Alison Blackwell

Electrophysiological investigation of larval water and potential oviposition chemo-attractants for Anopheles gambiae s.s.

Potential Anopheles gambiae s.s. breeding sites were sampled in rural sites in the Tanga region of Tanzania. Sites containing the largest numbers of An. gambiae larvae were small, shaded pools and rice fields. Water samples were collected from the larval sites and these, together with ether extracts of the water samples, were active in electro-antennogram (EAG) studies with female mosquitoes from a laboratory colony of Anopheles (Cellia) gambiae KWA. EAG response thresholds varied for 10 randomly selected water samples, from 0.01%–100% dilution for the whole water samples and from 0.001%–1.0% dilution for the ether extracts. Potential chemical components of the water samples were also electrophysiologically active with female An. gambiae antennae; thresholds varied from 1 ng (3-methylindole and indole) to 10 µg (m-cresol and 4-methylcyclohexanoI).

The role of 1‐octen‐3‐ol as a host‐odour attractant for the biting midge, Culicoides impunctatus Goetghebuer, and interactions of 1‐octen‐3‐ol with a volatile pheromone produced by parous female midges

Abstract. 1‐octen‐3‐ol, a component of the body odour of ruminants, is demonstrated to be an attractant for host‐seeking, parous female Culicoides impunctatus Goetghebuer (Diptera, Ceratopogonidae).This compound was electrophysiologically and behaviourally active and, in the field, released at 0.11 ± 0.01 mg/day, increased female C.impunctatus catches over a seven‐day period compared with control catches.When 1‐octen‐3‐ol was combined either with live female midges or an extract of female midges in an electroantennogram assay, additive effects were recorded.Behaviourally, the same combination resulted in both enhanced attraction and repellency, depending on concentration.Attraction was recorded mainly when sub‐threshold concentrations of the separate components were combined.The potential of such a combination as an effective field bait is discussed.

Diel flight periodicity of the biting midge Culicoides impunctatus and the effects of meteorological conditions.

Abstract The hourly flight periodicity of adults of the biting midge Culicoides impunctatus was sampled at a site in Western Scotland, using suction traps over 18 days in July/August 1994. In addition, meteorological conditions were logged continuously. Female but not male C. impunctatus had a bimodal pattern of activity, with peaks at dawn and dusk. The dawn peak (05.00–07.00 hours) was most distinct. Correlation analysis revealed significantly positive relationships between catches of female midges and both relative humidity and rainfall, and negative relationships with wind velocity. The calculation of partial correlation coefficients reinforced the influence of relative humidity on female activity, and highlighted a further positive relationship with air temperature. Male C.impunctatus activity was negatively correlated with air temperature, although the total male catch was relatively small (15% of total trap catches) and further data would be required to confirm this result. Overall, the results help to clarify previous confusion as to whether C.impunctatus has a circadian rhythm of activity, with the data matching closely predictions of a bimodal pattern. Clearly, this pattern will be damped by meteorological conditions, which may vary greatly on a local scale.

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture. Methodology/Principal Findings A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (Cq values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination. Conclusions/Significance Cq values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.

The spatial distribution of larvae of Culicoides impunctatus biting midges.

The spatial distribution of Culicoides impunctatus Goetghebuer (Diptera: Ceratopogonidae) larvae was sampled at a site in western Scotland, and geostatistical analyses were used to quantify spatial dependencies. Nested sampling and analysis indicated that most of the spatial variance occurred within distances of 160–640 cm, levelling off at distances > 640 cm. Semivariograms for transformed data from three 100 m × 100 m grids showed similar, isotropic patterns for larval counts, soil organic content, soil water content and the distribution of Juncus spp. rushes, with the variance increasing with separation distance. High levels of significance were associated with power models used to describe the semivariograms, which was indicative of the absence of a plateau (or ‘sill’) in the respective data. Correlation analysis of transformed data revealed significantly positive relationships between larval counts and soil pH, soil percentage organic content, soil percentage water content and also the distribution of Sphagnum spp., Juncus spp. and Myrica gale. There were also significantly negative relationships between larval counts and the distribution of Pteridium aquilinum and all mosses other than Sphagnum spp. The results suggest a far more structured and predictable pattern of C. impunctatus larval sites than previous studies and are discussed in relation to their application in localized Culicoides control and to studies of the mechanisms determining the spatial distribution of C. impunctatus larvae.

The vertical distribution of Culicoides impunctatus larvae.

