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J. Cell Set.

51, 15-23 (1981) 15

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Biology Department, University of Massachusetts at Boston, Boston, Mass. 02125,

Periodic acid-Schiff staining, acid phosphatase localization, and yellow autofluorescence
have been correlated with the PAS-body of Gonyaulax polyedra for the first time. PAS-
staining and acid phosphatase activity are both correlated with the PAS-body of Gonyaulax
tamarensis. These results suggest that the PAS-body of these marine dinoflagellate algae
functions in subcellular digestion.

It is important to know the subcellular localization of acid phosphatase activity in
algal cells. This enzyme is associated with digestive processes throughout the animal
kingdom, but there are fewer reports of subcellular localizations from plant cells, and
especially from the algae. Many of the algal organisms studied were in unusual
nutrient conditions or undergoing specific developmental changes. These include
2 species of Euglena under conditions of carbon starvation and aging (Brandes, Buetow,
Bertini & Malkoff, 1964; Malkoff & Buetow, 1964; Brandes, 1965; Sommer & Blum,
1965; Palisano & Walne, 1972; Gomez, Harris & Walne, 19740,6); 3 species of
Cryptomonas (Lucas, 1970); Polytomella caeca, a colourless heterotroph (Cooper,
Bowen & Lloyd, 1974); Viva mutabilis during gamete release and fertilization (Briten,
1975) and U.lactuca vegetative cells (Micalef, 1975); Micrasterias americana at
various stages of growth (Noguchi, 1976); aging Ectocarpus sp. (Oliveira & Bisalputra,
1977); and 2 wall-less volvocine algae, Dunaliellaprimolecta (Eyden, 1975) and Astero-
monas gracilis (Swanson & Floyd, 1979).
Cultured Gonyaulax polyedra cells possess a large membrane-bounded spherical
body, portions of which are stained by the periodic acid-Schiff (PAS) reaction.
Schmitter (1971) proposed an active digestive function for these PAS-bodies, either
in autophagy or in the use of stored metabolites, based in part on their ultrastructural
contents: aggregates of electron-dense material, fibrous areas, and membranous
vesicles. PAS-bodies are morphologically similar to the digestive granules in absorptive
cells of Hydra (Slautterback, 1967) and to the food vacuoles of the dinoflagellates
Ceratium hirundinella and Amyloodinium sp. (Dodge & Crawford, 1970; Lorn &
Lawler, 1973). They are also similar in many respects to the Corps de Maupas of
Cryptomonas reticulata (Lucas, 1970). They bear a lesser structural resemblance to the
'accumulation bodies' of symbiotic dinoflagellates (Taylor, 1968, 1971a, b\ Tomas &
16 R. E. Schmitter and A. J. Jurkiewicz
Cox, 1973; Trench, 1974), in which granular electron-dense material increases with
cell age until the contents often appear quite homogeneous in texture. Taylor (1968)
suggested that the structure is an accumulation site for waste materials and Tomas &
Cox (1973) proposed that the accumulation bodies of Peridinium balticum function in
waste storage or elimination. Lee (1977) has described accumulation bodies in free-
living colourless heterotrophic isolates of Gyrodinium lebouriae Herdman but these
resemble PAS-bodies structurally more than accumulation bodies.
In our study we found acid phosphatase activity to be localized in the PAS-bodies
of the red tide organisms, Gonyaulax polyedra and G. tamarensis. This investigation
also supports the proposal of Schmitter (1971, 1973) that PAS-bodies of G. polyedra
have a digestive function.


Growth of cells
G. polyedra and G. tamarensis were cultured in medium f/2 without silicate (Guillard &
Ryther, 1962) at 21 and 16 °C, respectively, under an alternating cycle of 12 h light-12 h dark.
G. polyedra strain GP60E was obtained from Dr R. R. L. Guillard, Woods Hole Oceanographic
Institution; G. tamarensis was isolated by Dr C. Martin, University of Massachusetts Marine
Station, Gloucester. All samples used were from cultures in the first hour of their light period
(CT 0-1) unless otherwise stated.

