designs were compared: (a) Centre for Disease Control (CDC) downdraft UV trap; (b) CDC trap with an incandescent bulb and (c) CDC trap with semiochemical lure consisting of R-(-)-1-octen-3-ol and CO2 (no bulb). Sweep netting was used to define diel periodicity. A total of 30,701 biting midges were collected using static traps, dominated by female Culicoides furens (>70% of trap collections across all three designs). There was no significant difference in the Margalef's index between the three traps; however, trap designs A and C collected a significantly greater number of individuals than trap B, and trap C gained highest species richness. The greatest species richness and abundance of Culicoides collected by sweep net was observed between 6:00 and 6:15 pm and notable differences in the crepuscular activity pattern of several species were identified. Comparative data on Culicoides species richness, abundance, sex and reproductive status is discussed and can be used to improve surveillance strategies, research designs and risk management.

The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Investigation of diel activity of Culicoides biting 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.

Identification and characterization of epizootic hemorrhagic disease virus serotype 6 in cattle co-infected with bluetongue virus in Trinidad, West Indies.

Epizootic hemorrhagic disease virus (EHDV) is an economically important virus that can cause severe clinical disease in deer and to a lesser extent cattle. This study set out to determine and characterize which EHDV serotypes were circulating in Trinidad. Serum and whole blood samples were collected monthly for six months from a cohort of cattle imported to Trinidad from the USA. Results revealed that all the cattle seroconverted to EHDV within six months of their arrival, with EHDV RNA being detected in the samples just prior to antibodies, as expected. Serotyping assays revealed that a single serotype (EHDV-6) was circulating in the cattle. Sequencing of the surface viral protein (VP2) of EHDV-6, followed by phylogenetic analysis, revealed that the Trinidad EHDV-6 strain was closely related to EHDV-6 viruses found in Guadeloupe (2010), Martinique (2010) and USA (2006), with 96-97.2% nucleotide identity. The Trinidad EHDV-6 VP-2 shared 97.2% identity with the Australian EHDV-6 prototype strain, classifying it within the eastern topotype clade. Bayesian coalescent analysis support Australia as the most probable source for the EHDV-6 VP2 sequences in the Americas and Caribbean region and suggests that the they diverged from the Australian prototype strain around 1966 (95% HPD 1941-1979).

Blood-feeding, susceptibility to infection with Schmallenberg virus and phylogenetics of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of internationally important arboviruses of livestock. In 2011, a novel Orthobunyavirus was discovered in northern Europe causing congenital malformations and abortions in ruminants. From field studies, Culicoides were implicated in the transmission of this virus which was subsequently named Schmallenberg virus (SBV), but to date no assessment of susceptibility to infection of field populations under standardised laboratory conditions has been carried out. We assessed the influence of membrane type (chick skin, collagen, Parafilm M®) when offered in conjunction with an artificial blood-feeding system (Hemotek, UK) on field-collected Culicoides blood-feeding rates. Susceptibility to infection with SBV following blood-feeding on an SBV-blood suspension provided via either (i) the Hemotek system or via (ii) a saturated cotton wool pledglet was then compared. Schmallenberg virus susceptibility was defined by RT-qPCR of RNA extractions of head homogenates and related to Culicoides species and haplotype identifications based on the DNA barcode region of the mitochondrial cytochrome c oxidase 1 (cox1) gene. RESULTS: Culicoides blood-feeding rates were low across all membrane types tested (7.5% chick skin, 0.0% for collagen, 4.4% Parafilm M®, with 6029 female Culicoides being offered a blood meal in total). Susceptibility to infection with SBV through membrane blood-feeding (8 of 109 individuals tested) and pledglet blood-feeding (1 of 94 individuals tested) was demonstrated for the Obsoletus complex, with both C. obsoletus (Meigen) and C. scoticus Downes & Kettle susceptible to infection with SBV through oral feeding. Potential evidence of cryptic species within UK populations was found for the Obsoletus complex in phylogenetic analyses of cox1 DNA barcodes of 74 individuals assessed from a single field-site. CONCLUSIONS: Methods described in this study provide the means to blood-feed Palaearctic Culicoides for vector competence studies and colonisation attempts. Susceptibility to SBV infection was 7.3% for membrane-fed members of the subgenus Avaritia and 1.1% for pledglet-fed. Both C. obsoletus and C. scoticus were confirmed as being susceptible to infection with SBV, with potential evidence of cryptic species within UK Obsoletus complex specimens, however the implications of cryptic diversity in the Obsoletus complex on arbovirus transmission remains unknown.

Quantification of within- and between-farm dispersal of Culicoides biting midges using an immunomarking technique.

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.

How often do mosquitoes bite humans in southern England? A standardised summer trial at four sites reveals spatial, temporal and site-related variation in biting rates.

