The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner.

Genome segmentation is mainly thought to facilitate reassortment. Here, we show that segmentation can also allow differences in segment abundance in populations of bluetongue virus (BTV). BTV has a genome consisting in 10 segments, and its cycle primarily involves periodic alternation between ruminants and Culicoides biting midges. We have developed a reverse transcription-quantitative PCR (RT-qPCR) approach to quantify each segment in wild BTV populations sampled in both ruminants and midges during an epizootic. Segment frequencies deviated from equimolarity in all hosts. Interestingly, segment frequencies were reproducible and distinct between ruminants and biting midges. Beyond a putative regulatory role in virus expression, this phenomenon could lead to different evolution rates between segments.IMPORTANCE The variation in viral gene frequencies remains a largely unexplored aspect of within-host genetics. This phenomenon is often considered to be specific to multipartite viruses. Multipartite viruses have segmented genomes, but in contrast to segmented viruses, their segments are each encapsidated alone in a virion. A main hypothesis explaining the evolution of multipartism is that, compared to segmented viruses, it facilitates the regulation of segment abundancy, and the genes the segments carry, within a host. These differences in gene frequencies could allow for expression regulation. Here, we show that wild populations of a segmented virus, bluetongue virus (BTV), also present unequal segment frequencies. BTV cycles between ruminants and Culicoides biting midges. As expected from a role in expression regulation, segment frequencies tended to show specific values that differed between ruminants and midges. Our results expand previous knowledge on gene frequency variation and call for studies on its role and conservation beyond multipartite viruses.

Crimean-Congo Hemorrhagic Fever Virus Genome in Tick from Migratory Bird, Italy.

We detected Crimean-Congo hemorrhagic fever virus in a Hyalomma rufipes nymph collected from a whinchat (Saxicola rubetra) on the island of Ventotene in April 2017. Partial genome sequences suggest the virus originated in Africa. Detection of the genome of this virus in Italy confirms its potential dispersion through migratory birds.

Climate change and the spread of vector-borne diseases: using approximate Bayesian computation to compare invasion scenarios for the bluetongue virus vector Culicoides imicola in Italy.

Bluetongue (BT) is a commonly cited example of a disease with a distribution believed to have recently expanded in response to global warming. The BT virus is transmitted to ruminants by biting midges of the genus Culicoides, and it has been hypothesized that the emergence of BT in Mediterranean Europe during the last two decades is a consequence of the recent colonization of the region by Culicoides imicola and linked to climate change. To better understand the mechanism responsible for the northward spread of BT, we tested the hypothesis of a recent colonization of Italy by C. imicola, by obtaining samples from more than 60 localities across Italy, Corsica, Southern France, and Northern Africa (the hypothesized source point for the recent invasion of C. imicola), and by genotyping them with 10 newly identified microsatellite loci. The patterns of genetic variation within and among the sampled populations were characterized and used in a rigorous approximate Bayesian computation framework to compare three competing historical hypotheses related to the arrival and establishment of C. imicola in Italy. The hypothesis of an ancient presence of the insect vector was strongly favoured by this analysis, with an associated P ≥ 99%, suggesting that causes other than the northward range expansion of C. imicola may have supported the emergence of BT in southern Europe. Overall, this study illustrates the potential of molecular genetic markers for exploring the assumed link between climate change and the spread of diseases.

Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV­8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV­8, sharing the same transmission patterns of BTV, can also spread to Europe.

Reoccurrence of West Nile virus lineage 1 after 2-year decline: first equine outbreak in Campania region.

West Nile virus (WNV) is the most widespread arbovirus worldwide, responsible for severe neurological symptoms in humans as well as in horses and birds. The main reservoir and amplifier of the virus are birds, and migratory birds seem to have a key role in the introduction and spread of WNV during their migratory routes. WNV lineage 1 (L1) has been missing in Italy for almost 10 years, only to reappear in 2020 in two dead raptor birds in southern Italy. The present study reports the first equine outbreak in the Campania region. A 7-year-old horse died because of worsening neurological signs and underwent necropsy and biomolecular analyses. WNV-L1 was detected by real-time RT-PCR in the heart, brain, gut, liver, and spleen. Next Generation Sequence and phylogenetic analysis revealed that the strain responsible for the outbreak showed a nucleotide identity of over 98% with the strain found in Accipiter gentilis 2 years earlier in the same area, belonging to the WNV-L1 Western-Mediterranean sub-cluster. These results underline that WNV-L1, after reintroduction in 2020, has probably silently circulated during a 2-year eclipse, with no positive sample revealed by both serological and biomolecular examinations in horses, birds, and mosquitoes. The climate changes that have occurred in the last decades are evolving the epidemiology of WNV, with introductions or re-introductions of the virus in areas that were previously considered low risk. Thereby, the virus may easily amplify and establish itself to reappear with sporadic evident cases in susceptible hosts after several months or even years.

Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

Direct and Indirect Role of Migratory Birds in Spreading CCHFV and WNV: A Multidisciplinary Study on Three Stop-Over Islands in Italy.

The annual movements of migratory birds can contribute to the spread of African ticks and tick-borne pathogens of potential public health concern across Europe. The aim of the study was to investigate their role in the possible introduction of African ticks and tick-borne pathogens into European countries during spring migration. A total of 2344 ticks were collected during three spring seasons from 1079 birds captured on three Italian stop-over islands during their northbound migration. Once identified, each tick was tested by RT-PCR for the presence of Crimean-Congo hemorrhagic fever (CCHFV), West Nile (WNV), and Usutu (USUV) viruses. Moreover, carcasses of birds found dead were collected and tested for the possible presence of WNV and USUV. Results confirmed a higher contribution of trans-Saharan migrants compared to intra-Palearctic ones and the prevalence of African tick species in the sample. CCHFV was detected for the second time in Italy in a Hyalomma rufipes, and WNV was found in two ticks of the same genus, all carried by trans-Saharan birds. WNV lineage 1 was also found in the organs of a Garden warbler. These results confirm the role of migratory birds in carrying African ticks, as well as viruses of zoonotic importance, from Africa into Europe.

Species of mosquitoes present in Abruzzo and Molise and their possible role as vector of Usutu and West Nile viruses.

In 2019, entomological survey on mosquitoes was carried out in Abruzzo and Molise regions in central Italy to obtain data on local mosquito fauna. Collection sites were selected based on a previous ecoregion classification of the territory.  From 2019 to 2021 virological surveillance for West Nile virus (WNV) and Usutu virus (USUV) on mosquitoes was carried out in the same regions, selecting ecoregions where virus circulation and vector presence were more likely,  all mosquitoes were collected and identified, and the female mosquitoes were sorted in 3046 pools and tested for the presence of WNV and USUV by Real-time PCR. All pools tested negative for WND, while USUV was detected in 7 pools of Aedes caspius collected in Molise region, 17 pools of Culex pipiens s.l. (2 collected in Molise, 15 in Abruzzo), and 1 pool of Culiseta longiareolata collected in Molise. These results suggests the presence of an USUV enzootic cycle, maintained by Culex pipiens s.l. and Aedes caspius in both Italian regions, as well as providing a useful picture in terms of species presence and abundance for both regions. Ecoregions proved to be a very valuable tool in determining high risk areas for vector borne diseases.

The Absence of Abdominal Pigmentation in Livestock Associated Culicoides following Artificial Blood Feeding and the Epidemiological Implication for Arbovirus Surveillance.

Culicoides midges (Diptera: Ceratopogonidae), the vectors of economically important arboviruses such as bluetongue virus and African horse sickness virus, are of global importance. In the absence of transovarial transmission, the parity rate of a Culicoides population provides imperative information regarding the risk of virus dispersal. Abdominal pigmentation, which develops after blood feeding and ovipositioning, is used as an indicator of parity in Culicoides. During oral susceptibility trials over the last three decades, a persistent proportion of blood engorged females did not develop pigment after incubation. The present study, combining a number of feeding trials and different artificial feeding methods, reports on this phenomenon, as observed in various South African and Italian Culicoides species and populations. The absence of pigmentation in artificial blood-fed females was found in at least 23 Culicoides species, including important vectors such as C. imicola, C. bolitinos, C. obsoletus, and C. scoticus. Viruses were repeatedly detected in these unpigmented females after incubation. Blood meal size seems to play a role and this phenomenon could be present in the field and requires consideration, especially regarding the detection of virus in apparent "nulliparous" females and the identification of overwintering mechanisms and seasonally free vector zones.

Species composition and relative abundance of the genus Culicoides (Diptera: Ceratopogonidae) in Romania.

