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.

Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors (tlr2, tlr5, tlr7, and tlr13), urokinase plasminogen activator surface receptor-like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1. Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1-like, macrophage mannose receptor-1, crem, il-10, and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.

The Isles of Scilly are an archipelago of islands in the far southwest of the UK which contain numerous beds of wild bivalve molluscs which are recreationally harvested for local consumption. However, the islands have never previously been assessed for the presence of harmful algae and their shellfish toxin metabolites which can cause serious human health impacts. This study sought to address these knowledge gaps through the analysis of seawater and shellfish tissues for microalgae and toxins utilizing portable and lab-based microscopy, nanopore sequencing, chemical analysis and immunoassay kits. The study design was affected by the national COVID-19 lockdown which enforced implementation of citizen-led sampling and in-field microscopy. Microscopy and sequencing approaches led to the confirmation of multiple HAB species of concern, including those potentially responsible for production of neurotoxic and diarrhetic shellfish toxins. A portable microscope was successfully utilized in the field for recognition of microalgae and for early warning of potential shellfish toxicity events. Chemical analysis of cockle, clam and mussel samples confirmed the detection of paralytic, diarrhetic and amnesic shellfish toxins, with an unusual okadaic acid group toxin profile reaching a maximum toxicity of approximately half the regulatory limit as defined by EU law. The Sensoreal Alert Lateral Flow Assay was used to screen and highlight samples containing higher concentrations of DSP toxins. Furthermore, Tetrodotoxin was detected for the first time in the UK in cockle and grooved carpet shells. Multiple saxitoxin analogues were also detected in two echinoderm species, with this providing the first ever report of paralytic shellfish toxins in the spiny starfish, Marthasterias glacialis. The toxin profiles in the two species varied significantly with a dominance of GTX4 in Luidia ciliaris as opposed to a dominance of STX in Marthasterias glacialis. Overall, the study showed that a multi-method assessment of a previously unexplored region within the UK territory contained microalgae and toxins of concern to human health, and that a citizen-led programme could be instigated using portable microscopy and rapid toxin testing to assess the early warning for potentially harmful microalgae and toxins in the region, with confirmatory analysis being conducted to establish actual levels of risk for local consumers of seafood.

Cyanobacteria are microorganisms that can produce natural toxins with significant impacts on human, animal and ecosystem health. Portishead Marine Lake is a small freshwater lake in southwest England frequented by thousands of people each year for recreational activities but was associated periodically with prolific cyanobacterial blooms with potential risk to visitor health. Water samples were tested weekly between January and December 2016 for cyanobacteria and cyanotoxins. Citizen scientists undertook testing for nutrients, dissolved oxygen, pH, temperature and meteorological parameters. Four major blooms of Microcystis spp. were observed in the warmer months between May and September and were associated with high concentrations of microcystins. Complex toxin congener profiles were dominated by MC-LR and MC-RR, with lower contributions from MC-WR and MC-YR. Cyanobacterial cell densities and summed microcystin concentrations ranged from 1600 to 5.6 × 10⁶ cells/mL and 0.1 to 18,069 µg/L, respectively, and correlated significantly. Toxin concentrations were positively correlated with nitrate levels and inversely correlated with pH, although mean pH levels did not fall below 7.0. There was little statistical evidence for influence of weather conditions overall, although significant intra-lake differences in cell densities and toxin concentrations were noted. Intervention measures employed by local authorities included the use of barley straw, hydrological manipulation, debris removal and ultrasound treatment. Whilst no statistical assessment of intervention effectiveness was possible without effective control samples, there was some limited evidence for the temporary reduction in toxin levels after hydrological intervention, but none following installation of ultrasound. Overall, the study utilized LC-MS/MS and microscopy to quantify microcystins and Microcystis spp., respectively, and evidenced risks to lake visitors and the importance of toxin testing in recreational waters and the use of appropriate risk mitigation approaches. Further work incorporating appropriate experimental controls would be needed to determine the effectiveness of hazard mitigation measures for reducing or eliminating human health threats.