Quantification of Diatom and Dinoflagellate Biomasses in Coastal Marine Seawater Samples by Real-Time PCR

Abstract

Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.Several diverse taxa are represented in the phytoplankton communities of coastal marine environments. These contribute to primary production and form the base of the marine food chain. Annual variation in primary production is caused by external factors such as nutrient access, light, and temperature (18). Seasonal patterns-including changes in phytoplankton diversity, community composition, and biovolumesalso affect the magnitude of primary production. Additionally, short-term fluctuations within the phytoplankton community are common and are due to the dynamics of local hydrographic conditions (1), zooplankton grazing (48), and exchange between sediment and the water column (6, 27). Thus, the structure of the phytoplankton community is influenced by several biotic and abiotic factors, and the taxonomic composition will, in turn, affect other functions of the marine ecosystem.Two of the most prominent and important phytoplankton classes in coastal marine waters and freshwater bodies are diatoms, Bacillariophyceae, and dinoflagellates, Dinophyceae. The taxonomic class Bacillariophyceae comprises approximately 100,000 extant species; planktonic species are predominantly autotrophic, and cell sizes range from 2 m to 5 mm (37). The number of dinoflagellate species in the marine phytoplankton is approximately 2,000, of which 50% are heterotrophic and the rest are auto-or mixotrophic. Their cell sizes range from 2 m to 2 mm (45). The variability within these classes is vast in many respects, but genetic, phy...

Seasonal and Interannual Changes in Ciliate and Dinoflagellate Species Assemblages in the Arctic Ocean (Amundsen Gulf, Beaufort Sea, Canada)

“…Ciliates, can also be fragile and tend to be either ignored or under sampled using standard Lugol's techniques (Lovejoy et al, 1993). Ciliate and dinoflagellate dominance in 18S rRNA gene libraries has also been attributed to their comparatively larger genomes and multiple copies of the rRNA gene (Godhe et al, 2008). Irrespective of the actual cell abundance, our analysis clearly revealed otherwise undetected changes in species over seasons (Supplementary Tables 4 and 5).…”

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

“…Interestingly, both Pearson and Spearman rank correlations were typically strongest for the nuclear 18S rDNA marker. Many studies have shown strong correlations between biovolume and 18S copy number for a range of eukaryotic taxa, including metazoans (Godhe et al., 2008; de Vargas et al., 2015; Zhu, Massana, Not, Marie, & Vaulot, 2005). Several factors could reduce the strength of the correlation between biomass and HTS reads in this study, including using separate samples for DNA‐based and morphology‐based identification, and estimating biomass using conversion factors rather than direct measurement.…”

Bioassessment of a Drinking Water Reservoir Using Plankton: High Throughput Sequencing vs. Traditional Morphological Method

“…Evans et al (2007) [54] assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model "morphospecies", Sellaphora pupula agg., and found that tree topologies were very similar although support values were generally lower for cox1. To assess the proportional biomass of diatoms and dinoflagellates along the Swedish west marine coast, Godhe et al (2008) [27] designed two real-time PCR assays with special primers to find that the linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. Thus, for diatoms, linear regression of the number of small subunit ribosomal DNA (SSU rDNA) copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates.…”

“…The method is faster in terms of sampling processing, and may become cheaper as the technology improves, and more importantly with the ability to provide more reliable and richer biological information than the traditional morphology-based method [22][23][24]. Different plankton assemblages as bioindicators were characterized using DNA sequencing including bacterioplankton [18,[24][25][26], and phytoplankton [27][28][29][30][31][32][33]. Baird and Hajibabaei (2012) [17] envisioned a new paradigm (i.e., Biomonitoring 2.0) in ecosystem assessment based on a HTS platform, though a complete paradigm shift may require more research.…”