An epifluorescence microscopy method for direct detection and enumeration of the fungilike marine protists, the thraustochytrids

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1 182 Notes Limnol. Oceanogr., 38(l), 1993, by the American Society of Limnology and Oceanography, Inc. An epifluorescence microscopy method for direct detection and enumeration of the fungilike marine protists, the thraustochytrids Abstract-An epifluorescence microscopy method using the fluorescent dye acriflavine is described for direct detection and enumeration of the fungilike protists, the thraustochytrids, in natural samples. The technique is based on the principles that the fluorochrome stains thraustochytrid cell walls containing sulfated polysaccharides red and the nucleus yellowgreen. Photosynthetic protists were distinguished by autofluorescence of pigments and protozoans by absence of cell-wall fluorescence. Several natural phytoplankton samples from the North Sea were examined with this technique, where up to 5.4 x 10h cells of thraustochytrids were found per gram dry weight of sample. The results suggest that thraustochytrids might be much more abundant in natural samples than previously suspected. Thraustochytrids are a common, fungilike group of protists in the sea. These eucaryotic, unicellular osmotrophs are characterized by the presence of an ectoplasmic net system and formation of biflagellate zoospores; they have been reported frequently from seawater and sediments, algae, and invertebrates both as saprotrophs and parasites. Their ubiquitousness and physiological capabilities to utilize a wide variety of complex organic substrates suggest that they play a definite role in the marine ecosystem. Most reports on detection and enumeration of these organisms have been based on cultural techniques such as the modified MPN method with pine pollen (Gaertner 1968) or membrane or glass-fiber filtration and plating on nutrient media (Bahnweg and Sparrow 1974; Raghukumar 1985). Although such techniques might yield a picture of the relative abundance and diversity of these protists in different habitats and substrata, they have dis- Acknowledgments We thank Victor Smetacek for his continuing interest in this research and also for providing a guest scientist fellowship for S.R. We also thank T. L. Tan for advice and encouragement, U. Riebesell for the aggregate material, and M. Luecker and A. Krack for technical assistance. Contribution 556 from the Alfred-Wegener-Institute for Polar and Marine Research, Bremerhaven, Germany. advantages. All species may not grow on the substrates or nutrient media currently used for isolation. A colony on an agar plate or growth in a positive MPN tube might have arisen from a clump of cells. Cells may die or multiply in numbers during the prolonged time of handling of the samples, resulting in inaccurate results upon culturing. Several replicates are needed to obtain statistically significant results, thereby conferring a demand on materials and manpower. The lack of a rapid direct detection technique has hampered a more accurate estimation of the biomass of thraustochytrids in the sea and a detailed search for these protists on various substrata. The major hurdle has been to distinguish thraustochytrids ranging from -2.0 to 20.0 pm in size from various protists in that size range in natural samples. In this note we describe results of an attempt to develop such a technique based on the principles that thraustochytrids possess cell walls made up of sulfated polysaccharides (Chamberlain 1980), that the living cells could be distinguished from detritus by staining their nuclei, and that they lack autofluorescing photosynthetic pigments. Acriflavine hydrochloride (Trypaflavin, Roth No. 9621) was used to simultaneously stain the sulfated polysaccharide cell walls and the nuclei according to the method of Pearse (1968). Staining was standardized with cultures of the following representative species: Thraustochytrium kinnei Gaertner (Isolate No. N-1709/c), Thraustochytrium multirudimentale Goldstein (N-2722/ 17), Thraustochytrium striatum Schneider (N-A II lo), Thraustochytrium pachydermum Scholz (N-267), Ulkenia visurgensis (Ulken) Gaertner (N- 5 1 /Type), and Schizochytrium aggregatum Goldstein et Belsky (N-557/b). Cultures were grown on pine pollen in seawater (Gaertner 1968) and a liquid medium of the following composition at ph : glucose, 1.0 g; peptone, 1.0 g; yeast extract, 2.0 g; disodium hydrogen phosphate,

