HaCeD-Seq: a Novel Method for Reliable and Easy Estimation About the Fish Population Using Haplotype Count from eDNA.

It is common to count the numbers of specified fish in the field after speciation of captured fish according to their morphology and to subject these counts to appropriate statistical analyses. In recent years, a non-invasive method to estimate the abundance of a particular fish species using environmental DNA (eDNA) has been developed. However, it is still difficult to determine accurate numbers of fish species using such method. We predict that the estimation of individuals of certain fish species in the field is more accurate and easier by using haplotypes of DNA in the fast evolutionary region. Therefore, we focused on the regulatory region (D-loop) in mitochondrial DNA, which is known to have a high genetic variation at the intraspecific level of the targeting eel. We investigated haplotype diversity in eel at first and then determined the number of D-loop haplotypes contained in their exfoliated cells in breeding water. Finally, we developed a novel analytical method, HaCeD-Seq, to estimate the number of individuals based on the abovementioned data.