Abstract. Soil cores were taken from three transects, representing the vegetation typical of much of the west coast and Highlands of Scotland, in an attempt to investigate the distribution of Culicoides impunctatus larvae. Sampling of soil cores was carried out at four periods throughout the day (00.00,05.00,12.00 and 17.00 hours). Each core sample was sliced into sections, 1 cm deep and Culicoides impunctatus larval numbers in the first 8 cm of the soil core were investigated, using a sugar‐flotation technique. At all sampling times, approximately 50% of the total numbers of larvae recovered were from the first 2cm of the soil. During the daylight sampling periods (05.00, 12.00 and 17.00 hours), there was a small but significant migration of larvae into the deeper layers (2–5 cm). The results are discussed in relation to those for other species from previous studies, and also to their application in larval collection for colonization techniques and in localized Culicoides control.

Identifying environmental drivers of insect phenology across space and time: Culicoides in Scotland as a case study

Interpreting spatial patterns in the abundance of species over time is a fundamental cornerstone of ecological research. For many species, this type of analysis is hampered by datasets that contain a large proportion of zeros, and data that are overdispersed and spatially autocorrelated. This is particularly true for insects, for which abundance data can fluctuate from zero to many thousands in the space of weeks. Increasingly, an understanding of the ways in which environmental variation drives spatial and temporal patterns in the distribution, abundance and phenology of insects is required for management of pests and vector-borne diseases. In this study, we combine the use of smoothing techniques and generalised linear mixed models to relate environmental drivers to key phenological patterns of two species of biting midges, Culicoides pulicaris and C. impunctatus, of which C. pulicaris has been implicated in transmission of bluetongue in Europe. In so doing, we demonstrate analytical tools for linking the phenology of species with key environmental drivers, despite using a relatively small dataset containing overdispersed and zero-inflated data. We demonstrate the importance of landcover and climatic variables in determining the seasonal abundance of these two vector species, and highlight the need for more empirical data on the effects of temperature and precipitation on the life history traits of palearctic Culicoides spp. in Europe.

Electrophysiological and behavioural studies of the biting midge, Culicoides impunctatus Goetghebuer (Diptera, Ceratopogonidae): interactions between some plant-derived repellent compounds and a host-odour attractant, 1-octen-3-ol

Abstract. Electroantennogram (EAG) and y‐tube bioassays have been used to demonstrate the repellent properties of five plant compounds with host‐seeking parous female Culicoides impunctatus Goetghebuer. The compounds were methyl salicylate and allyl‐, butyl‐, phenyl‐ and 2‐phenylethyl isothiocyanate. EAG thresholds were 1 times 10‐3 to 1 μg. In the bioassays, maximal repellencies occurred with 1 times 10˜2 to lOug. When each compound was combined with 1‐octen‐3‐ol, a confirmed host‐odour attractant for C.impunctatus females, additive effects were recorded in EAG assays and in bioassays, all of the compounds either reduced or reversed the attractancy of l‐octen‐3‐ol. Of the isothiocyanates, allyl isothiocyanate was the most potent and when combined with 1‐octen‐3‐ol in field trials, the attractant effect of l‐octen‐3‐ol was reduced.

Olfactory Cues for Oviposition Behavior in Toxorhynchites moctezuma and Toxorhynchites amboinensis (Diptera: Culicidae)

Abstract In laboratory tests gravid female Toxorhynchites moctezuma (Dyar & Knab) and Toxorhynchites amboinensis (Doleschall) were offered the choice of black oviposition jars containing a diethyl ether extract of water collected from a natural oviposition site for these species (i.e., used tires), a dilution series of 4-methylcyclohexanol, 3-methylindole, 2-methylphenol, 3-methylphenol and 4-methylphenol, or solvent. Tire water extract and all test compounds acted as oviposition attractants and stimulants for both species, with the threshold amounts required to elicit these behaviors varying between the species and among the compounds tested.

Electroantennogram studies of potential oviposition attractants for Toxorhynchites moctezuma and T. amboinensis mosquitoes

Abstract.Ether extracts of water samples taken from tyre sections in the field were shown to be active in electroantennogram studies with female Toxorhynchites moctezuma (Dyar & Knab) and Toxorhynchites amboinensis (Doleschall). In addition, electroantennograms were recorded from both Toxorhynchites species for seven compounds found commonly in water containing decaying leaves and known to be oviposition attractants for other mosquito species. The compounds were 4‐methylcyclohexanol, phenol, indole, 3‐methylindole, m‐cresol, o‐cresol and p‐cresol. The response thresholds for these compounds ranged between 1 × 10–4μg and 1 μg for female antennae (100 μg for male antennae). These results provide a basis for behavioural studies of oviposition attractants for the Toxorhynchites species.