Searching for a Vanishing Fish: Development of an eDNA-Based Digital PCR Assay and Field Surveys for the Critically Endangered Chinese Bahaba

Abstract

The critically endangered Chinese bahaba (Bahaba taipingensis) is difficult to monitor via conventional methods due to its low abundance and estuarine habitat. We developed a species-specific microfluidic digital PCR assay targeting the mitochondrial COI gene for environmental DNA (eDNA) detection. No cross-amplification was observed with seven sympatric non-target sciaenid species. The limit of quantification (LOQ) was 1.313 copies µL−1 and the limit of detection (LOD) was bounded between 0.162 and 1.313 copies µL−1 among the tested concentrations. All 12 aquaculture samples yielded quantifiable eDNA of 3.610 to 2109 copies µL−1, with CVs of 0.53–11.05% among technical triplicates. All putative field detections were below the LOQ and were classified as detected but not quantifiable. Among 21 paired stations, putative target-DNA detection frequencies were 19.05% (4/21) in April and 47.62% (10/21) in September, but the difference was not significant (exact McNemar test, p = 0.070). A stock-enhancement release between surveys confounded the comparison. Thus, the observed difference cannot be confidently attributed to natural seasonal variation. Given that only one water sample was collected per station and each field extract was analyzed in a single dPCR reaction, these field observations should be considered preliminary and require further sampling and analytical replication.