Development of a species-specific qPCR assay for the detection of the threatened dugong (Dugong dugon)

The global population of dugongs ( Dugong dugon ) has declined by 20% over the last ninety years, with some regional populations facing total extirpation. Effective conservation depends on the ability to reliably monitor population trends and habitat use through time. Hence, there is a growing need for the development of reliable, scalable, cost-effective and non-invasive monitoring techniques that could inform dugong conservation. To this end, we developed a species-specific, non-probe-based qPCR assay from the 16S rRNA mitochondrial gene region to enable the detection of D. dugon DNA in environmental samples. The assay was validated both in silico and in vitro using non-target sympatric species. Synthetic DNA was employed to evaluate assay sensitivity and potential inhibition effects. A total of 40 samples, including 28 water and 11 seagrass roller samples from the Saudi Arabian Red Sea coast (n=39), as well as one water sample from dugong exhibit at SeaWorld Abu Dhabi (n=1) were tested using this newly developed assay. Our results showed that the assay is specific to D. dugon and analytically sensitive under controlled conditions, with a modelled limit of detection of 2.88 copies/reaction and a limit of quantification of 66 copies/reaction. In field applications, D. dugon was detected in water samples from the aquarium and two field sites, while no amplification was observed in the seagrass roller samples. This qPCR assay enables targeted detection of D. dugon , which will improve eDNA biomonitoring and conservation efforts by enhancing understanding of the species’ distribution.