Fig. 1 A Le. frauenfeldi worker in Nanko Port carrying a Crematogaster matsumurai worker on 14 July 2018. S. INOUE and S. UEDA according to Terayama et al. (2014). We then calculated ant diversity using Simpson’s the Shannon-Weiner index (H’) and Pielou’s evenness index (J’) for each route, and compared these indices between the routes between April 2022 and January 2023. In November 2022, the eastern part of Nanko Port, including the monitoring survey sites (375 ha), was divided into 15 grids (Fig. 3). In each grid, ants observed on the ground and on vegetation were captured for 15 minutes using an aspirator. The species and number of individuals were recorded using the same methods described above. Fig. 2 Locations of Route A (500 m) and Route B (560 m) for monitoring Le. frauenfeldi in Nanko-Port, Osaka, on Japan’s main island of Honshu. The two routes are separated by a distance of 210 m. Fig. 3 Distribution survey map of Le. frauenfeldi in the eastern part of Nanko Port (375 ha), divided into 15 grids. The grid number is shown in the upper right of each grid. The pie chart shows the proportional abundance of Le. frauenfeldi (black) and other ant species (white) in each grid, with the sample size collected using the time-unit sampling method (Nakajima et al., 2013) for 15 minutes per grid. index (1-λ), rearing, As stated above, Ueda and Murakami (2022) demonstrated that Le. frauenfeldi had been introduced and had established colonies at Nanko Port, Osaka in 2018, with the numbers of this species increasing to the stage of producing reproductive castes in 2019. In addition, predation on other the protection of insects and agricultural pests, such as aphids, were also documented. At this site, however, monitoring surveys and chemical control measures have not yet been initiated. Therefore, to gather information needed for controlling this invasive alien ant, we investigated its distribution and seasonal population changes at this site. Ant monitoring surveys were conducted approximately once a month using the time-unit sampling method of Nakajima et al. (2013 and 2018), in Nanko Port, Osaka. Two survey sites were established: Route A (500 m), along a sidewalk adjacent to a roadway in the port area, and Route B (560 m), located 210 m away from Route A (Fig. 2). A total of 17 surveys were conducted along Route A between September 2021 and January 2023, and 10 along Route B between April 2022 and January 2023. Along each survey route, a researcher walked at a constant speed, capturing ants observed on the ground and on vegetation for approximately 30 minutes using an aspirator. The researcher did not explore or collect in ants the same spot for more than one consecutive minute (Nakajima et al., 2013). The collected ants were preserved in sealed containers (50 ml centrifuge tubes, 3.2 cm in diameter × 11.5 cm in length) filled with 80 percent ethanol and brought back to the laboratory. The species and numbers of individuals were then examined under a stereomicroscope. Species identification was conducted 126 reports of ecological damage, the Ministry of the Environment of Japan listed this species as an Invasive Alien Species in November 2020, and prohibited its import, possession, transport release and (Ministry of the Environment of Japan, 2020). 2. Study Site and Methods
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