MEMO: Monitoring of exotic mosquitoes in Belgium

‘MEMO: Monitoring of Exotic MOsquitoes in Belgium’ is a sampling event dataset published by the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. It forms part of the early detection of exotic mosquito species (EMS) along high-risk introduction routes in Belgium, where data are collected at defined points of entry (PoEs) using a standardised protocol. The MEMO dataset contains mosquito sampling counts performed between 2017 and 2020. MEMO+2020, an extension of the MEMO dataset, contains only Aedes albopictus mosquito trap counts performed in 2020. Here, we present these data published as a standardised Darwin Core archive, which includes, for each sampling event, an eventID, date, location and sampling protocol (in the event core); and an occurrenceID for each occurrence (tube), the number of collected individuals per tube, species status (present/absent), information on the identification and scientific name (in the occurrence extension).

. Overview of the MEMO points of entry (PoEs). Blue dots: MEMO; red diamonds: MEMO+2020. In total, 33 locations were sampled in MEMO and six locations in MEMO+2020.

MEMO (dataset 1) [2, 3]
In 2017, 2018 and 2019, active monitoring was implemented by researchers from ITM in 20-23 different PoEs ( Figure 1) with exact GPS coordinates. The risk of introduction and establishment of EMS at each PoE was re-evaluated annually to ensure that monitoring focused on the highest risk sites. Different sampling protocols were used, defined, planned and coordinated by ITM researchers, including BG-Sentinel, Mosquito Magnet ® and gravid traps to collect host-seeking female mosquitoes; oviposition traps to detect eggs; and larval sampling [6]. At each PoE, a combination of different trapping methods was used simultaneously. The collected specimens were sorted and identified using morphological characteristics [7][8][9][10]. The caught EMS, as well as 5% of all collected mosquitoes, were identified using DNA-based techniques to validate and confirm the morphological identification. Tissue and DNA were subsequently deposited in a molecular reference collection hosted at the Royal Belgian Institute of Natural Sciences (RBINS) [2].
A specific DNA-based identification pipeline was developed [11] to enable accurate identification to species (or biotype for Culex pipiens s.s.) level of all mosquitoes occurring in Belgium (native and potential EMS). Further, a morphological collection with a representation of 23 species and the most intact specimens sampled during the MEMO project was generated for future reference and is also hosted at RBINS [3]. Data management was done using VECMAP ® software [12] (Avia-GIS, Zoersel, Belgium).

MEMO+2020 (dataset 2) [4, 5]
In 2020, active monitoring was implemented at six different PoEs ( Figure 1). The focus was on parking lots along the highway as this pathway for exotic Aedes species is becoming more important. Four fixed parking lots (Aische-en-Refail, Raeren, Marke, Saint-Ghislain) were monitored at first, but following the detection of Ae. albopictus in the Netherlands, two further parking lots (Minderhout, Gierle) were added. Oviposition traps were used to collect eggs, and potential breeding sites were inspected for larvae [13]. MEMO+2020 sampling resulted in one positive identification of Ae. albopictus.

Taxonomic coverage of the two datasets
In the period 2000-2009, substantial changes were proposed for the Aedini tribe taxonomy, which resulted in almost tripling the number of genera in the entire Culicidae family. A recent publication [14] proposed a return to the taxonomy from before 2000, restoring a classification system useful for the operational community. This latter classification system was used during the MEMO and MEMO+2020 projects Most specimens collected of the Anopheles maculipennis complex were molecularly identified up to species level. Three adults could morphologically be identified as Culex torrentium, other specimens are grouped together with Culex pipiens s.l. (Figure 2).

Geographic coverage of the two datasets
Belgium is a small country in Western Europe. To the west, its 70-km coastline fronts the North Sea; to the north lies the Netherlands; to the east, Germany, and to the south, France   Belgian has a temperate maritime climate that is influenced by the North Sea and the Atlantic Ocean with substantial precipitation in all seasons. Summers are moderate and winters are mild.

Temporal coverage
The first phase of the project ran from July 2017 until June 2020 (MEMO dataset), and the second phase from July 2020 until November 2020 (MEMO+2020 dataset).
The overall distribution of the mosquito sampling events over time is illustrated in

Sample processing for the two datasets
On arrival at the laboratory, adult specimens were killed by freezing at −20 °C. Larvae were transported alive to the laboratory and killed by a thermal shock with hot water (70°C).
Next, larvae were transferred in 80% ethanol for preservation before morphological identification. In the case of EMS, larvae were then transferred in absolute ethanol for further DNA-based species validation. Morphological identification of adults and larvae was done with a stereomicroscope using dichotomic and digital keys [7][8][9][10]. DNA-barcoding technology [15] was applied to validate the morphological identification of EMS, of 5% of the annual sampling (quality control), and to identify damaged adults and larvae or species complexes (following the methodology of Smitz et al. [16]). The step-by-step procedures can be viewed in the Github MEMO repository [11].
Polystyrene pieces collected from oviposition traps were checked for EMS eggs in the laboratory using a stereomicroscope. A subsample of the eggs from the polystyrene piece (1-5 eggs per side) was always DNA barcoded [17]. In 2020, the positive polystyrene pieces were immersed in water in secured containers, which were stored in a climate-controlled to BioProject ID PRJNA837425 [16][17][18][19][20]. The generated microsatellites database for Aedes japonicus population genetic investigation [17] was deposited on the open-access Dryad Digital Repository [21].

MEMO and MEMO+2020 dataset creation
Collected data were entered into VecMap, exported and manually corrected by experts. A custom R & Grel (General Refine Expression Language) script was created to map the original data to Darwin Core as an event core with an occurrence extension [11].
Occurrence data from the MEMO (dataset 1) [2] and MEMO+2020 (dataset 2) [4] projects are extracted, standardised, and published as two separate Darwin Core Archives. The Darwin Core files are connected to the Belgian Biodiversity Platform IPT and documented with metadata. Datasets are published and registered with GBIF [3,5].
The Darwin Core terms [22] in the dataset at the time of publication are as follows.

DATA AVAILABILITY
This data paper is linked with two MEMO-related datasets; dataset 1 [3] and dataset 2 [5].
The database server uses Windows Server 2003 SBS R2 as operating system, and is running IIS with PHP for site development, MS SQL Server for database development and SQL Server Mobile Tools to allow remote access from a PDA.

The generated microsatellites are deposited on the open-access Dryad Digital
Repository [21]. The generated DNA barcodes are linked to NCBI BioProject ID: PRJNA837425.
The data are published under a Creative Commons CC0 waiver and we kindly ask you to notify the corresponding authors of the respective dataset if you use the data, especially for research purposes. Issues with the dataset can be reported at https://github.com/BelgianBiodiversityPlatform/data-publication-ITG/issues.