An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve

Abstract

The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples.

Nitrate addition has minimal short‐term impacts on Greenland ice sheet supraglacial prokaryotes

“…We note, however, that no steps were taken to reduce or quantify the amplification of extracellular DNA (e.g. Nielsen et al, 2007;Kim et al, 2016); therefore our values may overestimate abundance. The lack of response to nitrate addition indicates that the majority of the communities were either not nitrate limited, or they are co-limited by nitrate and another nutrient.…”

Meltwater export of prokaryotic cells from the Greenland ice sheet

“…2a), and we therefore believe our reported concentrations to be representative estimates. We however note that beyond the filtration of samples through 0.22 mm polyethersulfone filters (Liang and Keeley, 2013), no steps were taken to further reduce or quantify the amplification of extracellular DNA (e.g Nielsen et al, 2007, Kim et al, 2016, therefore our values may overestimate abundance.…”

Unexpected presence of the nitrogen‐fixing symbiotic cyanobacterium UCYN‐A in Monterey Bay, California

“…Light and scanning electron microscopy observations show that the coccolithophore is non-motile (do not present flagella) and presents an haptonema that can be used for adhesion to external substrates (Hagino et al 2016), which makes plausible the idea of sinking and/or adhering to sinking substrates or sediments. Moreover, the calcified form has been recently recovered from coastal sediment samples (Kim et al 2016, no specimen identification) and external oyster-shell scrapes (Jordan and Riaux-Gobin 2019) supporting the observations of Hagino et al (2016). We have not yet verified that B. bigelowii has a benthic life stage at coastal MB, but we hypothesize that after entering in the calcified stage (suspected to take place in late autumn in these waters) it sinks to the seafloor and is no longer detectable in the water column until the motile phase reappears in late spring/summer.…”