Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR

Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non -indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time -demanding, and thereby can hinder management efforts and restrict the "opportunity window" for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species -specific free-floating extra -cellular eDNA (free-eDNA) signals, i.e., detection of species -specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof -of -concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species -specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species -specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point -of -need detection and rapid management responses.