Histopathological effects of Aspergillus clavatus (Ascomycota: Trichocomaceae) on larvae of the southern house mosquito, Culex quinquefasciatus (Diptera: Culicidae).

Aspergillus clavatus (Ascomycota: Trichocomaceae) was previously found to be an opportunistic pathogen of mosquitoes (Diptera: Culicidae). In the present study, the mechanism leading to its insecticidal activity was investigated regarding histological damages on Culex quinquefasciatus larvae exposed to A. clavatus spores. Multiple concentration assays using spore suspensions (0.5-2.5 × 10(8) spores ml(-1)) revealed 17.0-74.3 % corrected mortalities after 48 h exposure. Heat-deactivated spores induced a lower mortality compared to nonheated spores suggesting that insecticidal effects are actively exerted. Spore-treated and untreated larvae were prepared for light microscopy as well as for scanning and transmission electron microscopy. Spores failed to adhere to the external body surface (except the mouth parts) of these aquatic immature stages but progressively filled the digestive tract where their metabolism seemed to activate. In parallel, the internal tissues of the larvae, i.e. the midgut wall, the skeletal muscles, and the cuticle-secreting epidermis, were progressively destroyed between 8 and 24 h of exposure. These observations suggest that toxins secreted by active germinating spores of A. clavatus in the digestive tract altered the larval tissues, leading to their necrosis and causing larval death. Fungal proliferation and sporulation then occurred during a saprophytic phase. A. clavatus enzymes or toxins responsible for these pathogenic effects need to be identified in further studies before any use of this fungus in mosquito control.

Diversity and ecology survey of mosquitoes potential vectors in Belgian equestrian farms: A threat prevention of mosquito-borne equine arboviruses.

Emergence of West Nile Virus was recently recorded in several European countries, which can lead to severe health problems in horse populations. Europe is also at risk of introduction of mosquito-borne equine alphavirus from Americas. Prevention of these arboviruses requires a clear understanding of transmission cycles, especially their vectors. To characterize mosquito fauna, their ecology and identify potential vectors of equine arboviruses in Belgium, entomological surveys of six equestrian farms located in the Wolloon Region were conducted during 2011-2012. The harvest of mosquitoes was based on larval sampling (272 samples from 111 breeding sites) and monthly adults trapping (CO2-baited traps, Mosquito Magnet Liberty Plus). Among 51,493 larvae and 319 adult mosquitoes collected, morphological identification showed the presence of 11 species: Anopheles claviger (Meigen), An. maculipennis s.l. (Meigen), An. plumbeus (Stephens), Culex hortensis (Ficalbi), Cx. territans (Walker), Cx. pipiens s.l. L., Cx. torrentium (Martini), Coquillettidia richiardii (Ficalbi), Culiseta annulata (Schrank), Aedes cantans (Meigen), Ae. geniculatus (Olivier). Molecular identification of Cx. pipiens species complex allowed the detection of three molecular forms, Pipiens (92.3%), Molestus (4.6%) and Hybrid (3.1%). Larvae of Cx. pipiens sl and Cx. torrentium were omnipresent and the most abundant species. Water troughs, ponds and slurry (liquid manure) were the most favorable breeding sites of mosquito larvae. Based upon behavior and ecology of the identified mosquito species, Studied Belgian equestrian farms seem to provide a suitable environment and breeding sites for the proliferation of potential vectors of arboviruses and those being a real nuisance problem for horses and neighboring inhabitants.

Orientation behaviour of Culicoides obsoletus (Diptera: Ceratopogonidae), a relevant virus vector in northern Europe, toward host-associated odorant cues.

