FASFC - Exotic vector and pathogen surveillance programme in Belgium - Mosquitoes & Culicoides

Study Extent

Mosquitoes During the previous project, the ExoSurv project (Deblauwe et al. 2012, 2014), 21 PoE’s were sampled. We reselected the 11 sites with the highest risk for import of exotic mosquitoes and midges for the FASFC project in 2013. The site at the port of Antwerp was moved to the Border Inspection Post of the FASFC as containers are opened there for the first time. Eleven of the 12 potential PoE surveyed in 2013 were also surveyed in 2014. One platform for imported used tyres, stopped its activities and was replaced by another close to the colonised area of Ae. koreicus. All 12 potential PoE surveyed in 2014 were also surveyed in 2015. Only two potential PoE surveyed in 2015 were also surveyed in 2016. A new second-hand tyre company was identified from customs data collected for another ITM project (HarmVect, Berkvens et al. 2017) and added to the selection. This company imported more than 10000000 tyres from high risk countries in both 2013 and 2014. The colonised site with Ae. koreicus at Maasmechelen was not selected as the population seemed to be stable at the moment. However, sporadic visits were made to the site and its environments to confirm its establishment and investigate its spread. The colonised site with Ae. j. japonicus at the second hand tyre company in Natoye was not surveyed as mosquito control and follow up were conducted by the Wallon government. Culicoides In 2013, Culicoides were sampled at seven of the 12 PoE where mosquitoes were surveyed. In total five farms (province of Antwerp) were selected for the monitoring of Culicoides in 2016. The farms were already sampled during the bluetongue project from FASFC between 2007 and 2012. Four sites are cattle farms, while one site was a cattle farm before 2012 but now only has horses.

