Noninvasive genetic diversity assessment using exuviae for reintroduction of the endangered tiger beetle Cicindela (Abroscelis) anchoralis in South Korea

The population of the endangered tiger beetle Cicindela (Abroscelis) anchoralis Chevrolat, 1845 in South Korea has drastically declined in recent years due to coastal development and human recreational activities within its habitats. To address this issue, a reintroduction strategy involving the mass release of larvae reared through ex-situ propagation into their natural habitat was implemented. However, although genetic diversity is a key factor to consider in conservation strategies for reintroduction into habitats, the genetic diversity of the propagation population of C. anchoralis has not been assessed due to limitations in extracting genetic material from larvae without physical harm. To overcome this limitation, we developed a noninvasive method using larval exuviae to evaluate genetic diversity prior to reintroduction. Under rearing conditions, over 85% of larval exuviae were successfully recovered, and mitochondrial DNA (mtDNA) could be reliably extracted within five days of moulting using a direct DNA extraction method. Genetic analysis of the mtDNA revealed three haplotypes in the propagation population, based on two sequence variations in the cytochrome c oxidase subunit I gene. Our findings establish a method for assessing and maintaining the genetic diversity of propagation population by utilizing larval exuviae, thereby avoiding physical harm to the larvae. This approach will enable the formation of genetically diverse, unbiased populations for reintroduction, supporting effective restoration of the tiger beetle species. This study employed a noninvasive mtDNA extraction method to assess the genetic diversity within the ex-situ propagation population of the endangered tiger beetle Cicindela (Abroscelis) anchoralis prior to reintroduction. Utilising larval exuviae to identify haplotypes and determine haplotype ratios without causing physical harm to the larvae provides a valuable approach for establishing and propagation population with enhanced genetic diversity. This strategy can improve the evolutionary potential of reintroduced populations and support the long-term conservation of insect species.