Nonlinear SAR Modelling of Mosquito Repellents for Skin Application.

Finding new marketable mosquito repellents is a complex and time-consuming process that can be optimized via modelling. In this context, a SAR (Structure-Activity Relationship) model was designed from a set of 2171 molecules whose actual repellent activity against Aedes aegypti was available. Information-rich descriptors were used as input neurons of a three-layer perceptron (TLP) to compute the models. The most interesting classification model was a 20/6/2 TLP showing 94% and 89% accuracy on the training set and test set, respectively. A total of 57 other artificial neural network models based on the same architecture were also computed. This allowed us to consider all chemicals both as training and test set members in order to better interpret the results obtained with the selected model. Most of the wrong predictions were explainable. The 20/6/2 TLP model was then used for predicting the potential repellent activity of new molecules. Among them, two were successfully evaluated in vivo.

Modelling the monthly abundance of Culicoides biting midges in nine European countries using Random Forests machine learning.

BACKGROUND: Culicoides biting midges transmit viruses resulting in disease in ruminants and equids such as bluetongue, Schmallenberg disease and African horse sickness. In the past decades, these diseases have led to important economic losses for farmers in Europe. Vector abundance is a key factor in determining the risk of vector-borne disease spread and it is, therefore, important to predict the abundance of Culicoides species involved in the transmission of these pathogens. The objectives of this study were to model and map the monthly abundances of Culicoides in Europe. METHODS: We obtained entomological data from 904 farms in nine European countries (Spain, France, Germany, Switzerland, Austria, Poland, Denmark, Sweden and Norway) from 2007 to 2013. Using environmental and climatic predictors from satellite imagery and the machine learning technique Random Forests, we predicted the monthly average abundance at a 1 km2 resolution. We used independent test sets for validation and to assess model performance. RESULTS: The predictive power of the resulting models varied according to month and the Culicoides species/ensembles predicted. Model performance was lower for winter months. Performance was higher for the Obsoletus ensemble, followed by the Pulicaris ensemble, while the model for Culicoides imicola showed a poor performance. Distribution and abundance patterns corresponded well with the known distributions in Europe. The Random Forests model approach was able to distinguish differences in abundance between countries but was not able to predict vector abundance at individual farm level. CONCLUSIONS: The models and maps presented here represent an initial attempt to capture large scale geographical and temporal variations in Culicoides abundance. The models are a first step towards producing abundance inputs for R0 modelling of Culicoides-borne infections at a continental scale.

Development and evaluation of a real-time quantitative PCR assay for Culicoides imicola, one of the main vectors of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe.

The current microscopy method for identifying the Culicoides imicola Kieffer, 1913 species can be time and labour intensive. There is a need for the development of a rapid and quantitative tool to quantify the biting midges C. imicola ss in light trap catches. A reproducible and sensitive real-time polymerase chain reaction method that targets the internal transcribed spacer (ITS-1) of ribosomal DNA of C. imicola ss species was developed. This real-time PCR assay was first performed on 10-fold serial dilutions of purified plasmid DNA containing specific C. imicola ss ITS-1. It was then possible to construct standard curves with a high correlation coefficient (r2=0.99) in the range of 10(-2)-10(-8) ng of purified DNA. The performances of this PCR were evaluated in comparison with morphological determination on Culicoides trapped along the Mediterranean coastal mainland France. ROC statistical analysis was carried out using morphology as gold standard and the area under the ROC curve had a satisfactory value of 0.9752. The results indicated that this real-time PCR assay holds promise for monitoring C. imicola ss population in both surveillance and research programmes because of its good specificity (92%) and sensitivity (95%).

Spatial and temporal variation in the abundance of Culicoides biting midges (Diptera: Ceratopogonidae) in nine European countries.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are vectors of bluetongue virus (BTV), African horse sickness virus and Schmallenberg virus (SBV). Outbreaks of both BTV and SBV have affected large parts of Europe. The spread of these diseases depends largely on vector distribution and abundance. The aim of this analysis was to identify and quantify major spatial patterns and temporal trends in the distribution and seasonal variation of observed Culicoides abundance in nine countries in Europe. METHODS: We gathered existing Culicoides data from Spain, France, Germany, Switzerland, Austria, Denmark, Sweden, Norway and Poland. In total, 31,429 Culicoides trap collections were available from 904 ruminant farms across these countries between 2007 and 2013. RESULTS: The Obsoletus ensemble was distributed widely in Europe and accounted for 83% of all 8,842,998 Culicoides specimens in the dataset, with the highest mean monthly abundance recorded in France, Germany and southern Norway. The Pulicaris ensemble accounted for only 12% of the specimens and had a relatively southerly and easterly spatial distribution compared to the Obsoletus ensemble. Culicoides imicola Kieffer was only found in Spain and the southernmost part of France. There was a clear spatial trend in the accumulated annual abundance from southern to northern Europe, with the Obsoletus ensemble steadily increasing from 4000 per year in southern Europe to 500,000 in Scandinavia. The Pulicaris ensemble showed a very different pattern, with an increase in the accumulated annual abundance from 1600 in Spain, peaking at 41,000 in northern Germany and then decreasing again toward northern latitudes. For the two species ensembles and C. imicola, the season began between January and April, with later start dates and increasingly shorter vector seasons at more northerly latitudes. CONCLUSION: We present the first maps of seasonal Culicoides abundance in large parts of Europe covering a gradient from southern Spain to northern Scandinavia. The identified temporal trends and spatial patterns are useful for planning the allocation of resources for international prevention and surveillance programmes in the European Union.

