Characterizing seasonal persistence of low levels of Pseudo-nitzschia australis in the Gulf of Maine with environmental DNA

The potent neurotoxin domoic acid is produced by some species of diatoms in the genus Pseudo-nitzschia, which can lead to amnesic shellfish poisoning (ASP) events in marine food webs. Of the toxin-producing Pseudo-nitzschia, P. australis has caused the most toxic and widespread events, making it a species of concern. Toxic and non-toxic Pseudo-nitzschia species cannot be distinguished easily by light microscopy, necessitating molecular testing. We designed a real-time quantitative PCR assay for rapid and accurate detection of P. australis in environmental DNA (eDNA) samples, targeting the intergenic region between rbcS and psaI in the chloroplast genome, providing species-level specificity and high sensitivity. Experimental determination of rbcS gene copies per cell revealed a limit of detection equivalent to 1 P. australis cell per liter of seawater, facilitating the early detection of this toxic species. This novel qPCR assay was applied to eDNA samples collected from Maine coastal waters between 2021 - 2024 to characterize the nearshore dynamics of P. australis within a regional hub for shellfish aquaculture. Though relatively rare, P. australis peaked in abundance in mid-October when conditions became favorable for this species following convective overturning. The eDNA assay was able to detect a low P. australis signal at a time when 'large' Pseudo-nitzschia spp. cell counts approached 8000 cells l^-1 (by microscopy) while qPCR-based estimates of P. australis remained below 10 cells l^-1. We demonstrate an eDNA approach for the early detection of HABs, calibration of qPCR assays to cell counts, and provide a new tool for coastal management.