2021 Volume 23 Issue 2 Pages 279-293
The Genji firefly (Luciola cruciata) is an important tourism resource, especially in Japan. Nevertheless, its habitat has decreased, mainly due to human activity. Until now, its population size has been studied primarily by visual observation of adults, which fly and flash during the night. Surveys of larvae have been avoided not to disturb its populations and habitats. In this study, we performed environmental DNA (eDNA) analysis to quantify firefly larvae by showing a correlation between larval numbers and eDNA concentration in the field. We then compared the eDNA concentrations and larval numbers to the adult numbers of two generations to evaluate the suitability of this analysis for exploring bottleneck events in firefly populations. We designed "primer set I" and "primer-probe set II" to specifically detect and quantify Genji firefly DNA fragments, respectively. The species specificities of the primer sets were validated by PCR using tissue-derived DNA samples from the target species and its most closely related species (Heike firefly, Luciola lateralis), which were captured in the field. The quantifiability of primer-probe set II was confirmed by quantitative PCR using artificially synthesized DNA of the two species. Quantitative PCR using water samples collected from areas where firefly larvae were captured (15 locations) showed a positive correlation with the number of firefly larvae. The relationship between the number of adults in the previous and focal generations and the number of captured larvae and eDNA concentration suggested that repeated measurements of eDNA concentration during the larval stage are necessary. In conclusion, we showed a positive relationshipbetween eDNA concentration and firefly larvae number in the field for the first time. It may be possible to identify causes or seasons of Genji firefly reduction by repeated measurements of eDNA concentration during the larval stage.