Monthly variation in the probability of presence of adult Culicoides populations in nine European countries and the implications for targeted surveillance.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are small hematophagous insects responsible for the transmission of bluetongue virus, Schmallenberg virus and African horse sickness virus to wild and domestic ruminants and equids. Outbreaks of these viruses have caused economic damage within the European Union. The spatio-temporal distribution of biting midges is a key factor in identifying areas with the potential for disease spread. The aim of this study was to identify and map areas of neglectable adult activity for each month in an average year. Average monthly risk maps can be used as a tool when allocating resources for surveillance and control programs within Europe. METHODS: We modelled the occurrence of C. imicola and the Obsoletus and Pulicaris ensembles using existing entomological surveillance data from Spain, France, Germany, Switzerland, Austria, Denmark, Sweden, Norway and Poland. The monthly probability of each vector species and ensembles being present in Europe based on climatic and environmental input variables was estimated with the machine learning technique Random Forest. Subsequently, the monthly probability was classified into three classes: Absence, Presence and Uncertain status. These three classes are useful for mapping areas of no risk, areas of high-risk targeted for animal movement restrictions, and areas with an uncertain status that need active entomological surveillance to determine whether or not vectors are present. RESULTS: The distribution of Culicoides species ensembles were in agreement with their previously reported distribution in Europe. The Random Forest models were very accurate in predicting the probability of presence for C. imicola (mean AUC = 0.95), less accurate for the Obsoletus ensemble (mean AUC = 0.84), while the lowest accuracy was found for the Pulicaris ensemble (mean AUC = 0.71). The most important environmental variables in the models were related to temperature and precipitation for all three groups. CONCLUSIONS: The duration periods with low or null adult activity can be derived from the associated monthly distribution maps, and it was also possible to identify and map areas with uncertain predictions. In the absence of ongoing vector surveillance, these maps can be used by veterinary authorities to classify areas as likely vector-free or as likely risk areas from southern Spain to northern Sweden with acceptable precision. The maps can also focus costly entomological surveillance to seasons and areas where the predictions and vector-free status remain uncertain.

Spatio-temporal genetic variation of the biting midge vector species Culicoides imicola (Ceratopogonidae) Kieffer in France.

BACKGROUND: Introduction of vector species into new areas represents a main driver for the emergence and worldwide spread of vector-borne diseases. This poses a substantial threat to livestock economies and public health. Culicoides imicola Kieffer, a major vector species of economically important animal viruses, is described with an apparent range expansion in Europe where it has been recorded in south-eastern continental France, its known northern distribution edge. This questioned on further C. imicola population extension and establishment into new territories. Studying the spatio-temporal genetic variation of expanding populations can provide valuable information for the design of reliable models of future spread. METHODS: Entomological surveys and population genetic approaches were used to assess the spatio-temporal population dynamics of C. imicola in France. Entomological surveys (2-3 consecutive years) were used to evaluate population abundances and local spread in continental France (28 sites in the Var department) and in Corsica (4 sites). We also genotyped at nine microsatellite loci insects from 3 locations in the Var department over 3 years (2008, 2010 and 2012) and from 6 locations in Corsica over 4 years (2002, 2008, 2010 and 2012). RESULTS: Entomological surveys confirmed the establishment of C. imicola populations in Var department, but indicated low abundances and no apparent expansion there within the studied period. Higher population abundances were recorded in Corsica. Our genetic data suggested the absence of spatio-temporal genetic changes within each region but a significant increase of the genetic differentiation between Corsican and Var populations through time. The lack of intra-region population structure may result from strong gene flow among populations. We discussed the observed temporal variation between Corsica and Var as being the result of genetic drift following introduction, and/or the genetic characteristics of populations at their range edge. CONCLUSIONS: Our results suggest that local range expansion of C. imicola in continental France may be slowed by the low population abundances and unsuitable climatic and environmental conditions.

Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events.

The role of the northward expansion of Culicoides imicola Kieffer in recent and unprecedented outbreaks of Culicoides-borne arboviruses in southern Europe has been a significant point of contention. We combined entomological surveys, movement simulations of air-borne particles, and population genetics to reconstruct the chain of events that led to a newly colonized French area nestled at the northern foot of the Pyrenees. Simulating the movement of air-borne particles evidenced frequent wind-transport events allowing, within at most 36 hours, the immigration of midges from north-eastern Spain and Balearic Islands, and, as rare events, their immigration from Corsica. Completing the puzzle, population genetic analyses discriminated Corsica as the origin of the new population and identified two successive colonization events within west-Mediterranean basin. Our findings are of considerable importance when trying to understand the invasion of new territories by expanding species.

