Background: Aedes japonicus japonicus (Theobald, 1901) and Aedes koreicus (Edwards, 1917) have rapidly spread in Europe over the last decades. Both species are very closely related and occur in sympatry. Females are difficult to distinguish, and no distinctive morphological characters are known for males. However, accurate species discrimination is important as both species may differ in their vectorial capacity and spreading behaviour. In this study, we assessed the potential of geometric wing morphometrics as an alternative to distinguish the two species. 

Methods: A total of 147 Ae. japonicus specimens (77 females and 70 males) and 124 Ae. koreicus specimens (67 females and 57 males) were collected in South-West Germany. The left wing of each specimen was removed, mounted and photographed. The coordinates of 18 landmarks on the vein crosses were digitalised by a single observer. The resulting two-dimensional dataset was used to analyse the differences in the wing size (i.e., centroid size) and wing shape between Ae. japonicus and Ae. koreicus by means of geometric morphometrics. To analyse the reproducibility of the analysis, the landmark collection was repeated for 20 specimens per sex and species by two additional observers.

Results: The wing size in female Ae. koreicus was significantly greater than in Ae. japonicus but did not differ significantly for males. However, the strong overlap in wing size for the females would not allow for discriminating the two species. In contrast, the wing shape clustered species-specific and a leave-one-out validation resulted in a reclassification accuracy of 95% for the females and 91% for the males. The data collected by different observers resulted in a similar accuracy, indicating a low observer bias for the landmark collection. 

Conclusions: Geometric wing morphometrics provide a reliable and robust tool to distinguish female and male specimens of Ae. japonicus and Ae. koreicus.

In total, 147 Ae. japonicus specimens (77 females and 70 males) and 124 Ae. koreicus specimens (67 females and 57 males) were included in the study. The left wing of each mosquito was removed and mounted in Euparal (Carl Roth, Karlsruhe, Germany) on microscopic slides, and dried. Subsequently, the mounted wings were photographed (Olympus DP23, Olympus GmbH, Tokyo, Japan) under 20x magnification with a stereomicroscope (Olmypus SZ61, Olympus GmbH, Tokyo, Japan).