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.

A deep dive into the ecology of Gamay (Botany Bay, Australia): current knowledge and future priorities for this highly modified coastal waterway

“…This research has also uncovered strong links between microbiota and host kelp health (Marzinelli et al 2015;Qiu et al 2019). However, it should be noted that much of this research has focused on bacteria and archaea, and so we still have only limited understanding of the role of other microorganisms such as fungi, microalgae and viruses in these systems (but see Ferrari et al 2021;McLennan et al 2021).…”

“…In comparison to Gamay's pelagic macrofauna, the microbiota of these habitats are less well documented. Planktonic microbe communities in Gamay are highly dynamic both temporally and spatially (McLennan et al 2021), with seasonality being a strong driver of this variability. For example, the 'red tide'-forming species Noctiluca scintillans is highly abundant in Gamay in spring and late summer, forming visible water discolouration at times, and is present only at low abundances in winter (Murray and Suthers 1999).…”

“…For example, the 'red tide'-forming species Noctiluca scintillans is highly abundant in Gamay in spring and late summer, forming visible water discolouration at times, and is present only at low abundances in winter (Murray and Suthers 1999). Riverine inflow, land runoff, and other processes influence phytoplankton communities in an episodic manner, as seen by the periodic blooms of the dinoflagellate Prorocentrum minimum, which was significantly correlated with decreased salinity and water-column CO 2 (McLennan et al 2021). Multiple species of potentially harmful phytoplankton taxa have been documented at Woolooware Bay, close to oyster production leases (Ajani et al 2013).…”

“…Multiple species of potentially harmful phytoplankton taxa have been documented at Woolooware Bay, close to oyster production leases (Ajani et al 2013). Species producing harmful toxins are also known to appear 10-30 times per year at detectable abundances (Ajani et al 2013) and high biomass blooms of other phytoplankton species (Murray and Suthers 1999;McLennan et al 2021) are also common. Blooms of Alexandrium pacificum (as A. catenella), which produce the paralytic shellfish toxin (PST), occur regularly in Gamay (Murray et al 2011).…”

“…The extensively modified nature and well-connectedness of Gamay also makes it highly susceptible to pathogens and disease (Glasby and Lobb 2008). Harmful algal blooms are known to occur in Gamay and have the potential to poison seafood or cause fish and invertebrate mortalities (Murray and Suthers 1999;Ajani et al 2013;McLennan et al 2021); however, we have little information on which to assess their ongoing threat. Last, the bay is the focus of intensive recreational fishing activity with anecdotal suggestions of large changes to the assemblage of catch over the past 20 years.…”