Some Culicoides biting midge species (Diptera: Ceratopogonidae) are biological virus vectors worldwide and have recently been associated with outbreaks of important epizootic diseases such as bluetongue and Schmallenberg in northern Europe. These diseases, which affect domestic and wild ruminants, have caused considerable economic losses. Knowledge of host preferences of these biting midges - especially of the relevant vectors of arboviruses near farms, such as Culicoides obsoletus in northern Europe - is essential to understand pathogen transmission cycles and the epidemiology of associated diseases. This study aimed to determine host preferences of C. obsoletus using an in-field flight tunnel containing pairs of calf, sheep, chicken, and human hosts (and controls) and a laboratory two-choice bioassay containing volatile extracts of host skin (and controls). Behavioural responses of nulliparous C. obsoletus females in the in-field flight tunnel showed a preference for human (but also calf and sheep) hosts, probably due to their exhalation of greater quantities of carbon dioxide. The laboratory experiment revealed that volatile organic compounds released from the skin of chicken and sheep seemed to attract this species. Culicoides obsoletus, thus, seems to have a wide host range and to be particularly attracted by humans under field conditions. A better understanding of vector-host interaction could enable the development of control strategies against adult biting midges, by exploiting insect-repelling or -attractive semiochemicals. Volatile extracts of chicken and/or sheep skin could be used to identify volatile compounds attractive to C. obsoletus, which in turn could be used in baited traps.

Larval development sites of the main Culicoides species (Diptera: Ceratopogonidae) in northern Europe and distribution of coprophilic species larvae in Belgian pastures.

Some Culicoides species of biting midges (Diptera: Ceratopogonidae) are biological virus vectors worldwide and have indeed been associated with outbreaks of important epizoonoses in recent years, such as bluetongue and Schmallenberg disease in northern Europe. These diseases, which affect domestic and wild ruminants, have caused considerable economic losses. Knowledge of substrates suitable for Culicoides larval development is important, particularly for the main vector temperate species. This study, realized during two years, aimed to highlight the larval development sites of these biting midge species in the immediate surroundings of ten Belgian cattle farms. Moreover, spatial distribution of the coprophilic Culicoides larvae (C. chiopterus and C. dewulfi) within pastures was studied with increasing distance from farms along linear transects (farm-pasture-woodland). A total of 4347 adult specimens belonging to 13 Culicoides species were obtained by incubation of 2131 soil samples belonging to 102 different substrates; 18 of these substrates were suitable for larval development. The Obsoletus complex (formed by two species) was observed in a wide range of substrates, including silage residues, components of a chicken coop, dung adhering to walls inside stables, leftover feed along the feed bunk, a compost pile of sugar beet residues, soil of a livestock trampling area, and decaying wood, while the following served as substrates for the other specimens: C. chiopterus, mainly cow dung; C. dewulfi, cow dung and molehill soil; C. circumscriptus, algae; C. festivipennis, algae and soil in stagnant water; C. nubeculosus, algae and silt specifically from the edge of a pond; C. punctatus, mainly wet soil between silage reserves; C. salinarius, algae; and C. stigma, algae and wet soil between silage reserves. We also recorded significantly higher densities of coprophilic larvae within pastures in cow dung located near forests, which is likely due to the localization of potential hosts; the presence of these larvae within cow dung is, however, uninfluenced by relative distance from farms. A better knowledge of the microhabitats of Culicoides biting midges and their spatial distribution may allow the development of targeted species-specific vector control strategies, and may help to prevent the creation of new larval development sites.

Reintroduction of the invasive mosquito species Aedes albopictus in Belgium in July 2013.

Since its first report in 2000, the invasive mosquito Aedes albopictus was not found any more during the different entomological inspections performed at its place of introduction in Belgium between 2001 and 2012. In July 2013, one adult male was captured at the same site (a platform of imported used tires located in Vrasene, Oost-Vlaanderen Province), during a monitoring using CO2-baited trap. This finding suggests the reintroduction of the species in Belgium via the used tire trade.

Are bogs reservoirs for emerging disease vectors? Evaluation of culicoides populations in the Hautes Fagnes Nature Reserve (Belgium).

