Invasion dynamics of the disease vector Aedes japonicus in Spain

Native to East Asia, the Asian bush mosquito (Aedes japonicus) has recently expanded its global range, with established invasive populations in Europe and North America. Given its potential role as a vector of various arboviruses, understanding its invasion process and ecological dynamics is crucial for managing its spread and mitigating public health risks. In the Iberian Peninsula, the species was first detected in Asturias in 2018 and has since expanded to neighbouring regions. Here, we elucidate the invasion pathways and possible origins of Ae. japonicus populations in Spain using mitochondrial and nuclear sequence data and microsatellite markers, and by screening for the presence of maternally transmitted bacteria of the genus Wolbachia, which is known to affect mitochondrial DNA diversity. We analysed 635 Ae. japonicus specimens collected between 2018 and 2023 from 14 countries, including Japan (native range), the United States, and 12 European countries including Spain. No clear association between haplotypes and geographical location was detected in any of the three genes analysed (nuclear ITS2, mitochondrial COI and ND4), providing limited insight into the origin of the Spanish Ae. japonicus. Wolbachia was not detected in any of the screened samples based on the wsp gene. In contrast, population structure analysis based on microsatellites revealed a clearer picture of geographic association. Most of the Spanish samples clustered closely with those from College Park, Maryland (USA), located near the Port of Baltimore, one of the largest ports in the United States and a recognised gateway for invasive species introductions. Northern Spain hosts major seaports such as Bilbao and Gijón, both ranked among the ten largest in the country, whereas the nearest established Ae. japonicus population in Europe lies over 1,000 km away in northeastern France. Taken together, these findings suggest that the most plausible invasion route of Ae. japonicus into Spain involves maritime transport of eggs from the eastern coast of the United States (Port of Baltimore, Maryland) to northern Spanish ports, likely accompanied by additional minor introductions of European origin. The inclusion of additional microsatellite loci originally developed for Ae. albopictus yielded results consistent with those obtained using Ae. japonicus-specific loci, reinforcing the robustness of the observed patterns. This work provides new insights into the invasion process of Ae. japonicus in Europe and highlights the need for continuous monitoring and tailored interventions at key ports of entry.