Comparison of Lipid Content in Nine Dinoflagellate Species Using Flow Cytometry

Abstract

The lipid content of nine dinoflagellates was analyzed using flow cytometry to compare lipid levels. Additionally, the correlation between lipid content, cell size, and carbon content in dinoflagellates was evaluated using BODIPY 505/515 staining. The flow cytometry side scatter (SSC) effectively represented relative cell size, showing a linear relationship with the equivalent spherical diameter (ESD). Larger cells exhibited higher relative lipid content; however, lipid accumulation was influenced by nutritional modes and habitats, with mixorophic and benthic species displaying higher lipid content than heterotrophic species. A comparison of fluorescent dyes revealed that Nile Red overestimated lipid content, suggesting overlap with chlorophyll autofluorescence. Flow cytometry analysis with BODIPY 505/515 demonstrated a linear correlation with the sulfo-phospho-vanillin (SPV) method, enabling determination of actual lipid content using FL1 fluorescence and the slope value. As the carbon content increased, the lipid content initially increased rapidly but plateaued at higher carbon levels, indicating saturation. These findings suggest that relative fluorescence via flow cytometry provides an effective means to estimate the lipid content and carbon content as a function of cell size.

Keywords: carbon content; cell size; dinoflagellate; flow cytometry; lipid content.

Figure 1

Gating of dinoflagellate Prorocentrum micans to exclude non-fluorescent particles, bacteria, and detritus. (A) P. micans labelled with Nile Red and fluorescence measurements using FL2. R1 was the selected gate that contained labelled P. micans only. (B) Measurement of the gated population (R1 only).

Figure 2

Flow cytometry analysis of lipid content in experimental dinoflagellate species with BODIPY 505/515 and Side Scatter (SSC) as size factors.

Figure 3

Correlation between experimental cell size measured with a Coulter counter (X-axis, ESD) and flow cytometry (SSC: Side Scatter Detector fluorescence). R² value is 0.86.

Figure 4

Correlation between the lipid content of nine dinoflagellate species labelled with BODIPY 505/515 and the size factor. (A) Size factor measured by flow cytometry (SSC); (B) size measured by Coulter Multisizer (ESD). Open circles indicate Ostreopsis ovata and open squares indicate Prorocentrum micans. R² value is 0.87 and 0.81, respectively.

Figure 5

Lipid bodies were labeled with a lipophilic fluorescent dye under an epifluorescence microscope. (A) Heterotrophic Oxyrrhis marina labelled with BODIPY 505/515 was observed using an FITC (green emission) long-pass filter. (B) O. marina labelled with Nile Red was observed using a rhodamine (red emission) filter. (C) Prorocentrum cordatum labelled with BODIPY 505/515, observed using an FITC (green emission) long-pass filter. Orange-red fluorescence indicates chlorophyll autofluorescence. (D) P. cordatum labelled with Nile Red using a rhodamine (red emission) filter. The lipid bodies showed different colors (orange) with chlorophyll autofluorescence (red). Scale bar = 10 µm.

Figure 6

Comparison of lipid content of nine dinoflagellate species labelled with BODIPY 505/515 and Nile Red. Red and green indicate Nile Red and BODIPY 505/515 measurements, respectively. The slope of the linear correlation between the size and lipid content was steeper with Nile Red stain. Open circles indicate Oxyrrhis marina, which has no pigment (heterotrophic), and the elongated rectangle indicates Scrippsiella acuminate. R² value is 0.85 and 0.83, respectively.

Figure 7

Correlation between BODIPY 505/515 fluorescence and lipid content estimated by the sulfo-phosphor-vanillin reaction in nine dinoflagellate species (three readings per species). The actual lipid content could be estimated multiplying FL1 fluorescence by 0.0003 and adding 0.06. R² value is 0.83.

Figure 8

Correlations between BODIPY 505/515 fluorescence (FL1) and size (ESD; (A)), lipid content ([20]; (B)) and carbon content (C) of nine dinoflagellate species, and correlation between lipid content and carbon content (D). Oo = O. ovata; Am.= A. minutum; Pmc, P. micans; Sa, S. acuminata; Om, O. marina.