Several species of Culicoides (Diptera: Ceratopogonidae) biting midges serve as biological vectors for the bluetongue virus (BTV) and the recently described Schmallenberg virus (SBV) in northern Europe. Since their recent emergence in this part of the continent, these diseases have caused considerable economic losses to the sheep and cattle industries. Much data is now available that describe the distribution, population dynamics, and feeding habits of these insects. However, little is known regarding the presence of Culicoides in unusual habitats such as peaty marshes, nor their potential vector capacity. This study evaluated Culicoides biting midges present in the bogs of a Belgian nature reserve compared to those residing at a nearby cattle farm. Culicoides were trapped in 2011 at four different sites (broadleaved and coniferous forested areas, open environments, and at a scientific station) located in the Hautes Fagnes Nature Reserve (Belgium). An additional light trap was operated on a nearby cattle farm. Very high numbers of biting midges were captured in the marshy area and most of them (70 to 95%) were Culicoides impunctatus, a potential vector of BTV and other pathogens. In addition, fewer numbers of C. obsoletus/C. scoticus species, C. chiopterus, and C. dewulfi were observed in the bogs compared to the farm. The wet environment and oligotrophic nature of the soil were probably responsible for these changes in the respective populations. A total of 297,808 Culicoides midges belonging to 27 species were identified during this study and 3 of these species (C. sphagnumensis, C. clintoni and C. comosioculatus) were described in Belgium for the first time.

Chemical composition of silage residues sustaining the larval development of the Culicoides obsoletus/Culicoides scoticus species (Diptera: Ceratopogonidae).

Culicoides (Diptera: Ceratopogonidae) are biological vectors of bluetongue virus (BTV). Bluetongue is a viral disease that affects domestic and wild ruminants. Since its recent emergence in northern Europe, this disease has caused considerable economic losses to the sheep and cattle industry. The biotopes, and more particularly the chemical characteristics which are suitable for larval development of the main vector species, are still relatively unknown. This study shows that the larvae of biting midges belonging to the species Culicoides obsoletus and Culicoides scoticus are able to breed in different types of silage residue (maize, grass, sugar beet pulp and their combinations). The chemical composition of substrates strongly influences the presence of the immature stages of these biting midges. Higher lignin and insoluble fibre contents seem to favour their presence and could play the role of a physical support for semi-aquatic larvae. In contrast, higher concentrations of magnesium and calcium are negatively correlated with the presence of these two species. These data will help to locate and monitor the breeding sites of these species and could contribute to the control of these insects on farms.

The impact of naturally-occurring, trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep.

Infection with bluetongue virus serotype (BTV)-8 occurred in ruminants in 2006 in Central-Western Europe. The trans-placental passage of this virus has been demonstrated in naturally- and experimentally-infected cattle and in experimentally-infected sheep. Trans-placental transmission is potentially important in the 'over-wintering' of this virus and its subsequent impact on reproductive performance. This epidemiological study was carried out on a sheep flock in Belgium that had experienced a severe outbreak of BTV-8 infection, and where the seroprevalence had increased from 1.3% to 88% between January and November 2007. In total, 476 lambs and 26 aborted fetuses from 300 ewes, lambing at four distinct time periods, were investigated between November 2007 and May 2008. The following evidence suggested that BTV-8 infection occurred in utero: (1) positive PCR results from splenic tissue from aborted fetuses (n=4); (2) fetal malformations suggestive of BTV infection (n=10); (3) positive PCR results from red blood cells in-lambs (n=7), and (4) the presence of antibody at birth in viable lambs prior to the intake of colostrum (n=9). The evidence provided by this investigation strongly suggests that trans-placental BTV-8 infection occurs in naturally-infected sheep and the impact of infection on the reproductive performance of such a naïve flock was considerable, with up to 25% of ewes aborting and with flock fertility reduced by 50%. The contribution of in utero-infected lambs to the over-wintering of BTV appears limited.