Sampling

Mosquitoes At each of the 12 PoE adult mosquitoes were captured using one CO2-trap (Mosquito Magnet™ Liberty Plus or Executive (MMLP)), which produces CO2 by burning propane. Attracted mosquitoes are then pulled in by a fan. Each PoE was sampled almost continuously (collections every two weeks) from April until October-November. MMLP traps were placed as close as possible to the breeding places (e.g. tyres, lucky bamboo vessels), the place where containers are opened with Lucky bamboo, fresh flowers, fruit or vegetables, and where cars or trucks are opened at parking lots. The captured mosquitoes and by-catch were transported in their MMLP net enclosed in a sealable, fully labelled plastic bag in a cool box. At the lab they were put in the freezer (-20°C) for at least 2h to kill the mosquitoes and to store for later sorting and identification. Five oviposition traps (ovitraps) were placed at each of the 12 PoE at a perimeter of 100m around the MMLP. They consist of a small black plastic bucket (0.5 to 2.0l, 2/3 filled with an oak infusion and sometimes with a hole preventing overflowing and flooding of the eggs) and an oviposition support (piece of polystyrene). Ovitraps were sampled every month. In the lab polystyrenes were stored at room temperature or in a fridge. The pieces of polystyrene were checked for eggs under a stereomicroscope. In 2013 and 2014, positive polystyrenes were immerged in water in secured containers, which were stored in a secured mosquito breeding room at ITM. Hatched larvae (3rd or 4th instar (L3 or L4)) were stored in 80% ethanol for identification. In case the eggs did not hatch, molecular analysis was performed on the eggs for identification. In 2015 and 2016 molecular analysis was performed immediately on the eggs for identification. At each of the 12 PoE, 1 up to 20 potential larval breeding sites (PBS) (depending on the available type of potential breeding site (tyres or other containers)), if present, were inspected. A potential breeding site is a single vessel or a group of the same vessels (e.g. a stock of tyres, lucky bamboo containers in the same shelter) in which mosquito larvae can develop. More precisely, small man-made water containers that accumulate dead leaves, algae and/or other organic matter. Also natural containers like tree holes and other similar water bodies were inspected. Larval sampling was done every two months. Each potential breeding site was sampled to maximize collection. For small sites, one dip with a plastic tray or total clearance of sites (e.g. bucket, tree hole, small water containers) was performed. For larger sites (e.g. big tyres, animal water trough) four to six dips were made with an aquarium net. Larvae were collected with a plastic pipette and put in a fully labelled vial with water (for rearing the larvae to 4th instar (L4) or to adults) or directly in 80% ethanol for later species identification. During intensified surveillance, once an IMS was detected, other traps were used to catch adult mosquitoes, the BG-Sentinel trap (Biogents) (2013 - 2016) and Frommer updraft gravid trap (John W. Hock company) (only in 2013). Also 0-19 additional oviposition traps were set-up at the PoE (if necessary indoor and outdoor) and/or in a 200-300m buffer zone around the PoE, and inspected every two or four weeks depending on the PoE. And larval sampling frequency increased to once or twice a month and up to 312 potential breeding sites were checked for larvae. The ECDC guidelines for the placement of all trap types (ECDC 2012) were taken into account. Morphological identification of adult mosquitoes and larvae was carried out using taxonomic keys (Schaffner et al. 2001, Schaffner 2003, Becker et al. 2010, ECDC 2012). Identified mosquitoes were stored dry in boxes with silica-gel. Morphological identification of adults, eggs and larvae of Ae. albopictus was confirmed by PCR and sequencing. The ITS2 region of the rDNA gene was amplified using 5.8S (5’-ATCACTCGGCTCGTGGATCG-3’) and 28S (5’-ATGCTTAAATTTAGGGGGTAGTCAC-3’) universal primers which flank the ITS2 gene (Djadid et al. 2007). A reverse primer specific for the Ae. albopictus 28S sequence was used to perform a semi-nested PCR (R28Salbo: 5’-TTGCGGGTGTTTTGTGTGTCGTC-3’) (De Jong et al. 2009). DNA was run on 2% agarose gel. Gels were stained with ethidium bromide and bands were visualized by UV trans-illumination. PCR products were cloned and DNA sequencing of selected clones was carried out by VIB (Vlaams Instituut voor Biotechnologie, Department of Molecular Genetics, University of Antwerp) on an automated sequencer. The obtained sequences were compared to sequences submitted to GenBank. Culicoides At each of the PoE (2013) or farms (2016) one OVI 220V down-draught black light (8W) trap (ARC-Onderstepoort Veterinary Institute, Onderstepoort, SA) was placed at 1.5 to 2 m above ground level to capture adult Culicoides. Traps were operated twice per month during two consecutive days (48h) from April until November 2013 and 2016 (except in August 2016 only once was sampled and in July 2016 no sampling took place), and weekly from December 2016 until January 2017. Midges were collected into a 500ml cup with 60% ethanol. A few drops of soap were added to break the surface tension. After retrieval, insects were transferred to 80% ethanol, Culicoides sorted and identified up to species level (2013) or up to sub-genus level (2016). In 2013, the same MMLP trap and frequency used to capture adult mosquitoes was also used to capture adult Culicoides. Midges were transported together with the mosquitoes and by-catch in their MMLP net enclosed in a sealable, fully labelled plastic bag in a cool box. At the lab they were put in the freezer (-20°C) for at least 2h to kill the midges and to store for later sorting and identification. At least 30m distance was kept between the light trap and MMLP trap to reduce interference, as the range of attraction for female Culicoides is about 30m (Rigot & Gilbert 2012) and the MMLP trap attracts species from about 36m away (Versteirt 2012). Morphological identification of adult Culicoides was carried out using taxonomic keys (Delécolle 1985, Mathieu et al. 2012). Identified midges were stored in 80% ethanol. Culicoides samples collected in the end of May 2016 (1 sample week) and in June 2016 (3 sample weeks) were not identified as priority was given to the intensive surveillance of the invasive mosquito Aedes albopictus at three PoE at the same time during summer 2016.

Quality Control

Data are collected using a predefined sampling protocol and checked by professionals

Method steps

1. Researchers from ITM defined the appropriate sampling protocol for the target species.
2. Fieldwork was planned and coordinated by ITM
3. Data was collected in the field by specialized personnel.
4. The collected data was entered into an Access database
5. The data was exported and manually corrected by experts
6. A custom R script was created to map the original data from the Access database to Darwin Core as an event core with an occurrence extension.(https://github.com/BelgianBiodiversityPlatform/data-publication-ITG)
7. The Darwin Core files are connected to the BBPF IPT and documented with metadata.
8. The dataset is published and registered with GBIF.

Culicoidea

1. Animalia
   rank: kingdom
2. Arthropoda
   rank: phylum
3. Insecta
   rank: class
4. Diptera
   rank: order
5. Culicoidea
   rank: superfamily
6. Culicidae
   rank: family
7. Ceratopogonidae
   rank: family

Belgium