Acid phosphatase localization

Cells were collected by centrifugation at 80-100g in an IEC clinical centrifuge for 4 min,
then fixed with 2-5% glutaraldehyde in 005 M-sodium cacodylate buffer (pH 7-2), for 30 min
at 3-4 °C. Subsequent steps were carried out in 12-ml centrifuge tubes; centrifugation be-
tween steps was at 80-100g for 4 min. Acid phosphatase was localized within whole cells by
a modification of Trelease's method (1980) for algae. Fixed cells were rinsed 3 times in caco-
dylate buffer at room temperature before preincubating in 005 M-sodium acetate buffer
(pH 5-o) for 20 min at room temperature. The acid phosphatase medium was mixed as follows
to minimize precipitation: 0-36 M-lead nitrate was prepared using cooled, freshly boiled
distilled water. One ml of lead nitrate solution was added in 20-fi\ aliquots with gentle sirring
to n o ml of 0-05 M-sodium acetate buffer (pH 5-0) in which 0-3 g of sodium /?-glycerophosphate
had just been dissolved. The complete medium was preincubated at 37 °C for 1 h and any
precipitate removed using Whatman no. 1filterpaper. Controls consisted of incubation medium
lacking substrate (Pb1+ control) and complete incubation medium containing the enzyme
inhibitor sodium fluoride (NaF control). In the latter, sodium fluoride was added to the buffer-
substrate mixture to give a final concentration of 001 M before lead nitrate was added. Control
solutions were also preincubated and filtered. All incubations were carried out as 5 -ml volumes
in stoppered centrifuge tubes for 30 min at 37 °C with occasional gentle stirring. The ensuing
steps were at room temperature. Cells were rinsed 3 times in distilled water, soaked for 5 min
in i - o% acetic acid to remove unprecipitated lead, and rinsed again in distilled water. Sites of
lead phosphate deposition were revealed by conversion to lead sulphide using i - o% aqueous
ammonium sulphide for 10 min. Cells were rinsed thoroughly with distilled water before
mounting in glycerol for light microscopy. Some preparations were stained with acetocarmine,
so that nuclei were also clearly visible.
Cells were examined by light microscopy for acid phosphatase localization at a magnification
of x 400. Cells from several adjacent fields were scored for lead sulphide deposition within
PAS-bodiea; a minimum of 500 cells was counted from each slide. Photographs were recorded
on Kodak Plus-X-Pan or Ektachrome 200 film.
Acid phosphatase in Gonyaulax 17

Periodic acid-Schiff staining

Periodic acid-Schiff (PAS) staining was carried out as described by Grimstone & Skaer (1972);
their directions for Schiff's reagent were also used. Cells were fixed as for the acid phosphatase
studies, or at room temperature in ethanol/glacial acetic acid (3:1, v/v) for 30 min. Cells were
rinsed thoroughly after either fixation. Non-specific staining of glutaraldehyde-fixed cells was
prevented by blocking with aniline/glacial acetic acid (1 :<), v/v) for 20 min. Pellets were gently
resuspended throughout using a Pasteur pipette. Oxidation, staining, and rinsing times were
as described by Grimstone & Skaer, except that rinses were done by repeated centrifugation.
Cells were mounted in glycerol and examined for PAS staining at a magnification of x 400.

Fluorescence microscopy
Autofluorescence of the PAS-bodies was studied using either unfixed cells, or cells fixed in
the ethanol/acetic acid fixative just described. Fixed cells were rinsed in distilled water twice
and mounted in glycerol for study with a Zeissfluorescencemicroscope equipped with a high-
intensity illuminator and superpressure mercury vapor lamp HBO 200 W/4. Excitation filters
BG3 and UGi were used in conjunction with a no. 53 barrier filter. Photographic records were
made using Kodak Technical Pan Film 2415 (ESTAR-AH base) and developed for 4 min with
Kodak D-19 developer for maximum contrast. At least 500 cells were examined at a magni-
fication of x 400 in all studies. The autofluorescence is yellow.