2 Notes g; ferric chloride, g; seawater, 1,000 ml. Cells were harvested from l-week-old cultures by filtering onto a sterile 0.45pm Sartorius membrane filter and fixed in 3.7% formaldehyde in seawater and Lugol s iodine for at least 1 week. Staining was carried out by collecting the fixed thraustochytrid cells on a 0.22-pm Nuclepore filter. The material was rinsed with filter-sterilized seawater, and 3-4 ml of 0.05% acriflavine in 0.1 M citrate buffer at ph 3.0 (prepared from a 0.5% stock solution of acriflavine in distilled water) was added. The stain was vacuum drained after 4 min. Differentiation was carried out for 1 min with 75% isopropyl alcohol. The material was then rinsed with sterile distilled water after draining the isopropanol. The filter was placed on a microscope slide, a drop of water placed over it, and a cover slip added prior to epifluorescence microscopy. Some of the preparations were poststained with 0.025% of the optical brightener Calcofluor White (Sigma F 6259) (Gahan 1984) for 1 min before mounting. Whenever a combination of epifluorescence and transmitted light microscopy was deemed necessary, the material was scraped from the filter with a blunt spatula and mounted in a drop of water. Epifluorescence microscopy was carried out with a Leitz Aristoplan microscope, preferably with a nm violet-to-blue (Leitz Filterblock H3) or a nm blue excitation (Leitz Filterblock I 2/3) filter mounted in a dichroic prism, together with the barrier filter LP 515. Various protists were examined as controls to verify the specificity of the staining technique to thraustochytrids. Presence of autofluorescence in photosynthetic protists was checked under blue and UV excitation. All detections and enumerations were subsequently carried out with a prism holder containing the UV and violet-blue excitation filters. Phytoplankton and detritus samples were collected from the North Sea with an Apstein net of 100~pm-mesh size (Hydro-Bios No ). Total volumes of 10 liters were filtered, made up to 100 ml, and fixed in 3.7% formaldehyde in seawater to yield concentrations of 100 : 1. Following the technique of Velji and Albright (1986), we suspended small subsamples of such samples in 100 ml of 0.1 M tetra-sodium diphosphate (pyrophosphate) for 30 min, followed by ultrasonication at 30 W for 1 min with a Bransonic 250 ultrasonicator. Triplicate subsamples of this homogenate were used for staining and another triplicate for dry weight determinations. Relative to the volume used for staining over the filter area, the total area of grids counted, and the corresponding dry weight of the samples, we determined the number of thraustochytrids per unit weight. Bacteria were counted similarly with the acridine orange direct count method. Vegetative cells of all the thraustochytrids revealed distinct orange-to-red fluorescing cell walls and yellow-to-green fluorescing nuclei (Fig. l A-D). However, zoospores and very young, small vegetative cells of -3-pm size, apparently representing freshly encysted zoospores, did not reveal a fluorescing cell wall, but only green fluorescing nuclei (Fig. IA). There was no difference between cells grown in the two different media, namely pine pollenseawater and nutrient broth. Poststaining with Calcofluor White significantly increased the staining intensity. Fresh, unfixed material showed rapid quenching of the stain. Lugol s iodine-preserved samples cannot be used, because the cell walls do not take up the acriflavine stain. Fixation with Formalin in seawater yielded good results. The following protists, stained with acriflavine and examined under violet-blue excitation, could be differentiated from thraustochytrid cells. l Naked amoebae, flagellates and ciliates which were present in decomposing or aggregating samples of the diatom Thalassiosira nordenskiiildii Cleve and Odontella sp. and also in formaldehyde-preserved phytoplankton net samples from the North Sea proved negative for a red-fluorescing cell wall. Cells of the above diatoms as well as those in natural samples occasionally revealed a thin layer of reddish fluorescence of the cell wall but could be distinguished from the thraustochytrids by their shape and morphology. Besides, the diatoms revealed a red autofluorescence of their cell contents under violet-blue excitation which was absent in thraustochytrids. l Cells of the photosynthetic protists, namely the prymnesiophyte Phaeocystis sp., and fresh, 6-d-old Dunaliella sp. from cultures and Ceratium sp. in natural samples proved negative

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4 Notes 185 for red cell-wall fluorescence after acriflavine staining. Cells of Dunaliella sp. from 15-d-old cultures revealed an overall orange-red fluorescence. Cell walls of spores of an unidentified red alga obtained from contaminated algal culture tanks as well as outer mucous sheaths of an unidentified filamentous cyanobacterium stained orange-red, more or less similar to thraustochytrid cell walls (Fig. 1J). Thus, in Dunaliella sp., the red alga, and the cyanobacterium, red autofluorescence of cell contents could not be distinguished from acriflavine-stained cell-wall fluorescence under violet-blue excitation, and the cells vaguely resembled those of thraustochytrids in appearance under the epifluorescence microscope. In such cases, the material was examined under UV excitation. The latter clearly revealed autofluorescence of the photosynthetic pigments in these cells (Fig. lj), which could