The planktonic marine diatom genus Pseudo-nitzschia is synonymous with the neurotoxin domoic acid (DA), which causes the syndrome amnesic shellfish poisoning (ASP). DA can bioaccumulate in shellfish during blooms of specific, toxin-producing Pseudo-nitzschia, harming wildlife and human health. Phenotypic discrimination of variants requires electron microscopic observation of the frustule, a process too time-consuming for routine monitoring, which invariably uses light microscopy. This process is less specific and has poor correlation with accumulation of DA in shellfish. The assay outlined here targets the ribosomal DNA gene, providing species resolution of Pseudo-nitzschia. It combines recombinase polymerase amplification (RPA) with nanopore sequencing and is effective on almost all known Pseudo-nitzschia species. It provides preliminary, real-time indication of environmental species composition, and further bioinformatic processing confirms results. Validation experiments correctly identified 13 reference cultures, with sufficient sensitivity (2500 cells l-1). Two environmental case studies highlighted efficacy on complex natural Pseudo-nitzschia blooms, including detection of low abundance species. The first study consisted of 37 samples collected from Scottish waters between May 2022 and August 2023, which identified 13 species. The second consisted of 10 samples from Aotearoa (New Zealand) collected February 2025, which found 17 species. Both studies identified species previously undetected in the regions. Statistical analysis of complementary toxin data from Scotland, although limited, highlighted that relative abundance of toxic species was a better predictor of toxic events than genus level cell counts. The RPA assay was also adapted to a lateral flow dipstick, providing a rapid screening tool for toxic Pseudo-nitzschia seriata and Pseudo-nitzschia australis.

Harmful Algal Blooms (HABs) are a hazard for coastal environments worldwide; identifying screening thresholds of chlorophyll-a (chl-a) associated with increased risk of HABs is a management priority. Molecular surveillance of coastal phytoplankton and bivalve biotoxins could be used to link chl-a with HAB risk, but requires an understanding of whether the HAB risks increase uniformly as chl-a rises, or whether some taxa are disproportionately favored, and if these relationships vary by season. In this study, we present a novel use of molecular abundance data to investigate the scientific bases for estuarine chl-a thresholds protective against HABs. In San Francisco Bay (SFB), California, the relationship between molecular relative abundance (as measured by 18S metabarcoding) of nine different HAB taxa, absolute quantitative polymerase chain reaction (qPCR) abundance, and mussel toxin concentrations of a subset of the taxa were investigated for thresholds as a function of increasing chl-a. Our results show most HAB taxa did not increase in absolute or relative abundance during SFB's spring bloom interval, when chl-a levels were highest (>10 µg/L) but the assemblage was dominated by non-harmful diatoms. However, several flagellated, mixotrophic taxa did increase above their molecular baseline in fall, and the combined probability of any HAB occurring above baseline was elevated when chl-a reached ∼4.6 µg/L in the fall. This work demonstrates the promise of molecular approaches in disentangling the seasonally complex interplay between stressors and phytoplankton/HAB community responses and has the potential to provide clearer, more cost-effective monitoring and mitigation strategies for managers.