Assessing the Use of Molecular Barcoding and qPCR for Investigating the Ecology of Prorocentrum minimum (Dinophyceae), a Harmful Algal Species.

Kate Mclennan,Rendy Ruvindy,Martin Ostrowski,Shauna Murray

doi:10.3390/microorganisms9030510

Abstract

Prorocentrum minimum is a species of marine dinoflagellate that occurs worldwide and can be responsible for harmful algal blooms (HABs). Some studies have reported it to produce tetrodotoxin; however, results have been inconsistent. qPCR and molecular barcoding (amplicon sequencing) using high-throughput sequencing have been increasingly applied to quantify HAB species for ecological analyses and monitoring. Here, we isolated a strain of P. minimum from eastern Australian waters, where it commonly occurs, and developed and validated a qPCR assay for this species based on a region of ITS rRNA in relation to abundance estimates from the cultured strain as determined using light microscopy. We used this tool to quantify and examine ecological drivers of P. minimum in Botany Bay, an estuary in southeast Australia, for over ~14 months in 2016–2017. We compared abundance estimates using qPCR with those obtained using molecular barcoding based on an 18S rRNA amplicon. There was a significant correlation between the abundance estimates from amplicon sequencing and qPCR, but the estimates from light microscopy were not significantly correlated, likely due to the counting method applied. Using amplicon sequencing, ~600 unique actual sequence variants (ASVs) were found, much larger than the known phytoplankton diversity from this region. P. minimum abundance in Botany Bay was found to be significantly associated with lower salinities and higher dissolved CO2 levels.

Highlights

A sensitive, specific, and efficient P. minimum quantitative polymerase chain reaction (qPCR) assay was successfully developed and will allow for high-throughput information to be collected on the distribution and abundance of this species
P. minimum was found to be significantly correlated to total CO2 and to a decrease in salinity at the sites in Botany Bay
Further field and laboratory studies may be useful to determine more detailed information on the environmental variables associated with blooms of P. minimum in southeast Australia

Summary

Introduction

There has been an apparent global increase in the range, intensity, and frequency of harmful algal blooms (HABs) linked to a variety of factors, including range expansions, increases in anthropogenic nutrients into coastal water bodies, and increased aquaculture [1,2,3,4,5,6]. P. minimum blooms are most common in eutrophic coastal waters of the northern hemisphere; they have been reported in tropical and subtropical regions globally [1,7,8,9,10]. Few studies have been conducted on P. minimum in Australia, it is known to occur in high abundances in some regions, with frequent blooms in the Hawkesbury River in New South

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