Acid phosphatase
The highest levels of acid phosphatase activity were recorded when 2-5% glutar-
aldehyde was employed. Lower concentrations of glutaraldehyde resulted in relatively
low levels of localization in identifiable PAS-bodies. For example, after 1*5%

Table 1. Acid phosphatase localization in Gonyaulax

% Acid phosphatase in PAS-bodies

Organism (days) Experimental Pb 1+ control NaF control
G. polyedra 14 24 0 0
17 23 0 0
29 33 0 0

3° 24 0 0
35 45 0 0

G. tamarensis 21 13 0 1

glutaraldehyde fixation a maximum of 9% of cells examined showed acid phosphatase

localization in PAS-bodies; Pb 2+ controls gave up to 1% positive results. In our
hands methods other than that of Trelease (1980) resulted in a considerable degree of
lead deposition within nuclei (e.g. see Gomori, 1952; Lewis & Knight, 1977). We did
not use any substrates other than /?-glycerophosphate in our studies (see Beaufay,
Table 1 lists the results of several acid phosphatase localization experiments using
G. polyedra and one using G. tamarensis. The percentage of cells with lead sulphide
deposits in identifiable PAS-bodies is given for cultures of several ages. Figs. 4 and 7
R. E. Schmitter and A. J. Jurkicuricz
Acid phosphatase in Gonyaulax 19
show typical examples of localizations in G. polyedra; Fig. 3 gives a comparable
picture of G. tamarensis. Figs. 1 and 2 depict typical cells from NaF and Pb 2+ controls
in G. tamarensis and G. polyedra, respectively.
The data do not allow any conclusions about the relative amounts of acid phos-
phatase activity present in PAS-bodies of cells from cultures of different ages. Some
of the reasons for this are discussed below.
A portion of cells in all experimental preparations stained non-specifically. We do
not know whether this reflects intracellular disruption or some other undetermined
variable. Such ' overstaining' was never observed in the NaF or Pb 2+ controls.
Cells from the 29-day culture experiment were stained by the PAS reaction after
aniline blockage. A total of 35% of the experimental cells contained PAS-reactive
PAS-bodies, as did 35% of the Pb2+ control cells.
The reasons for the lower percentage of localization in G. tamarensis are unknown.
That experiment was done using the same incubation mixture as the 29-day G. poly-
edra culture.

Fluorescent PAS-bodies were seen in a high percentage of G.polyedra cells examined
from 3 times of day (14-day culture). Circadian times o and 7 h (light cycle), and
16 h (dark cycle) had 90, 99, and 89% yellow autofluorescent PAS-bodies,
respectively. Cells from o h showed no fluorescence at all if subjected to the PAS
reaction before viewing. A total of 98 % of these same cells had PAS-bodies when
viewed by conventional illumination. Fig. 5 depicts cells of G. polyedra as viewed by
conventional illumination; Fig. 6 shows the same cells as viewed by fluorescence
microscopy. The PAS-bodies are clearly visible as discrete sites of fluorescence
surrounded by indistinct cytoplasm.

Fig. 1. G. tamarensis; NaF control. Only 1 % of NaF control cells showed any lead
sulphide deposition. Arrow indicates girdle region of cell. Clear area within is the
nucleus. Figs. 1-5, bar 20/tm.
Fig. 2. G. polyedra; Pb l + control. None of the Pb l + control cells showed lead sulphide
deposition in any of the experiments described.
Fig. 3. G. tamarensis; acid phosphatase experimental preparation of 21-day culture.
A total of 13 % of cells had lead sulphide localized in a PAS-body. Arrows indicate
Fig. 4. G. polyedra; acid phosphatase preparation of 35-day culture. A total of 45 %
of cells had lead sulphide localized in PAS-bodies. Arrows indicate 3 deposits.
Fig. 5. G. polyedra viewed by conventional illumination after fixation with ethanol/
acetic acid.
Fig. 6. Same cells as those of Fig. 5, viewed by fluorescence microscopy; 7-5 min
photographic exposure time. Discrete sites of fluorescence (PAS-bodies) are clearly
visible, as are outlines of surrounding cytoplasm.
2O R. E. Schmitter and A. J. Jurkieioicz


Fig. 7. Two cells of G. polyedra with acid phosphatase localization in PAS-bodies.

