Sheep breed and shearing influences attraction and blood-feeding behaviour of Culicoides (Diptera: Ceratopogonidae) on a UK farm.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of arboviruses of international importance between ruminant livestock. These arboviruses include bluetongue virus (BTV) and Schmallenberg virus (SBV), which have emerged in unprecedented outbreaks in northern Europe. The impact of breed and shearing of sheep on Culicoides: host contact rates has not been investigated in detail and has the potential to influence arbovirus transmission and control measures employed to limit spread. METHODS: Attraction of Culicoides to Hartline and Hartline/Suffolk cross-breed sheep was compared using 224 drop trap collections over 22 nights and 181 catches from sheared or unsheared Hartline/Suffolk ewes were made over 17 nights to compare Culicoides activity and rates of blood engorgement. RESULTS: A total of 31,314 Culicoides was collected in the two trials and females of the subgenus Avaritia represented over 96.9% of individuals collected. Attraction to breed was dependent upon species of Culicoides and physiological status, with a significantly greater number of individuals collected on the cross-breed sheep. Shearing of sheep did not significantly increase or decrease the number of Culicoides attracted but increased the rate of successful engorgement. CONCLUSIONS: Both breed and shearing were shown to influence Culicoides biting rate on sheep. These data are useful in a direct context in understanding the likely impact of control measures against arboviruses including BTV and SBV and additionally in providing data from field-based studies to enable modelling exercises of arbovirus transmission and spread.

Assessment of a Cross-Vane Trap as a Tool for Sampling the Invasive Megacopta cribraria (Hemiptera: Plataspidae) in Soybean with Associated Evaluations of Female Reproductive Status.

Megacopta cribraria (F.) (Hemiptera: Plataspidae) is an established pest of soybean, Glycine max (L.) Merrill, in the southeastern United States. Populations of this pest in soybean are typically estimated using sweep nets, a time-consuming procedure. Cross-vane traps may provide an alternative to using sweep nets. The relationship between trap and sweep-net sampling, and the effect of distance from field edges on M. cribraria densities were studied in 2013 and 2014. Adults were detected in the traps up to 3 wk before they were present in the sweep-net samples. Adults and nymphs collected from trap and sweep-net sampling decreased from the field edge toward the field interior. Across all dates and distances, there was a strong positive association between the two sampling methods (r = 0.60), but only 36% of location analyses by week showed significant association. Females collected using both sampling methods were dissected and rated based on egg development. Female populations of all reproductive maturities dispersed into soybean, but proportions varied with sampling method. Results provide a foundation for work into understanding dispersal cues, with implications for management in soybean. Cross-vane traps may serve as an early-season tool to monitor populations, but more studies are needed before robust sampling recommendations can be defined.

Agamermis (Nematoda: Mermithidae) Infection in South Carolina Agricultural Pests.

Native and invasive stink bugs (Hemiptera: Pentatomidae) and the closely related invasive Megacopta cribraria (Hemiptera: Plataspidae) are agricultural pests in the southeastern United States. Natural enemies, from various phyla, parasitize these pests and contribute to population regulation. We specifically investigated Nematoda infections in pentatomid and plataspid pests in one soybean field in South Carolina in 2015. Nematodes were identified through molecular and morphological methods and assigned to family Mermithidae, genus Agamermis. This study reports mermithid nematode infection in immature M. cribraria for the first time and provides the first mermithid host record for the stink bugs Chinavia hilaris, Euschistus servus, and another Euschistus species, and a grasshopper (Orthoptera: Acrididae) in South Carolina. The same Agamermis species infected all hosts. The broad host range and prevalence suggests that Agamermis may be an important contributor to natural mortality of pentatomid and plataspid pests. Previous mermithid host records for the Pentatomidae and Plataspidae worldwide are summarized. Further work is needed to assess the impact of infection on populations over a broader range of agricultural fields and geographic localities.

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.

A comparison of commercial light-emitting diode baited suction traps for surveillance of Culicoides in northern Europe.

