Development of methodologies for rapid identification of harmful algal bloom causative agents

Abstract

Harmful algal blooms (HABs) caused by paralytic shellfish toxins (PST) producing dinoflagellates are highly toxic to humans, fishes and other sea creatures. Quick identification of HABs causative agents can help facilitate timely and adequate response to subsequent events brought by HABs and thus it is an effective means to reduce the risk of economic losses and food poisoning caused by PST. In this study, five global invasive toxic dinoflagellates, Alexandrium lusitanicum (CCMP1888), A. minutum (CCMP113), A. tamarense (ATCI03), Gymnodinium catenatum (CCMP1937) and Karenia mikimotoi (K1), were selected as the subjects of study. The aim of this study is to characterize these selected toxic dinoflagellates, identify their on-surface species-specific proteins and novel saxitoxins-related gene expressions by proteomic and transcriptomic approaches. Subsequently, this study also aims to develop rapid identification methodologies for toxic dinoflagellates. There are four result chapters in this study: (i) verification of the properties and identity of five selected toxic dinoflagellate species, (ii) transcriptomic assembly and analysis for the discovery of novel saxitoxins-related expressions in toxigenic dinoflagellates, which also support subsequent proteomic studies, (iii) species-specific extracellular-facing surface proteomes in toxigenic dinoflagellates, and (iv) development of laymen-friendly methodology for quick detection of harmful dinoflagellates. To begin with, the basic physical and biological characters of the five dinoflagellates were studied with respect to their morphologies, growth patterns, DNA sequences of internal transcribed spacers, and productivity of saxitoxins (STX). Transcriptomic assembly and orthogroup inference were then carried out for these species. Hence, orthologous gene expressions from different species were grouped in a non-reference-based manner to allow cross-species comparisons. Transcriptome-wide differential gene expressions as well as pathways analysis were compared in relation to the productivity of STX in these dinoflagellates. Potential correlation between the metabolic pathways upregulated and the STX biosynthesis in dinoflagellates was discussed. The constructed cDNA libraries also served as in-house databases to support the subsequent proteomic search for the discovery of useful protein biomarkers. In order to refine the scope of proteomic study for discovering cell-surface species-specific proteins, cell-surface labelling technique and novel surface protein enrichment were applied. With the aid of the state-of-the-art LC-Orbitrap-MS/MS and Mascot search engine, surface proteomes of the five dinoflagellates were successfully established. Based on the gene ontology and the results of transcriptome-guided orthogroup inference, surface proteins with significant species-specificity among the other dinoflagellates were revealed. As a proof of concept, novel methods for cell harvest and protein extraction were ameliorated for laymen-friendly on-site application. Together with the aid of proteomic supports, surrogate species-specific proteins were found and antibodies against these proteins were produced. Rapid immunodetection using the custom antibodies was successfully demonstrated for the five harmful dinoflagellates.