BACKGROUND: This field-based study examined the abundance and species complement of mosquitoes (Diptera: Culicidae) attracted to humans at four sites in the United Kingdom (UK). The study used a systematic approach to directly measure feeding by mosquitoes on humans at multiple sites and using multiple volunteers. Quantifying how frequently humans are bitten in the field by mosquitoes is a fundamental parameter in assessing arthropod-borne virus transmission. METHODS: Human landing catches were conducted using a standardised protocol by multiple volunteers at four rural sites between July and August 2013. Collections commenced two hours prior to sunset and lasted for a total of four hours. To reduce bias occurring due to collection point or to the individual attractiveness of the volunteer to mosquitoes, each collection was divided into eight collection periods, with volunteers rotated by randomised Latin square design between four sampling points per site. While the aim was to collect mosquitoes prior to feeding, the source of blood meals from any engorged specimens was also identified by DNA barcoding. RESULTS: Three of the four sites yielded human-biting mosquito populations for a total of 915 mosquitoes of fifteen species/species groups. Mosquito species composition and biting rates differed significantly between sites, with individual volunteers collecting between 0 and 89 mosquitoes (over 200 per hour) of up to six species per collection period. Coquillettidia richiardii (Ficalbi, 1889) was responsible for the highest recorded biting rates at any one site, reaching 161 bites per hour, whilst maximum biting rates of 55 bites per hour were recorded for Culex modestus (Ficalbi, 1889). Human-biting by Culex pipiens (L., 1758) form pipiens was also observed at two sites, but at much lower rates when compared to other species. CONCLUSIONS: Several mosquito species are responsible for human nuisance biting pressure in southern England, although human exposure to biting may be largely limited to evening outdoor activities. This study indicates Cx. modestus can be a major human-biting species in the UK whilst Cx. pipiens f. pipiens may show greater opportunistic human-biting than indicated by earlier studies.

DNA barcoding and surveillance sampling strategies for Culicoides biting midges (Diptera: Ceratopogonidae) in southern India.

BACKGROUND: Culicoides spp. biting midges transmit bluetongue virus (BTV), the aetiological agent of bluetongue (BT), an economically important disease of ruminants. In southern India, hyperendemic outbreaks of BT exert high cost to subsistence farmers in the region, impacting on sheep production. Effective Culicoides spp. monitoring methods coupled with accurate species identification can accelerate responses for minimising BT outbreaks. Here, we assessed the utility of sampling methods and DNA barcoding for detection and identification of Culicoides spp. in southern India, in order to provide an informed basis for future monitoring of their populations in the region. METHODS: Culicoides spp. collected from Tamil Nadu and Karnataka were used to construct a framework for future morphological identification in surveillance, based on sequence comparison of the DNA barcode region of the mitochondrial cytochrome c oxidase I (COI) gene and achieving quality standards defined by the Barcode of Life initiative. Pairwise catches of Culicoides spp. were compared in diversity and abundance between green (570 nm) and ultraviolet (UV) (390 nm) light emitting diode (LED) suction traps at a single site in Chennai, Tamil Nadu over 20 nights of sampling in November 2013. RESULTS: DNA barcode sequences of Culicoides spp. were mostly congruent both with existing DNA barcode data from other countries and with morphological identification of major vector species. However, sequence differences symptomatic of cryptic species diversity were present in some groups which require further investigation. While the diversity of species collected by the UV LED Center for Disease Control (CDC) trap did not significantly vary from that collected by the green LED CDC trap, the UV CDC significantly outperformed the green LED CDC trap with regard to the number of Culicoides individuals collected. CONCLUSIONS: Morphological identification of the majority of potential vector species of Culicoides spp. samples within southern India appears relatively robust; however, potential cryptic species diversity was present in some groups requiring further investigation. The UV LED CDC trap is recommended for surveillance of Culicoides in southern India.

Spatial and temporal distribution of Culicoides species in mainland Portugal (2005-2010). Results of the Portuguese Entomological Surveillance Programme.

Bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes an infectious, non-contagious disease of ruminants. It has been rapidly emerging in southern Europe since 1998. In mainland Portugal, strains of BTV belonging to three serotypes have been detected: BTV-10 (1956-1960), BTV-4 (2004-2006 and 2013) and BTV-1 (2007-2012). This paper describes the design, implementation and results of the Entomological Surveillance Programme covering mainland Portugal, between 2005 and 2010, including 5,650 caches. Culicoides imicola Kieffer was mostly found in central and southern regions of Portugal, although it was sporadically detected in northern latitudes. Its peak activity occurred in the autumn and it was active during the winter months in limited areas of the country. Obsoletus group was present at the highest densities in the north although they were found throughout the country in substantial numbers. Culicoides activity occurred all year round but peaked in the spring. A generalized linear mixed model was developed for the analysis of the environmental factors associated with activity of the species of Culicoides suspected vectors of BTV in the country. For C. imicola Kieffer, the most important variables were month, diurnal temperature range (DTR), the number of frost days (FRS) and median monthly temperature (TMP). For the Obsoletus group, the most important factors were month, diurnal temperature range (DTR), and linear and quadratic terms for median monthly temperature (TMP). The results reported can improve our understanding of climatic factors in Culicoides activity influencing their distribution and seasonal pattern.

Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization.

BACKGROUND: Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR). FINDINGS: Two colony lines of Cx. pipiens were created in the UK ('Brookwood' and 'Caldbeck') from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line. CONCLUSIONS/SIGNIFICANCE: This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.