CELLULAR DNA CONTENT OF MARINE PHYTOPLANKTON USING TWO NEW FLUOROCHROMES: TAXONOMIC AND ECOLOGICAL IMPLICATIONS¹

Abstract

Two new fluorochromes, PicoGreenm and SYTOX Green@ stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument con.iguration of modern lozupower Pow cytomters (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains @om 12 taxonomic classes) examined. The major advantages of these dyes over others are 1) suitability for direct use in seawater, 2) green fluorescence emission of the DNA-dye complex (wavelength 525 2 15 nm) showing no overlap with the autojuorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence > 1 00-fold compared to the unstained cell, and 4) dyes can be used to quanta2 double-stranded DNA. The high sensitivity allowed the quanta$cation of the DNA of the smallest known phytoplankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustagmatophyceae) stained poorly, and a f e u members ofthe Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PzcoGreen dye stained some living phytoplankton species, preservation is recommended f m quantitation. SYTOX Green did not stain live cells. The combination ofthe dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Parafaaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence.Total DNA values measured in 90 algal species (ca. 121 strains) varied a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinojagellate (Prorocentrum micans). DNA content appears to be a scaleable cell component covarying with the carbon and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent estimate of total cell carbon biomass in unicellular pelagtc phytoplankton.

Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana: Cell staining, chlorophyll alterations, and dimethylsulfoniopropionate (DMSP) metabolism

“…Two of these have been described as ''live'' and ''dead'' stains; SYTOX Green can be used to measure changes in membrane permeability (Veldhuis et al 1997;''dead'' cells) and 5-chloromethylfluorescein diacetate (CMFDA) is cleaved by a variety of enzymes, indicating hydrolytic enzymatic activity (D. J. Franklin and J.…”

“…Some progress has been made in recognizing cell state in the laboratory: the morphological changes associated with nutrient limitation in batch cultures have been studied, and similarities with metazoan programmed cell death (PCD; Bidle and Falkowski 2004;Franklin et al 2006) have been described in certain phytoplankton (e.g., Dunaliella tertiolecta; Segovia and Berges 2009). An improved ability to recognize senescent, quiescent, moribund, and dead cells within microbial populations is important because a substantial fraction of natural phytoplankton biomass may be nonviable (Veldhuis et al 2001;Agustí 2004) and yet viability will be a major driver of primary production and biogeochemistry. Accurate estimation of phytoplankton primary production through remote sensing could be improved by practical recognition of different physiological states.…”

“…In general, increased concentrations of chlorophyll oxidation products have been observed in nutrient-depleted cells, but it is likely that specific chlorophyll transformation pathways vary between species (Bale 2010). Initial investigations into pigment alteration and cell viability in natural phytoplankton assemblages (using SYTOX Green staining) have used pigment fluorescence to assess chlorophyll loss (Veldhuis et al 2001). Such approaches have been useful, but miss vital information on the early alteration of chlorophyll.…”

Goose-mediated nutrient enrichment and planktonic grazer control in arctic freshwater ponds

“…TP, ortho-phosphate, ammonium, nitrate and nitrite levels were measured using a segmented Xow analyzer (Skalar, type SA 400). For Wve ponds in the surroundings of Ny-Ålesund, 100 ml was preserved with 1% gluteraldehyde; of these samples, the total bacterial number count determined by staining with pico-green, followed by Xow cytometric counting (Veldhuis et al 1997).…”

Phytoplankton growth inhibited by the toxic and bacterivorous ciliate Uronema marinum (Protozoa, Ciliophora)

“…Flow cytometer and bi-plot axes in which cell clusters were counted are listed in Table 2. Bacteria were enumerated with the same flow cytometer 15 min after adding to 400 µl of TE-buffer: 10 µl TX-100 (40 × diluted), 100 µl sample and 10 µl Pico Green™ (Invitrogen, 500× diluted) (Veldhuis et al 1997). Phytoplankton was measured using chlorophyll autofluorescence as trigger (Table 2).…”