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

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.

Keywords: Prorocentrum minimum; harmful algae; next-generation sequencing.

Figure 1

Map of Botany Bay in southeast Australia, highlighting the two sampling points used for the Marine Microbes project, Bare Island and Towra Point.

Figure 2

(a) Amplification plot showing dilution series using P. minimum culture DNA using primer set 20 and (b) DNA standard curve with R² value = 0.997 and E = 101%. (c) Amplification plot showing dilution series using P. minimum gBlocks using primer set 20 and (d) gBlock standard curve with R² value = 0.991 and E = 99.3%.

Figure 3

Towra Point P. minimum cell counts L⁻¹ with amplicon sequencing, qPCR, and microscope counts across the BPA sampling period. Standard error bars are included for qPCR (too small to detect) and count data.

Figure 4

Bare Island P. minimum cell counts L⁻¹ with amplicon sequencing and qPCR across the BPA sampling period. Standard error bars are included for qPCR (too small to detect).

Figure 5

Relative abundance of the top 50 phytoplankton spp. at the Bare Island and Towra Point time series stations from April 2016 to June 2017. ASVs were assigned to phytoplankton species in the PR2.12 database. For clarity, only the top 50 by ASV abundance are shown. The less abundant taxa were lumped into the category “Other,” while those without taxa assignments to the genus level were omitted.