BACKGROUND: Culicoides biting midges are vectors involved in the biological transmission cycle of important animal diseases such as bluetongue and African horse sickness. In Romania, the first outbreaks of bluetongue were reported in 2014, leading to increased activities within the existing entomological surveillance network. The main goals of the surveillance activities were the establishment of the vector free period in relation to animal trade and the identification of Culicoides species involved in the transmission of the pathogen. This study was conducted on the composition and relative abundance of the species belonging to the genus Culicoides (Diptera: Ceratopogonidae) in certain regions of Romania and provided the opportunity to update the existing checklist of Culicoides species of this country. METHODS: The study was conducted in 33 of the 42 administrative units (counties), including a total of 659 catches, in 102 locations. The collections were carried out with UV blacklight suction traps (OVI type). The collected insects were preserved in 70% ethanol. Morphological insect identification was carried out using a stereomicroscope, according to established identification keys. In ten localities the relative abundance of the cryptic species of the Obsoletus complex was determined by multiplex PCR assay based on the ITS2 segment. The identification of the Culicoides chiopterus (Meigen) species by morphological examination was confirmed by PCR assay based on the ITS1 segment. RESULTS: Eleven species were identified using morphological and PCR tools. The rest of the individuals were separated into five taxa. The species of the Obsoletus complex (grouping Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle) were the most abundant, accounting for 59% of the total number of captured Culicoides spp. Three of the identified species are mentioned, according to our knowledge, for the first time in Romania: Culicoides newsteadi Austen, Culicoides flavipulicaris Dzhafarov and Culicoides bysta Sarvasová, Kocisová, Candolfi & Mathieu. CONCLUSIONS: Our study demonstrates that the Culicoides species most commonly cited as being involved in the transmission of arboviruses in Europe (i.e. bluetongue and Schmallenberg viruses) make up a high proportion of adult Culicoides trapped in Romania.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Defining ecological regions in Italy based on a multivariate clustering approach: A first step towards a targeted vector borne disease surveillance.

Ecoregionalization is the process by which a territory is classified in similar areas according to specific environmental and climatic factors. The climate and the environment strongly influence the presence and distribution of vectors responsible for significant human and animal diseases worldwide. In this paper, we developed a map of the eco-climatic regions of Italy adopting a data-driven spatial clustering approach using recent and detailed spatial data on climatic and environmental factors. We selected seven variables, relevant for a broad set of human and animal vector-borne diseases (VBDs): standard deviation of altitude, mean daytime land surface temperature, mean amplitude and peak timing of the annual cycle of land surface temperature, mean and amplitude of the annual cycle of greenness value, and daily mean amount of rainfall. Principal Component Analysis followed by multivariate geographic clustering using the k-medoids technique were used to group the pixels with similar characteristics into different ecoregions, and at different spatial resolutions (250 m, 1 km and 2 km). We showed that the spatial structure of ecoregions is generally maintained at different spatial resolutions and we compared the resulting ecoregion maps with two datasets related to Bluetongue vectors and West Nile Disease (WND) outbreaks in Italy. The known characteristics of Culicoides imicola habitat were well captured by 2/22 specific ecoregions (at 250 m resolution). Culicoides obsoletus/scoticus occupy all sampled ecoregions, according to its known widespread distribution across the peninsula. WND outbreak locations strongly cluster in 4/22 ecoregions, dominated by human influenced landscape, with intense cultivations and complex irrigation network. This approach could be a supportive tool in case of VBDs, defining pixel-based areas that are conducive environment for VBD spread, indicating where surveillance and prevention measures could be prioritized in Italy. Also, ecoregions suitable to specific VBDs vectors could inform entomological surveillance strategies.

Vector Competence of Italian Populations of Culicoides for Some Bluetongue Virus Strains Responsible for Recent Northern African and European Outbreaks.

The distribution of Bluetongue virus (BTV) in Europe can be represented by two distinct and interconnected epidemiological systems (episystems), each characterized by different ecological characteristics and vector species. This study investigated the vector competence of Italian populations of Culicoides imicola and Culicoides obsoletus/scoticus to some representative BTV strains after artificial oral infection. The BTV strains were selected according to their ability to spread to one or both episystems and included BTV-4 ITA, responsible of the recent Italian and French BTV-4 outbreaks; the BTV-2 strain which caused the first BTV incursion in Italy, Corsica, and Balearic Islands; BTV-4 MOR, responsible for the epidemic in Morocco; and BTV-8, the strain which spread through Europe between 2006 and 2008. Blood-soaked cotton pledgets and Hemotek membrane feeder using Parafilm® membrane were used to artificially feed midges. For each population/strain, recovery rates (positive/tested heads) were evaluated using serogroup- and serotype-specific RT-PCR. The trial demonstrated that, except for the Abruzzo population of C. obsoletus/C. scoticus, which was refractory to BTV-4 MOR infection, all the investigated Culicoides populations are susceptible to the selected BTV strains and that, if prompt vaccination programs and restriction measures had not been implemented, BTV-2 and BTV-4 MOR could have spread all over Europe.

