Green autofluorescence in dinoflagellates, diatoms, and other microalgae and its implications for vital staining and morphological studies

Abstract

Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.

FIG. 1.

Micrographs of GAF observed in microalgae, obtained using an epifluorescence microscope installed with filter cubes U-MNB (for chlorophyll RAF and green to orange autofluorescence) and U-N31001 (for GAF only). Unless noted otherwise, all images are of live cells. (a to g, m) Gymnodinium catenatum vegetative cells under bright field (a; arrow indicates nucleus), the same cells exhibiting RAF (b), the same cells exhibiting GAF (c; the arrow indicates eyespot), cyst under bright field (d), the same cyst observed with U-MNB filter (e), GAF of the cyst (f; the arrow indicates the lipid accumulation body), a vegetative cell indicating stronger GAF in eyespot and accumulation bodies (g; arrows), GAF of vegetative cells in a long chain after fixation with formaldehyde (m). (h to k) Corethron hystrix GAF (h; note the absence of GAF in the nucleus, indicated by the arrow), a cell observed under bright field (i; the arrow indicates an unidentified endosymbiont microalga in the C. hystrix cell), RAF (j), or GAF (k). (l) GAF of Takayama acrochocha. (n to p) Akashiwo sanguinea cell observed under bright field (n; the arrow indicates the nucleus), GAF (o; the arrow indicates the eyespot), or GAF of the same cell after breakage (p; the arrow indicates the absence of GAF in the nucleus). (q) GAF of a colony of Woronichinia sp. (the arrow indicates the aerotopes). (r) GAF of Pediastrum sp. (s) GAF of Desmodesmus sp. (the arrow indicates the spines). (t) GAF of a cyst from ballast tank sediment stored at 4°C for about 4 years. (u) GAF of Prorocentrum micans (note the absence of GAF in the spine and plates). Bars: 20 μm (a to k, m to p, t, and u) or 10 μm (l and q to s).

FIG. 2.

Relative intensity of GAF in stained, live, or formaldehyde-fixed cells of (A) Gymnodinium catenatum and (B) Corethron hystrix. CMFDA and SYTOX green stains were applied to live and dead cells, respectively. The relative intensity of chlorophyll-induced RAF in live G. catenatum cells is shown for comparison. Bars represent means ± 1 standard deviation for n = 10 to 35 cells. Corresponding data are presented in Table 1. AU, arbitrary units.

FIG. 3.

Temporal dynamics in the relative intensity of GAF and chlorophyll-induced RAF in (A) Gymnodinium catenatum (vegetative cells) and (B) Corethron hystrix. Individual cells were exposed continuously to blue light (470 to 490 nm; filter cube U-MNB). From 40 (C. hystrix) or 45 (G. catenatum) spectra evenly spaced over 10 (C. hystrix) or 15 (G. catenatum) minutes, 12 for each are shown here in chronological order (numbers indicate the order of measurements). These spectra are representative of those observed emanating from other cells (for G. catenatum, n = 5 cells; for C. hystrix, n = 4 cells). AU, arbitrary units.

FIG. 4.

For an individual cell of G. catenatum, the time course of additional GAF elicited by exposure to UV light (330 to 385 nm; filter cube U-MWU) is shown. Prior to all but the first measurement, the cell was exposed for 5 seconds to UV light and then to blue light (470 to 490 nm; filter cube U-N31001) while the intensity of GAF was determined. Within 1 to 2 min, the next exposure to UV light and subsequent measurement of GAF was begun. The first measurement, however, represents the relative intensity of GAF elicited by blue light alone. Compare these values with those shown in Fig. 3A. The time course shown here is representative of those seen in other cells of this species (G. catenatum; n = 4 cells) and another species (C. hystrix; n = 4 cells). AU, arbitrary units.