Abstract

Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations “one health” approach to marine monitoring.

Highlights

  • Aquatic microalgae fix carbon, release oxygen and provide a source of food for grazing organisms, and as such are essential components of the trophic web supporting healthy freshwater and marine environments

  • The following cultures were acquired from Culture Collection of Algae and Protozoa (CCAP, Oban, Scotland), Marine Biological Association (MBA), Plymouth, UK and Culture collection of Marine Protozoa (CCMP), Bigelow, Maine, United States.: Alexandrium tamarense (CCAP1119/31), Alexandrium tamutum (CCAP1119/51), Alexandrium minutum (MBA733), Alexandrium catenella∗ (CCAP1119/52), Gonyaulax spinifera (CCAP1118/2) and Lingulodinium polyedrum (CCAP1121/2), and Alexandrium fundyense (CCMP1719). ∗Alexandrium catenella and Alexandrium fundyense are accepted as the same species and catenella is the accepted name the synonym fundyense is present on a substantial amount of online material, in this paper catenella is used but A. fundyense appears where data for sequences were generated under this name, making identification of source material straight forward

  • This study examines, for the first time, the applicability of nanopore sequencing for the detection of marine eukaryotic harmful algal blooms (HABs) species

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Summary

Introduction

Release oxygen and provide a source of food for grazing organisms, and as such are essential components of the trophic web supporting healthy freshwater and marine environments. Many species of microalgae, including those that form blooms, have the potential to produce a range of toxins. These toxins bioaccumulate in bivalve shellfish, which if consumed pose a threat to human health (Bauder et al, 2001; Kwong et al, 2006). These toxins are commonly categorized by their symptomatic manifestations and include, Paralytic, Amnesic and Diarrhetic shellfish poisons or PSP, ASP and DSP respectively (Hallegraeff et al, 2004)

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