The Culicoides 'snapshot': a novel approach used to assess vector densities widely and rapidly during the 2006 outbreak of bluetongue (BT) in The Netherlands.

A novel method was developed and implemented during the recent outbreak of bluetongue (BT) in sheep and cattle in The Netherlands to obtain rapidly a 'snapshot' of Culicoides vector densities at the national level. The country was divided into 110 raster cells, each measuring 20 km x 20 km; within 106 of these cells, a farm was selected with a minimum of 10 cattle and sampled for Culicoides for one night only using the Onderstepoort-type blacklight trap. Prior to deployment of the light traps in the field, local veterinarians were trained in their use and in the preservation of captured Culicoides. The collections were dispatched daily by courier to a field laboratory where the Culicoides were counted and identified. The 'snapshot' commenced on 12 September 2006 and was completed on 28 September coinciding with the 5-7 weeks of BT virus (BTV) activity in The Netherlands and when the number of weekly cases of disease was on the rise. Analysis of the 106 collections was completed on 5 October. The number of grid cells in which a taxon occurred is represented by the index 20(2) gFR (=20 km x 20 km grid Frequency Rate); this index essentially reflects the percentage of examined raster cells found to contain the potential vector in question. The 'snapshot' results can be summarised as follows: A total of >35,000 Culicoides were captured (mu=333 midges/light trap) representing 16 species; Not a single specimen of C. imicola, the principal Afro-Asiatic vector of BTV in southern Europe, was captured; The three European species of Culicoides implicated previously as potential vectors in the Mediterranean region and the Balkans also occur in The Netherlands; The Obsoletus Complex (represented by both potential vectors Culicoides obsoletus and Culicoides scoticus) was the most prevalent taxon (20(2) gFR: 93.4%) followed by the Pulicaris Complex (76.4%); The Pulicaris Complex comprised at least six species. One of these, Culicoides pulicaris sensu stricto (ss), a potential vector in southern Europe, had a low 20(2) gFR of 17.9% indicating it played no role in the transmission of BTV; The next most prevalent taxa were Culicoides dewulfi (70.8%) and Culicoides chiopterus (67.0%) both of which breed in cattle dung. This close association with livestock raises their potential as vectors of BTV; Culicoides achrayi, Culicoides circumscriptus, C. dewulfi, Culicoides halophilus, Culicoides lupicaris and Culicoides stigma are new species records for The Netherlands and demonstrates that the extant knowledge on the Culicoides fauna of northern Europe is incomplete; Cooling trends in the weather induced a >50-fold decrease in overall Culicoides numbers but with subsequent warming these would rebound rapidly indicating that more intense cold is required before heightened mortalities are induced amongst adult biting midges. The northward advance of BT in Europe compels the competent authorities in affected and in neighbouring territories to acquire rapidly baseline information around which to plan sound vector surveillance and livestock movement strategies. The Culicoides 'snapshot' is a tool well suited to this purpose. It is stressed that a vector surveillance program must be built upon a firm taxonomic base because misidentifications will flaw the mapped seasonal and geographic distribution patterns upon which veterinary authorities depend.

Combined larvicidal and adulticidal treatments to control Culicoides biting midges (Diptera: Ceratopogonidae): Results of a pilot study.

Culicoides biting midges can transmit viruses such as bluetongue virus and Schmallenberg virus to ruminants and African horse sickness virus to equines. The control of Culicoides population can be based on physical and/or chemical methods. This study aims to evaluate the efficacy of larvicides and adulticides combined treatments to control the vector Culicoides populations. Field trials have been conducted during 2014 on seven farms in northern Sardinia (Italy). Three insecticide treatments (larvicides and adulticides) have been carried out on three farms, whereas four farms remained unsprayed as untreated controls. For the larval control two commercial formulations, based on diflubenzuron and Bacillus thuringiensis var. israelensis (H-14), have been used. Against adults a deltamethrin based product has been sprayed on the walls and roofs of animal shelters. Adult Culicoides have been collected on each farm twice a week with an Onderstepoort blacklight suction trap. After each treatment a significant reduction in the abundance of total Culicoides, C. imicola and C. newsteadi s.l., has been observed. The Obsoletus complex density has been affected by the first two treatments. Our results suggest the possibility to control the Culicoides populations with a combination of adulticide and larvicide treatments.