BACKGROUND: The response of Culicoides biting midges (Diptera: Ceratopogonidae) to artificial light sources has led to the use of light-suction traps in surveillance programmes. Recent integration of light emitting diodes (LED) in traps improves flexibility in trapping through reduced power requirements and also allows the wavelength of light used for trapping to be customized. This study investigates the responses of Culicoides to LED light-suction traps emitting different wavelengths of light to make recommendations for use in surveillance. METHODS: The abundance and diversity of Culicoides collected using commercially available traps fitted with Light Emitting Diode (LED) platforms emitting ultraviolet (UV) (390 nm wavelength), blue (430 nm), green (570 nm), yellow (590 nm), red (660 nm) or white light (425 nm - 750 nm with peaks at 450 nm and 580 nm) were compared. A Centre for Disease Control (CDC) UV light-suction trap was also included within the experimental design which was fitted with a 4 watt UV tube (320-420 nm). Generalised linear models with negative binomial error structure and log-link function were used to compare trap abundance according to LED colour, meteorological conditions and seasonality. RESULTS: The experiment was conducted over 49 nights with 42,766 Culicoides caught in 329 collections. Culicoides obsoletus Meigen and Culicoides scoticus Downes and Kettle responded indiscriminately to all wavelengths of LED used with the exception of red which was significantly less attractive. In contrast, Culicoides dewulfi Goetghebuer and Culicoides pulicaris Linnaeus were found in significantly greater numbers in the green LED trap than in the UV LED trap. The LED traps collected significantly fewer Culicoides than the standard CDC UV light-suction trap. CONCLUSIONS: Catches of Culicoides were reduced in LED traps when compared to the standard CDC UV trap, however, their reduced power requirement and small size fulfils a requirement for trapping in logistically challenging areas or where many traps are deployed at a single site. Future work should combine light wavelengths to improve trapping sensitivity and potentially enable direct comparisons with collections from hosts, although this may ultimately require different forms of baits to be developed.

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.

Species determination of Culicoides biting midges via peptide profiling using matrix-assisted laser desorption ionization mass spectrometry.

BACKGROUND: Culicoides biting midges are vectors of bluetongue and Schmallenberg viruses that inflict large-scale disease epidemics in ruminant livestock in Europe. Methods based on morphological characteristics and sequencing of genetic markers are most commonly employed to differentiate Culicoides to species level. Proteomic methods, however, are also increasingly being used as an alternative method of identification. These techniques have the potential to be rapid and may also offer advantages over DNA-based techniques. The aim of this proof-of-principle study was to develop a simple MALDI-MS based method to differentiate Culicoides from different species by peptide patterns with the additional option of identifying discriminating peptides. METHODS: Proteins extracted from 7 Culicoides species were digested and resulting peptides purified. Peptide mass fingerprint (PMF) spectra were recorded using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peak patterns analysed in R using the MALDIquant R package. Additionally, offline liquid chromatography (LC) MALDI-TOF tandem mass spectrometry (MS/MS) was applied to determine the identity of peptide peaks in one exemplary MALDI spectrum obtained using an unfractionated extract. RESULTS: We showed that the majority of Culicoides species yielded reproducible mass spectra with peak patterns that were suitable for classification. The dendrogram obtained by MS showed tentative similarities to a dendrogram generated from cytochrome oxidase I (COX1) sequences. Using offline LC-MALDI-TOF-MS/MS we determined the identity of 28 peptide peaks observed in one MALDI spectrum in a mass range from 1.1 to 3.1 kDa. All identified peptides were identical to other dipteran species and derived from one of five highly abundant proteins due to an absence of available Culicoides data. CONCLUSION: Shotgun mass mapping by MALDI-TOF-MS has been shown to be compatible with morphological and genetic identification of specimens. Furthermore, the method performs at least as well as an alternative approach based on MS spectra of intact proteins, thus establishing the procedure as a method in its own right, with the additional option of concurrently using the same samples in other MS-based applications for protein identifications. The future availability of genomic information for different Culicoides species may enable a more stringent peptide detection based on Culicoides-specific sequence information.

Developing Sampling Plans for the Invasive Megacopta cribraria (Hemiptera: Plataspidae) in Soybean.

Since its discovery in the southeastern United States, the invasive plataspid Megacopta cribraria (F.) (Hemiptera: Plataspidae) has infested soybean (Glycine max (L.) Merrill) fields in often very high numbers. To optimize sampling plans, sweep-net and beat-cloth sampling was conducted in soybean fields in South Carolina during 2012 and 2013. Across all fields, densities averaged 7.2 ± 0.5 (SEM) adults and 4.5 ± 0.4 nymphs per 20 sweeps and 5.5 ± 0.3 adults and 4.5 ± 0.3 nymphs per 1.83 m of row. Coefficients of Taylor's power law were used to generate sampling plans for population estimates and sequential sampling plans for pest management decision making. At an economic threshold of one nymph per sweep, optimum sample sizes were 184, 48, and 22 within 10, 20, and 30% of the mean with the sweep-net method. At the corresponding threshold for the beat cloth (24.7 nymphs per 1.83 m of row), optimum sample sizes were 239, 62, and 29 within 10, 20 and 30% of the mean, respectively. At all adult and nymph densities, fewer sweep-net samples were required for population estimations compared with the number of beat-cloth samples. Sequential sampling reduced the sample size required to reach a management decision for the sweep net and beat cloth compared with a fixed sampling plan. The sweep-net method was more cost reliable for population estimation at low densities of both life stages, with the beat cloth becoming more cost reliable as populations increased. The beat-cloth method was more cost reliable than sweep-net sampling across all densities and life stages for pest management practices. These results may be used by researchers, county Extension agents, consultants, and farm managers to both facilitate sampling and improve reliability of M. cribraria estimates for research purposes and for pest management.