PAS-body in cell at right shows typical compact morphology. That at upper left has
several large granules at its periphery (arrows). Bar, 10 fim.
Fig. 8. PAS-body of G. polyedra by electron microscopy. Methods were as given by
Schmitter (1971). Two membrane-bound elements (1, 2) are closely associated with
the PAS-body, and another (3) is fusing with it. These elements may correspond to
the granular localizations noted in Fig. 7. ch, chloroplast; st, starch; er, endoplasmic
reticulum. Bar, 1 fim.
Acid phosphatase in Gonyaulax 21

Results of the acid phosphatase studies show substantial localization within PAS-
bodies. These results are not completely quantitative, however. First, it is known that
glutaraldehyde inhibits a portion of acid phosphatase activity, while at the same time
preserving sufficient activity for cytochemical localization (Sabatini, Bensch &
Barrnett, 1963; Brunk & Ericsson, 1972). Swanson & Floyd (1979) determined from
in vitro studies of the /?-glycerophosphatase from Asteromonas that about 70% of the
activity of the enzyme was lost after the glutaraldehyde fixation method they employed
for cytochemistry. We have not done such a study, but the amounts of activity
localizable in G. polyedra are comparable to the amount of activity remaining in their
Second, we have not determined the pH optimum for acid phosphatase activity in
G. polyedra. If it differs very much from pH 5-0, localizable activity would be lower
than the optimum. Although we have not seen a study in which the pH optimum for
acid phosphatase activity was much higher, Miiller (1973) has described acid phos-
phatase activity from a trichomonad flagellate, with substantial activity above pH 6.
Third, because we used whole cells in our study, we cannot be certain that small
deposits of lead sulphide would be visible by light microscopy. If elements such as
those associated with the PAS-body in Fig. 8 (labelled 1, 2, 3) contained activity and
the body-proper did not, we would not have been able to resolve them. Similarly, the
contents of PAS-bodies as seen by electron microscopy are not homogeneous. Acid
phosphatase activity might be present only in certain portions at any given time.
Small localizations would be visible only by electron microscopy. It is possible that the
granular deposits associated with the PAS-body in Fig. 7 do represent large mem-
branous elements similar to those seen at the periphery of the PAS-body in Fig. 8.
The vegetative nucleus of G. polyedra is C-shaped, and the Golgi dictyosomes are
arranged within the inner curve of the nucleus (Schmitter, 1971). Occasionally, we
observed granular deposits between the PAS-body and the inner curve of the nucleus.
We are currently doing electron microscopic studies to investigate this point.
Correlations between autofluorescent cytoplasmic granules and lysosomal activity
have been made in a number of animal tissues (Koenig, 1963; Strehler, 1977). Pearse
(1968) tentatively attributed this autofluorescence either to lipid or lipoprotein of
lysosomal membranes, or to lipid material dissolved inside the organelles. Correlations
have also been made between lysosomal activity and PAS-reactive material (Koenig,
1962; Strehler, 1977), and Koenig reported the disappearance of autofluorescence of
lysosomes in certain tissues after the PAS-reaction. Studies on lipofuscin pigments in
aging mammalian tissues (summarized by Strehler, 1977) have linked lysosomal PAS-
reactivity and autofluorescence with the presence of partially oxidized, non-carbo-
hydrate-containing lipids (Pearse, 1972). The fragments of membrane seen within
PAS-bodies in our study (Fig. 8; also by Schmitter, 1971) support the notion that
lipid composition of PAS-bodies is related to their autofluorescence and PAS-
PAS-staining and acid phosphatase localization were observed by us in the same
22 R. E. Schmitter and A. J. Jurkievdcz
PAS-bodies of G. polyedra when cells were processed sequentially for acid phosphatase
localization and PAS-staining. Moreover, the size and unique location of PAS-bodies
within G. polyedra make it possible to correlate PAS-staining, acid phosphatase
activity, and autofluorescence unequivocally. Gomez et al. (1974 a) unsuccessfully
attempted a similar correlation in aging Euglena gradlis. Their Euglena cells had been
treated with benzpyrene, so they were observing secondary lipid fluorescence, not
autofluorescence (Pearse, 1968).
Our studies have shown that the PAS-bodies are a relatively permanent fixture in
G. polyedra. Their acid phosphatase activity is probably, as originally proposed by
Schmitter (1971), employed in autophagic processes recycling cellular materials. The
only other alga so far reported to possess a permanent site for digestive processes, as
opposed to the more usual transitory lysosomal activity, is Cryptomonas. Lucas (1970)
localized acid phosphatase activity in the Corps de Maupas and has suggested that the
structure is permanent, since it can be observed in natural populations and cultures
under a variety of conditions. Lucas (1970) did not report any studies of PAS-staining
or autofluorescence of this structure.
Our study is the first report in which PAS-staining, acid phosphatase localization,
and autofluorescence have all been correlated with the same subcellular algal organelle.

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(Received 18 March 1981)