be more clearly distinguished from the acriflavinestained red cell-wall fluorescence. Such an autofluorescence was absent in thraustochytrids. l Detritus in natural samples often stained orange to red, similar to the thraustochytrid cell wall (Fig. le,g). However, thraustochytrids could be distinguished amidst detritus by virtue of their green-fluorescing nuclei; their cell walls also fluoresced more intensely than detritus. Six samples of net phytoplankton and detritus from the North Sea were examined with the above staining technique and thraustochytrids were enumerated (Table 1; Fig. le,g,h). Presence of thraustochytrids in each sample was verified by Nomarski interference contrast microscopy (Fig. lf,i). Cells of thraustochytrids in these samples were globose to subglobose and ranged from 3.5 to 19.7 pm in diameter; cells were typically granular (Fig. 1F). The cells occurred mostly in the detrital matrix and rarely on diatom or dinoflagellate cells themselves (Fig. le-i). No ectoplasmic nets could be detected in any of these cells. Table 1. Numbers of thraustochytrids and bacteria per gram dry weight of phytoplankton sample from 2 m deep at six locations in the North Sea, July Sample July Location l/ N, 08 OO E 2/ N, E 3/ N, E 4/ N, E 5/A N, E 6/B N, E Thraustochytrids Bacteria 1.4x lo6 5.6~ lo8 5.4 X X x x x x x lo5 5.0~ lo9 0.9 x x lolo Acriflavine (3,6-diamino- 1 O-methyl acridinium chloride mixed with 3,6-acridine diamine) is a cationic acridine dye (Windholz 1983) and has been used as a fluorescent stain for DNA (Gahan 1984). It is also used to stain sulfated mucopolysaccharides and sulfolipids (Pearse 1968), whereby a yellow fluorescence is produced under UV light. In this study, a distinct orange-to-red fluorescence of the fluorochrome-stained thraustochytrid cell walls was observed under violet-to-blue excitation as well as with UV. Haas ( 1982) used proflavine (3,6-acridine diamine) for staining flagellates and other eucaryotic nonphotosynthetic organisms. He described only a green fluorescence of eucaryotic heterotrophic pro- tists, due to the staining of the nucleus. Problems in microscopic detection of the typically unicellular, hyaline, and globose to subglobose thraustochytrids, ranging from -2.0 to 20.0 pm in size in natural samples, could be caused by certain protists. These protists include heterotrophic eucaryotes such as flagellates, ciliates, and amoebae, and photosynthetic organisms such as cyanobacteria, phytoflagellates, dinoflagellates, diatoms, coccolithophorids, and unicellular chlorophytes. Using the acriflavine direct detection (AfDD) technique described here, we can distinguish the protozoans by the absence of cell-wall fluorescence as was also observed by Haas ( 1982). We have not examined a sufficient number of c Note absence of cell-wall fluorescence in smaller cells in panel A. C, D. Cells of Thraustochytrium striatum. E. Phytoplankton-detritus sample (sample 2/6) from the North Sea, revealing a large number of thraustochytrid cells. F. Same as panel E, viewed in Nomarski interference contrast microscopy. G. Phytoplankton-detritus sample (sample 6/B) from the North Sea stained with acriflavine, revealing thraustochytrids. H. Acriflavine-stained cell with multilayered wall. I. Same as panel H, under Nomarski interference contrast. J. Spores of an unidentified red alga under UV excitation. Bar in panel F applies to all panels and equals 20 pm.

5 186 Notes Cell walls fluorescing red Cell walls not fluorescing red under violet-blue under violet-blue excitation excitation 4l 4l Cell contents fluorescing Cell contents fluorescing Cell contents fluorescing Cell contents fluorescing blue-green under orange-red under orange-red under blue-green under violet-blue excitation violet-blue and/or violet-blue and/or violet-blue excitation UV excitation UV excitation THRAUSTOCHYTRIDS PHOTOSYNTHETIC PROTISTS PHOTOSYNTHETIC PROTISTS NAKED HETEROTROPHIC PROTISTS Fin. 2. Recommended scheme for distinguishing thraustochytrids from other protists with the acriflavine direct detection (AfDD) method. specimens with respect to thecate protozoans. However, choanoflagellates stained exactly the same as thraustochytrids, but could be distinguished by their shape. Among photosynthetic protists, many, including diatoms and dinoflagellates, can be distinguished by morphology. Sulfated polysaccharides form a component of cell walls of certain algae (McCandless 198 1). Such organisms might yield a cell-wall fluorescence similar to thraustochytrids, but can be distinguished by their orange-to-red cytoplasmic autofluorescence (Fig. 1 J). The conventional blue-light excitation for autofluorescence (Booth 1987) may not always differentiate this from acriflavine cell-wall fluorescence. Acriflavine may even mask chlorophyll autofluorescence under blue light (Booth 1987). Therefore, we recommend the use of UV besides the blue-light excitation to differentiate autofluorescence in samples stained with acriflavine (Fig. 1E). We