Ecology of Megacopta cribraria (Hemiptera: Plataspidae) and Implications for Management on Soybean

Megacopta cribraria (Fabricius) (Hemiptera: Plataspidae) has spread through thirteen states and the District of Columbia since accidental introduction into the United States in 2009. The potential impacts to soybean production necessitated research into the ecology and management of the plataspid in its new invasive range. A two year study in South Carolina using sweep-net and beat-cloth sampling generated sampling plans for M. cribraria population estimates and sequential sampling plans for pest management decision making. 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 both sampling methods compared with a fixed sampling plan. The sweep net method was more cost reliable for population estimation at low densities of both life stages; the beat cloth became 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. White cross-vane traps as a sweep-net sampling alternative on soybean were evaluated. Adults were first collected in traps before the sweep net. Adults collected from trap and sweep-net sampling decreased from the field edge towards the field interior. There was a positive association between the two sampling methods, but only 36% of weekly, by location analyses were significantly correlated. Collected females were rated based on egg development. Female populations of all reproductive maturities dispersed into soybean but proportions varied with sampling method. Kudzu, Pueraria montana Loureiro (Merrill) variety lobata (Willdenow) plays an important role in the bivoltine life cycle of M. cribraria so sweep-net sampling was employed to monitor seasonal activity over three time periods, in two kudzu patches in South Carolina. Adults colonized kudzu in early April and were present until October. The majority of females were intermediate or fully reproductive in early spring, whereas non-reproductive females dominated as the season progressed. Soybean stem-feeding behavior was conducted on adult females using electropenetrography and was the first time a member of the Plataspidae has been recorded using this technique. Waveforms were described and correlated with stylet insertion by staining for salivary sheaths. Adult females performed a stereotypical set of feeding behaviors and stylet sheaths terminated in vascular tissue during ingestion. Scanning electron microscopy images of M. cribraria mouthpart structures were produced and described. In 2014, nematodes were reported infecting M. cribraria adults for the first time, and were subsequently found in nymphs of M. cribraria, Euschistus servus (Say), Chinavia hilaris (Say) and adults of Euschistus spp. in 2015. Infection in less mobile nymphs suggests the insects were parasitized directly in the soybean field from which they were collected. Morphological and molecular analyses confirmed the nematode belongs to the family Mermithidae, genus Agamermis. Overall, the research presented in this dissertation presents insights into M. cribraria biology and ecology and provides information to facilitate sampling practices for improved reliability of M. cribraria estimates for research and pest management purposes. Entomoparasitic nematode infection in hemipteran pests emphasizes the need to acknowledge covert, under-studied natural enemies which may contribute to population regulation

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-0846-x) contains supplementary material, which is available to authorized users

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

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

Abstract 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

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1756-3305-7-392) contains supplementary material, which is available to authorized users

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

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).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.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

Frequency histogram of responses of <i>Cx</i>. <i>pipiens</i> lines to attempted infection with Schmallenberg virus.

<p>Black = Intrathoracically inoculated Brookwood line; Cross-hatch = Orally fed Brookwood line; White = Orally fed Caldbeck line; Grey = Orally fed Netherlands line. Samples of >40 C_q are excluded.</p

Polymerase chain reaction (PCR)-based assays used for genetic characterization of Brookwood and Caldbeck <i>Culex pipiens</i> lines.

<p>Polymerase chain reaction (PCR)-based assays used for genetic characterization of Brookwood and Caldbeck <i>Culex pipiens</i> lines.</p

Parameter estimates and 95% credible intervals for the fixed effects within the best-fitting model identified for each seasonal metric for the subgenus <i>Avaritia</i> female <i>Culicoides</i>.

<p>Asterix denotes significance (95% credible interval does not bridge zero).</p><p>Parameter estimates and 95% credible intervals for the fixed effects within the best-fitting model identified for each seasonal metric for the subgenus <i>Avaritia</i> female <i>Culicoides</i>.</p