Novel Assay for Endangered Hong Kong Grouper (Epinephelus akaara) to Assess eDNA Shedding, Decay, and Population Status

The Hong Kong Grouper (Epinephelus akaara) is a commercial fish species that suffered at least 50%–80% population declines in the past 40 years throughout its distribution range due to overexploitation. Yet, limited research on distribution and habitat utilization resulted in a lack of species-specific management strategies. Here we aim to utilize environmental DNA (eDNA) analysis as a potential tool for species detection and to provide both shedding and decay rate for future application in persistence prediction models. We first develop a novel, species-specific, sensitive, quantitative PCR (qPCR) assay amplifying 71 bp of the mitochondrial ND2 gene. From a mesocosm experiment, we found the decay rate (0.131 ± 0.0111/h) of E. akaara to be similar to other reported marine fish species. However, the shedding rate of E. akaara (1.94 × 10⁴ ± 2.07 × 10³ copies/h/g) is generally lower than reported values of other species, likely due to the relatively low activity and energy use from solitary and sedentary behavior of groupers. This highlights the importance of empirically determining species or taxon-specific shedding and decay rates to inform accurate abundance estimates with modeling tools for eDNA concentrations. Out of 88 water filters collected across four sampling seasons and 11 sites in Hong Kong, six samples from four different sites showed positive amplification. No samples were measured at a concentration above limit of detection of the assay, and this rarity is consistent with current observational data. Overall, we demonstrate that eDNA with qPCR assay is a promising tool to fill in knowledge gaps of endangered species with insufficient species management and conservation across its distribution. © 2026 The Author(s). Environmental DNA published by John Wiley & Sons Ltd.