The armoured dinoflagellate Alexandrium can be found throughout many of the world’s temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant threat to animal and environmental health. Moreover, the consumption of bivalve molluscs contaminated with saxitoxin poses a threat to human health. The identification of Alexandrium cells collected from sea water samples using light microscopy can provide early warnings of a toxic event, giving harvesters and competent authorities time to implement measures that safeguard consumers. However, this method cannot reliably resolve Alexandrium to a species level and, therefore, is unable to differentiate between toxic and non-toxic variants. The assay outlined in this study uses a quick recombinase polymerase amplification and nanopore sequencing method to first target and amplify a 500 bp fragment of the ribosomal RNA large subunit and then sequence the amplicon so that individual species from the Alexandrium genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different Alexandrium species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of A. minutum in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the A. catenella, A. minutum, A. tamutum, A. tamarense, A. pacificum, and A. ostenfeldii species from environmental samples, with just the alignment of the reads being sufficient to provide accurate, real-time species identification. By using sequencing data to qualify when the toxic A. catenella species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 (p ≤ 0.05). Furthermore, a McNemar’s paired test performed on qualitative data highlighted no statistical differences between samples confirmed positive or negative for toxic species of Alexandrium by both phylogenetic analysis and real time alignment with the presence or absence of toxins in shellfish. The assay was designed to be deployed in the field for the purposes of in situ testing, which required the development of custom tools and state-of-the-art automation. The assay is rapid and resilient to matrix inhibition, making it suitable as a potential alternative detection method or a complementary one, especially when applying regulatory controls.

High rates of mortality of the common cockle, Cerastoderma edule, have occurred in the Wash Estuary, UK, since 2008. A previous study linked the mortalities to a novel genotype of Marteilia cocosarum, with a strong correlation between cockle moribundity and the presence of M. cocosarum. Here, we characterize a novel iridovirus, identified by chance during metagenomic sequencing of a gradient purification of Marteilia cells, with the presence also correlated to cockle moribundity. The novel 179,695 bp iridovirus, bivalve iridovirus 1 (BiIV1), encodes 193 predicted ORFs and has a G+C content of 41 mol%. BiIV1 clusters together with other aquatic invertebrate iridoviruses in phylogenetic analyses and has a similar genome size to other invertebrate iridoviruses. Comparative analysis revealed that BiIV1 has lost three genes that were previously thought to be common amongst all iridoviruses but has also gained genes, potentially from horizontal transfer from its bivalve mollusc host(s). Electron microscopy showed 158 nm icosahedral virions present in the haemocytes of cockles, typical of those observed in host tissues infected with viruses of the family Iridoviridae. Prevalence of BiIV1 in moribund cockles was higher than that in apparently healthy cockles at most sites in the Wash Estuary, with up to 100% PCR prevalence in moribund cockles. Our findings provide the first genome for a bivalve-infecting iridovirus and identify a second bivalve-associated iridovirus in publicly available genomic datasets, adding to the knowledge of invertebrate iridovirus genomics and diversity.

Globally, Marteilia parasites have been associated with significant mass mortality events in populations of commercially important bivalve molluscs, frequently resulting in large-scale fishery collapses and substantial socio-economic impacts. The Wash Estuary, UK, supports several bivalve fisheries, and among these, common cockles Cerastoderma edule have suffered unusually high mortalities since 2008. We investigate potential causes of these mortalities, and confirm infection with Marteilia cocosarum , strongly associated with cockle moribundity, also confirming its presence in archived samples collected in 2009. Molecular and light microscopy screening of samples collected during mortality events in 2021, including healthy (buried) and moribund (weak, unable to bury) cockles, indicated high prevalence of M. cocosarum in moribund cockles (PCR incidence up to 95%) in contrast to healthy cockles (up to 42%), suggesting an association between cockle moribundity and Marteilia infection. Analysis of the full ribosomal RNA array identified consistently different nucleotides between M. cocosarum infections in The Wash (denoted as genotype WE) and those in Wales (denoted genotype BI). 83% of infections in The Wash could be identified as M. cocosarum WE and 12% as M. cocosarum BI, with both genotypes recovered from 5% of infected animals. Histopathologically, M. cocosarum WE infects the gill, mantle and connective tissues, identical to observations of M. cocosarum infecting Welsh cockles. Ongoing cockle mortalities in The Wash raise concerns regarding the sustainability of this resource ecologically and economically. Additional measures may be required to reduce the spread of this pathogen, noting that its distribution beyond The Wash and Wales is currently unknown.