New species of the genus Culicoides (Diptera Ceratopogonidae) for Tunisia, with detection of Bluetongue viruses in vectors.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are double-stranded RNA orbiviruses of the Reoviridae family. Bluetongue virus and EHDV infect domestic and wild ruminants and they are transmitted by biting midges of the genus Culicoides (Diptera: Ceratopogonidae). Since 1999, BTV outbreaks have occurred in Tunisia and 4 serotypes, BTV2, BTV1, BTV4 and BTV3, were involved in 2000, 2006, 2009, and 2016, respectively. Epizootic haemorrhagic disease was detected for the rst time in Tunisia and in other Northern African countries in 2006. These incursions have caused considerable economic losses. Our study had the goal to describe diversity, distribution, and seasonal dynamics of Culicoides. Fourteen sampling sites were chosen throughout the country and 2-night trapping of midges was performed monthly from June 2006 to July 2008. A total of 11,582 Culicoides specimens were collected from 336 light traps, comprising 25 species, of which 7 were identi ed for the rst time in Tunisia, increasing to 35 the total number of Culicoides species now reported in this country. Twenty-three pools of parous females belonging to the Culicoides imicola and Culicoides kingi were tested for detection of BTV and EHDV by molecular assays. Both BTV1 and BTV4 were detected in C. imicola.

Finding of Anopheles (Anopheles) hyrcanus (Pallas, 1771) (Diptera, Culicidae) during the entomological surveillance for West Nile virus in Umbria, Italy.

This study reports the mosquito collections conducted in July-August 2013 in Castiglione del Lago (Umbria Region, Italy) during the entomological surveillance within the West Nile national control program. The mosquito collections showed the noteworthy occurrence of Anopheles hyrcanus s.l. (n = 156; 35.8% of the whole sample), a relatively rare mosquito species in Italy, with molecular analyses con rming that these mosquitoes belong to An. hyrcanus s.s. The same catching site was characterized by a relevant richness in mosquito species, in particular the following were found: Anopheles maculipennis s.l. (n = 146; 33.5% composed by 80% Anopheles melanoon, 13.3% Anopheles maculipennis s.s. and 6.7% Anopheles atroparvus), Culex pipiens s.l. (n = 116; 26.6% composed by 90% Cx. p. pipiens, 5% Cx. p. molestus and 5% Cx. p. pipiens/molestus), Culiseta annulata (n = 10; 2.3%), Aedes albopictus (n = 3; 0.7%), and Anopheles claviger (n = 5; 100%).

Mosquito species involved in the circulation of West Nile and Usutu viruses in Italy.

Usutu (USUV) and West Nile (WNV) are mosquito-borne Flavivirus emerged in Italy in 1996 and 1998, respectively, and reappeared 10 years later. The aim of this work is to review the Italian mosquito species found positive for WNV and USUV between 2008 and 2014. Moreover, the role of mosquitoes in promoting the overwintering of these viruses is discussed, as a result of the mosquito collections performed in Molise region between September 2010 and April 2011. Overall 99,000 mosquitoes were collected: 337 and 457 mosquito pools tested positive by real time reverse transcriptase polymerase chain reaction (real time RT-PCR) for WNV and USUV, respectively. West Nile virus was detected in pools of Culex pipiens s.l. (329), Ochlerotatus caspius (4), Culex modestus (2), and Culex spp. (2). Positive USUV pools were from Cx. pipiens s.l. (435), Aedes albopictus (12), Oc. caspius (5), Culex spp. (2), Anopheles maculipennis s.l. (1), Culiseta annulata (1), and Ochlerotatus detritus (1). In Molise region, 1,694 mosquitoes were collected, and USUV was identi ed in Cx. pipiens s.l., Cs. annulata, and Oc. detritus pools. This paper shows that Cx. pipiens s.l. is the mosquito species most involved in the WNV and USUV circulation in Italy, although other species would also support the spread of both the viruses during Winter.

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.