Mosquito collection

To examine signs of introduction of Ae. albopictus from abroad, we compared the genetic structure of adult mosquitoes collected in three cities on Kyushu Island, Fukuoka, Kitakyushu, and Nagasaki, the locations of the busiest international terminals. Accordingly, it was sought to observe genetic differences between ship terminals and distant areas due to a population introduced via maritime transportation. In each city, three areas were designated for sampling: two areas around the terminals and one distant area. Fukuoka has two large container terminals; Island City Terminal and Kashii Park Terminal. These terminals are located next to each other in the northern part of Hakata Port (Fig 1). The former terminal imported 3.5 million tons and the latter imported 5.7 million tons of international cargo in 2018 (Hakata Port annual report 2018). Approximately 65% of the combined cargo from the terminals was imported from China and South Korea. Adults of Ae. albopictus were sampled from five vicinity sites of the two terminals using sweep nets in September 2019. A vicinity was considered as an area within 4 km of a terminal. At each collection site, one to three collectors swept nets for a maximum of 20 minutes or until 20–30 adults were obtained, typically around shaded places near bushes for efficient collection. Mosquitoes were also sampled from five vicinity sites of Chuo Terminal approximately 5 km south of the container terminals in September 2018, September 2019, and June 2020. This terminal imported 0.4 million tons of non-container cargo mainly from China and South Korea in 2018. The terminal also handled international passenger ships, mostly from South Korea, and 276 cruise ships in 2018. For further comparison, mosquitoes were sampled in June 2020 from eight inland sites distanced at least 6 km from each terminal (Fig 1 and Table 1). These sites were selected based on accessibility and available sampling time.

Created by processing Free vector and raster map data @ naturalearthdata.com and National Land Numerical Information (Administrative Area Data) @ Ministry of Land, Infrastructure, Transport and Tourism, Japan (https://nlftp.mlit.go.jp/ksj/gml/datalist/KsjTmplt-N03-v3_0.html).

N/A: not applicable

Kitakyushu has three container terminals, the adjacent Tachiura Terminals 1 and 2, and Hibiki Terminal 22 km distant (Fig 1). Hibiki Terminal and the two Tachiura terminals imported 1.3 and 1.8 million tons in 2018, respectively. Nearly 75% of containers were imported from China and South Korea. Adult mosquitoes were sampled in September 2019 from four vicinity sites of the Tachiura terminals and two vicinity sites of Hibiki Terminal. For reference, mosquitoes were sampled from one site at the middle point between the terminals (Fig 1 and Table 1).

Nagasaki has two international terminals, Yanagi Terminal and Matsugae Terminal, which are separated by a distance of over 4 km (Fig 1). Matsugae Terminal hosts cruise ships, and Yanagi Terminal hosts container ships and cruise ships. The latter imported 0.1 million tons of cargo that was mainly from Australia (32%) and South Korea (23%) in 2017. The terminals hosted 220 cruise ships, which in 2018 was the second largest port in Japan after Fukuoka. Adult mosquitoes were sampled in July 2019 from three vicinity sites of Yanagi Terminal and four vicinity sites of Matsugae Terminal. For comparison, mosquitoes were sampled from another two coastal sites and two inland sites distanced at least 6 km from each terminal in July and August of 2019 (Fig 1 and Table 1).

Adult mosquitoes were also sampled from Goto Island and Tsushima Island which are located between Kyushu Island and the Korean Peninsula. As Tsushima Island has sea connectivity with both sides and Goto Island has sea connectivity with Kyushu Island, the genetic information of the island mosquito populations was used for inferring the origin of mosquitoes found in the ports on Kyushu Island. Mosquitoes were sampled in September 2019 from two local ports, five vicinity sites, and one middle site on Tsushima Island; and one port and one vicinity site on Goto Island (Fig 1 and Table 1). For reference sites outside of Kyushu, adult mosquitoes were sampled from Honshu (Tottori, Tokyo, and Ibaraki) and Thailand (Ranong) during 2016 to 2020 (Fig 1 and Table 1). The present study also included genetic samples of adult Ae. albopictus from the Philippines (unknown locality) collected in 2013 and from Ryukyu Islands (four islands) collected in 2017 and 2019, which deposited in the Institute of Tropical Medicine, Nagasaki University (Fig 1 and Table 1).

Collected mosquitoes were immediately placed in 100% alcohol or silica beads and stored in freezers under -25°C. Before molecular analysis, the samples were identified morphologically using the key by Tanaka et al. [37].