also suggest that fixed natural samples be examined early enough to avoid loss of autofluorescence of the photosynthetic protists. The recommended scheme of differentiating thraustochytrids from other protists occurring in natural samples is presented in Fig. 2. We did not encounter major problems in distinguishing thraustochytrids in natural samples examined here. Proper evaluation of this technique, however, must await wider application with various natural samples. Up to 5.4 x 1 O6 cells of thraustochytrids were estimated per gram dry weight phytoplankton sample (Table 1). No published work on enumeration of thraustochytrids in phy- toplankton detrital samples is available and comparisons can therefore be made only with data obtained by cultural techniques for seawater and sediments. Typically, a few hundred of these protists have been reported per liter of seawater and up to 73,000 for sediments (Raghukumar 1990). It is well known for bacteria that direct enumeration yields numbers several orders of magnitude higher than culturing techniques (Daley 1979). Some of the samples we observed (Fig. le,g) showed very dense colonization by thraustochytrids. We suspect that thraustochytrids might be more prevalent in marine samples than has been believed hitherto, using cultural techniques. The apparently nondescript nature of thraustochytrid cells in nature, as observed with transmitted light microscopy (Fig. lf), might have resulted in their being passed off vaguely as eucaryotic protists or protozoans by workers until now. Ectoplasmic net elements, typically found in cultures of thraustochytrids, were not detected in natural samples from the North Sea by our staining technique, possibly because they were too fine for detection. However, it is also likely that thraustochytrids do not produce them when growing in nutrientrich surroundings as in detritus and as also happens in liquid culture media. Raghukumar (1988) reported the absence of ectoplasmic net elements in U. visurgensis associated with hydroid polyps. We do not claim that this technique will stain all thraustochytrid cells. Zoospores of most species of thraustochytrids lack a cell wall (Moss 1986). These and cells with a thin wall may not be recognizable by this technique.

6 Notes 187 Phagotrophic amoeboid stages have recently R. R. Colwell [eds.], Native aquatic bacteria. Am. Sot. been reported for thraustochytrids (Raghu- Testing Mater. GAERTNER, A Eine Methode des quantitativen kumar 1992), and if they lack cell walls, they Nachweises niederer mit Pollen koederbarer Pilze im also will be indistinguishable. The AfDD Meerwasser und im Sediment. Veroeff. Inst. Meeresmethod described here, however, may result forsch. Bremerh. 3: in much better recognition and more accurate GAHAN, P. B Plant histochemistry and cytochemenumeration of these enigmatic protists in na- istry. An introduction. Academic. HAAS, L. W Improved epifluorescence microsture than hitherto. copy for observing planktonic microorganisms. Ann. Seshagiri Raghukumar Inst. Oceanogr. Paris 58: MCCANDLESS, E. L Polysaccharides in seaweeds, Karsten Schaumann p Zn C. S. Lobban and M. J. Wynne [eds.], Biology of seaweeds. Blackwell. Alfred-Wegener-Institute for Polar and Moss, S. T Biology and physiology of the Laby- Marine Research rinthulales and Thraustochytriales, p. 105-I 29. In S. Am Handelshafen 12 T. Moss [ed.], The biology of marine fungi. Cam- D-2850 Bremerhaven, Germany bridge. PEARSE, A. G. E Histochemistry. Theoretical and applied. V. 1. Churchill. RAGHUKUMAR, S Enumeration of the thrausto- References chytrids (heterotrophic microorganisms) from the BAHNWEG,G.,AND F. KSPARROW, JR Fournew Arabian Sea. Mahasagar, Bull. Natl. Inst. Oceanogr. species of Thraustochytrium from Antarctic regions, 18: with notes on the distribution of zoosporic fungi in Detection of the thraustochytrid Protist the Antarctic marine ecosystems. Am. J. Bot. 61: 754- Ulkenia visurgensis in hydroids using immunofluo rescence. Mar. Biol. 97: BOOTH, B. C The use of autofluorescence for an Speculations on niches occupied by fungi alyzing oceanic phytoplankton. Bot. Mar. 30: 10 l- in the sea with relation to bacteria. Proc. Indian Acad Sci. (Plant Sci.) 100: CHAMBERLAIN, A. H. L Cytochemical studies on Bacterivory: A novel dual role for thrausthe cell walls of Thruustochytrium spp. Bot. Mar. 23: tochytrid fungi in the sea. Mar. Biol. 113: VELJI, M. I., AND L. J. ALBRIGHT Microscopic DALEY, R. J Direct epifluorescence enumeration enumeration of attached marine bacteria of seawater, of native aquatic bacteria: Uses, limitations and com- marine sediment, fecal matter, and kelp blade samples parative accuracy, p In J. W. Costerton and following pyrophosphate and ultrasonic treatments. Can. J. Microbial. 32: WINDHOLZ, M The Merck index. Merck & Co., Inc. Present address: Biological Oceanography Division, National Institute of Oceanography, Dona Paula, Goa , India. Submitted: 28 February 1992 Accepted: 7 May 1992 Revised: 31 August 1992