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe.

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus.

Development and validation of IIKC: an interactive identification key for Culicoides (Diptera: Ceratopogonidae) females from the Western Palaearctic region.

BACKGROUND AND METHODS: The appearance of bluetongue virus (BTV) in 2006 within northern Europe exposed a lack of expertise and resources available across this region to enable the accurate morphological identification of species of Culicoides Latreille biting midges, some of which are the major vectors of this pathogen. This work aims to organise extant Culicoides taxonomic knowledge into a database and to produce an interactive identification key for females of Culicoides in the Western Palaearctic (IIKC: Interactive identification key for Culicoides). We then validated IIKC using a trial carried out by six entomologists based in this region with variable degrees of experience in identifying Culicoides. RESULTS: The current version of the key includes 98 Culicoides species with 10 morphological variants, 61 descriptors and 837 pictures and schemes. Validation was carried out by six entomologists as a blind trial with two users allocated to three classes of expertise (beginner, intermediate and advanced). Slides were identified using a median of seven steps and seven minutes and user confidence in the identification varied from 60% for failed identifications to a maximum of 80% for successful ones. By user class, the beginner group successfully identified 44.6% of slides, the intermediate 56.8% and the advanced 74.3%. CONCLUSIONS: Structured as a multi-entry key, IIKC is a powerful database for the morphological identification of female Culicoides from the Western Palaearctic region. First developed for use as an interactive identification key, it was revealed to be a powerful back-up tool for training new taxonomists and to maintain expertise level. The development of tools for arthropod involvement in pathogen transmission will allow clearer insights into the ecology and dynamics of Culicoides and in turn assist in understanding arbovirus epidemiology.

Simultaneous quantification of the relative abundance of species complex members: application to Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae), potential vectors of bluetongue virus.

The two sympatric sibling species Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle (Diptera: Ceratopogonidae), are known to be competent vectors for bluetongue virus in the Palaearctic region. However, morphological identification of constituent species is only readily applicable to adult males and these two species distinguishing traits have overlapping character states. As their vector competence may differ in space and time, the correct identification and quantification of specimens of both species are essential for understanding bluetongue epidemiology. However, no molecular tools are available for high-throughput identification of the two species. We therefore developed a quantitative duplex real-time PCR assay to determine the relative abundance of each sibling species in a sample using TaqMan probes. For each species, standard curves were constructed from serial dilutions of purified plasmid DNA containing ITS1-5.8S-ITS2 (rDNA) in the range of 10(-1) to 10(-5)ng/µL. Standard curves were used to quantify samples of mixed C. obsoletus/C. scoticus specimens. Specificity was evaluated with 5156 specimens representing 62 species. Based on the DNA quantities detected according to the standard curves, a quadratic model developed on 1100 males and validated on 555 females was able to predict the relative abundance of each species simultaneously in a one-shot reaction (Pearson coefficient of 0.999). Our assay showed a requirement of two specimens or less for 95% of the predictions, making it highly applicable to field collections. Extensive use of this real-time PCR assay will provide a better understanding of geographical distribution, dynamics, and bionomics on a species level, which is essential for risk assessment. This approach is an important contribution to medical entomology for investigating the vector role of arthropod sibling species.

Low persistence of Bacillus thuringiensis serovar israelensis spores in four mosquito biotopes of a salt marsh in southern France.

We studied the persistence of Bacillus thuringiensis serovar israelensis (Bti) in a typical breeding site of the mosquito Ochlerotatus caspius in a particularly sensitive salt marsh ecosystem following two Bti-based larvicidal applications (Vectobac 12AS, 1.95 L/ha). The treated area was composed of four larval biotopes that differed in terms of the most representative plant species (Sarcocornia fruticosa, Bolboschoenus maritimus, Phragmites australis, and Juncus maritimus) and the physical and chemical characteristics of the soil. We sampled water, soil, and plants at various times before and after the applications (from spring to autumn, 2001) and quantified the spores of B. thuringiensis (Bt) and Bacillus species. The B. cereus group accounted for between 0% and 20% of all Bacillus spp. before application depending on the larval biotope. No Bti were found before application. The variation in the quantity of bacilli during the mosquito breeding season depended more on the larval biotope than on the season or the larvicidal application. More bacilli were found in soil (10(4)-10(6) spores/g) than on plant samples (10(2)-10(4) spores/g). The abundance in water (10(5) to 10(7) spores/L) appeared to be correlated to the water level of the breeding site. The number of Bti spores increased just after application, after declining; no spores were detected in soil or water 3 months after application. However, low numbers of Bti spores were present on foliage from three of the four studied plant strata. In conclusion, the larvicidal application has very little impact on Bacillus spp. flora after one breeding season (two applications).