Discovery of unknown new ponds occupied by the endangered giant water bug Kirkaldyia deyrolli (Hemiptera: Heteroptera: Belostomatidae) by combining environmental DNA and capture surveys

Basic ecological information (e.g. habitat or distribution) is indispensable for conserving endangered species. The giant water bug Kirkaldyia deyrolli is an aquatic insect in the large stink bug family (Belostomatidae). It inhabits ponds and is a critically endangered species. In this study, we developed a species-specific primer-probe set (the cytochrome c oxidase I region in mitochondrial DNA) that was used in real-time polymerase chain reaction (PCR) assays to detect K. deyrolli environmental DNA (eDNA). Next, using eDNA analysis, we investigated the presence of K. deyrolli in 89 study ponds (including one pond that had already been identified as the habitat of this species) in Shimane Prefecture, Japan. The eDNA of K. deyrolli was detected in 11 of these 89 ponds. Furthermore, when the traditional method of direct capture survey was carried out in four of the 10 ponds where K. deyrolli eDNA had been detected and no prior occupancy information for this species was available, the capture of one K. deyrolli (female) was successful in only one pond. This study showed that combining eDNA analysis and direct capture methods can lead to discovering previously unknown habitats essential for conserving an endangered species.