The first record of the invasive mosquito species Aedes albopictus in Chişinӑu, Republic of Moldova, 2020

Background In Europe, Aedes albopictus is an important vector of chikungunya virus and Dirofilaria nematodes and has been involved in local autochthonous circulation of dengue and Zika viruses. Due to the ongoing spread, targeted field surveillance at potential points of entry of invasive Aedes mosquitoes was initiated by the Republic of Moldova in 2020 as part of the transboundary “Invasive Aedes Mosquitoes COST-Action project.” Methods In 2020, ovitraps were positioned at each of three locations: the border crossing to Romania in Leuşeni (Hancesti region), Chişinӑu International Airport and Chişinӑu Botanical Garden. Results A total of 188 Aedes spp. eggs were collected at the Chişinӑu International Airport between August and September 2020. Twenty-three adults reared in the laboratory were identified morphologically as Ae. albopictus (Skuse, 1895), and 12 selected specimens were confirmed by molecular barcoding of the cytochrome oxidase subunit I gene region. In addition, one adult Ae. albopictus female at the same site was caught with a manual aspirator. Conclusions This is the first documented report of Ae. albopictus in the Republic of Moldova. The presence of immature and adult stages indicates the local reproduction of the species in the country. Therefore, it is crucial to extend and strengthen surveillance of the invasive Aedes mosquitoes to prevent Ae. albopictus and other exotic mosquito species from becoming established in the Republic of Moldova. Graphical abstract

Aedes albopictus (Skuse, 1895), commonly known as Asian tiger mosquito, is an invasive mosquito species native to tropical and subtropical regions of Southeast Asia and the Indian Ocean [1]. Over the last 4 decades, the species rapidly expanded its distributional range worldwide including in the Americas, Africa, Australia and Europe [2]. Experimental and field data demonstrated that Ae. albopictus is a potential vector of > 30 different pathogens [1,3,4]. In Europe, Ae. albopictus is an important vector of chikungunya virus with several outbreaks having occurred in Italy [5,6] and France [7][8][9] and Dirofilaria nematodes [10]. In addition, the species was involved in local autochthonous circulation of dengue virus [11][12][13] and Zika virus [14] in Europe.
Globalization, with increasing international trade and travel, facilitates the spread of Ae. albopictus. Due to its ecological plasticity [1], Ae. albopictus has invaded and become established in 30 countries in Europe including the neighboring regions of the Republic of Moldova in the Mediterranean Basin, the Thrace region of Turkey and the eastern Black Sea coast [15][16][17]. Recently, the species was introduced to the the northern Black Sea coast of the Crimean Peninsula [18]. The first report of Ae. albopictus in Romania, a neighboring country to the Republic of Moldova, was in Bucharest in 2012 [19]. Further sampling efforts demonstrated the spread of Ae. albopictus in the country, including the Constanta region, where positive sampling sites were close to the border of Moldova [20].
The global trade of lucky bamboo (Dracaena sp.) and tires is the most important pathway for the global dispersal of invasive Aedes species [21,22]. Subsequently, public and private transport especially along highways is considered one of the main drivers of Ae. albopictus's spread in Europe [23][24][25][26][27]. Thus, targeted field surveillance of potential points of entry (PoE) for Ae. albopictus and other invasive Aedes mosquitoes (AIM) at a highway, botanical garden and airport was carried out in 2020 in the Republic of Moldova as part of the "AIM-COST action project" [28].
Field surveys were conducted at three locations that were potential routes of entry to the Republic of Moldova for invasive Aedes mosquito species. These included the border crossing to Romania in Leuşeni, Hânceşti region (10 June-16 October 2020), where the border inspection post is located in an agricultural environment and international vehicular transport regularly enters the country, Chişinӑu International Airport (5 July-31 October 2020) and Chişinӑu Botanical Garden (10 July-16 October 2020), known for introducing and growing tropical plants (Fig. 1). Conicalshaped black plastic containers (height: 13 cm, lower diameter: 9 cm, upper diameter: 13 cm) with approximately 1-l volume were used as ovitraps [15]. Each trap was filled two thirds with clean water, and scratched tongue depressors (1.7 × 15 cm) were added as an egglaying substrate for invasive Aedes species. Five ovitraps were positioned in each location in the shaded sites at a distance not less than 20 m from each other, and the maximum distance between traps was about 400 m. (Fig. 1). The tongue depressors and water were collected from the traps and replaced with clean water and new tongue depressors every 2 weeks. All samples were transported to the Entomology Laboratory, Institute of Zoology, in Chişinӑu for rearing of eggs and larvae to adults in trays containing dechlorinated water. Hatched larvae were fed with aquarium fish food (ASTRA Aquaristik GmbH, Osnabrück, Germany). Morphological species identification of larvae and adults was conducted with the keys in Becker et al. [29].
Two mosquito taxa were collected from the ovitraps during the entomological surveys: Culex pipiens (sensu   (Fig. 2). Morphological identification of Ae. albopictus was confirmed by molecular barcoding of the cytochrome oxidase subunit I gene region of 12 randomly selected specimens [30]. All sequences were entered into GenBank (accession no. MZ069031-MZ069042). In addition, one Ae. albopictus female was caught by manual aspirator during ovitrap inspection at the airport on 27 September (Fig. 1b). Three additional ovitraps (A6, A7, A8) were placed at the Chişinӑu International Airport at the end of September and surveillance continued through to 31 October, but no further Aedes spp. eggs were collected. Surveillance of the presence/absence of invasive Aedes species at the potential PoE in the Republic of Moldova demonstrated the presence of Ae. albopictus at the Chişinӑu International Airport. In the past, only few studies have been dedicated to the role of European airports in importing exotic mosquito species with Ae. albopictus recorded at Schiphol Airport, The Netherlands [31][32][33][34].
The introduction of Ae. albopictus in Europe was facilitated by passive dispersion through the global transportation of tires [35,36] and the import of Dracaena plants known as "lucky bamboo, " e.g., in The Netherlands [21] and Bulgaria [37]. Further dispersal in Europe inside vehicles via highway systems was documented in Switzerland [38], Germany [23], Spain [24] and the UK [26]. However, no exotic mosquito species were detected at the border crossing between Romania and Chişinӑu and at the botanical garden.
This study emphasizes the importance of air transportation for the dispersal of Ae. albopictus in Europe. This is the first documented report of Ae. albopictus in the Republic of Moldova to our knowledge, and the presence of adult and immature stages indicates the local reproduction of the species. Further investigations with greater trapping efforts are necessary to clarify whether this is a stable, established population. This is especially important for determining future mosquito control measures; public health authorities were informed, but insecticidal control has not yet been implemented. In addition, with the increasing spread and population densities of Ae. albopictus in Europe, additional introductions have to be expected via air traffic and other routes of entry, which may allow long-term establishment. Therefore, it is crucial to extend and strengthen surveillance of invasive Aedes mosquitoes to prevent their establishment and future arbovirus transmission in the Republic of Moldova.

Abbreviations
PoE: Points of entry; AIM: Aedes invasive mosquitoes.

Acknowledgements
The work was done within the framework of the AIM-COST Action CA17108.
Authors' contributions TȘ designed the study. TȘ, GB, UL and RL conducted the field and laboratory work. TȘ and RL prepared the manuscript. GB, UL and JSC contributed to the discussion and improvement of the manuscript. All authors read and approved the final manuscript.

Funding
Open Access funding enabled and organized by Projekt DEAL. This work was financially supported by the Institute of Zoology, Republic of Moldova under the project EVOLANTER (project no. 20.80009.7007.02) and the Federal