Mycological Society of America

Ultrastructure and Cytochemistry of Dormant Basidiospores of Psilocybe cubensis

Author(s): Donald G. Ruch and Jerome J. Motta
Source: Mycologia, Vol. 79, No. 3 (May - Jun., 1987), pp. 387-398
Published by: Mycological Society of America
Stable URL: http://www.jstor.org/stable/3807461
Accessed: 22-06-2016 13:21 UTC

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Mycologia, 79(3), 1987, pp. 387-398.
© 1987, by The New York Botanical Garden, Bronx, NY 10458

ULTRASTRUCTURE AND CYTOCHEMISTRY OF DORMANT

BASIDIOSPORES OF PSILOCYBE CUBENSIS1

DONALD G. RUCH2 AND JEROME J. MOTTA

Department of Botany, University of Maryland,

College Park, Maryland 20742

ABSTRACT

The ultrastructure of dormant basidiospores of Psilocybe cubensis is described. The spore wall is
characterized by three distinct layers and a germ pore. A pore cap is described for the first time in a
species of Psilocybe. The protoplast is surrounded by a well defined plasma membrane with many
distinct invaginations. Internally, large numbers of nonmembrane-bound lipids occur at both ends of
the spore. Two nuclei are typically present and the nuclear envelope has many nuclear pores. Mito-
chondria with only a few, but well delineated, plate-like cristae are present. There is scant endoplasmic
reticulum. Ribosomes occur regularly attached to the ER and outer mitochondrial membrane, as well
as being densely packed throughout the cytoplasm. Variously sized vacuoles were demonstrated cy-
tochemically to contain acid phosphatase. Microbody-like organelles are described but cytochemical
tests to determine if these organelles are functionally similar to those of higher plants were unsuccessful.
Cell-free extracts of basidiospores exhibit catalase and isocitrate lyase activity but no malate synthase
activity.
Key Words: Psilocybe cubensis, ultrastructure, cytochemistry, basidiospore.

Basidiospores of the species of Psilocybe have MATERIALS AND METHODS

been the subject of few ultrastructural studies

The isolate of Psilocybe cubensis was obtained
(Olah, 1973; Stocks and Hess, 1970). Stocks and
from cap tissues of freshly collected basidiocarps
Hess (1970) described the ultrastructure of dor-
and was designated 78-1. The collection was
mant and germinated basidiospores of a species
identified by Dr. David Farr. Stock cultures were
of Psilocybe, later identified as P. mutans
maintained on Potato Dextrose Agar (Difco)
McKnight (McKnight, 1971). These authors uti-
supplemented with 0.1% yeast extract. Basid-
lized the freeze-etching technique primarily, ne-
iocarp production was induced by the method
cessitated by the impenetrability of the spore,
of Oss and Oeric (1976). Discharged basidio-
and only the structure of the wall, germ pore and
spores from fresh fruitbodies were collected in
hilar appendage were described from fixed and sterile 100 x 15 mm Petri dishes in a moist
embedded material. Olah (1973), experiencing chamber.
similar difficulties with penetration, also de-
Nuclei were stained using the mithramycin
scribed only the wall structure of P. quebecensis
technique of Slater (1976) as modified by Heath
Olah & Heim. Owing to this general lack of in-
(1980) and photographs were made using a Leitz
formation concerning the dormant spore in
SM-Lux Vertical Fluorescent Microscope em-
species of Psilocybe and the availability of a tech-
ploying a KP550 excitation filter and a K510/
nique to overcome the problems of tissue prep- 530 combination barrier filter.
aration presented by thickened walls (Olson,
For transmission electron microscopy, basid-
1978), we have examined the ultrastructure of
iospores were fixed in 3% glutaraldehyde in 0.05
Psilocybe cubensis Singer and carried out select-
M cacodylate buffer, pH 7.4. Spores were fixed
ed, correlative cytochemical studies.
for 6 h at 25 C, then washed in three 15 min
changes of buffer. Basidiospore walls were frac-
'This paper represents a portion of a dissertation tured by a modification of Olson's (1978) tech-
submitted to the Graduate School of the University of nique. A suspension of basidiospores was mixed
Maryland by Donald G. Ruch in partial fulfillment of with glass beads, 0.45-0.50 mm diam, in a ratio
the requirements for the degree of Doctor of Philoso-
phy.
of 2:5 (v:v, packed cell suspension: glass beads)
2 Current address: Physical and Life Sciences, Wil- in a 15 x 100 mm conical centrifuge tube. The
son College, Chambersburg, Pennsylvania 17201. tube was then agitated on a vortex mixer at the
387

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388 MYCOLOGIA

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 RUCH AND MOTTA: PSILOCYBE BASIDIOSPORES 389

highest speed for 30 s. The mixture was resus-
pended in buffer and the spores were separated
from the glass beads. After separation, the spores
were pelleted and post-fixed for 2 h at 25 C in
1% OsO4. They were then washed in distilled
water, centrifuged and the pellet mixed with a
drop of warm 3% agar. After solidifying, the agar-
spore mixture was cut into small pieces, dehy-
drated, embedded in Epon 812, sectioned and
stained with uranyl acetate followed by lead ci-
trate. Electron micrographs were made in a Hi-
tachi HU-11C electron microscope.
For light microscopy, lipids were visualized by
staining spores, previously fixed in 50% ethanol,
in a saturated solution of Sudan Black B in 70%
ethanol (Jensen, 1962). Insoluble polysaccha-
rides and proteins were demonstrated using the
periodic acid-Schiff's and aniline blue-black
method (Motta, 1971).
For acid phosphatase localization, basidio-
spores were fixed for 1 h at 15 C in 1% glutar-
aldehyde in 0.05 M cacodylate buffer, pH 7.4,
before fracturing the walls. After fracturing, the
spore suspension was washed three times, 15 min
each, in 0.05 M citrate buffer, pH 5, followed by
three 10 min washes in 0.05 M acetate buffer pH
5. Basidiospores were preincubated for 60 min
in the reaction medium minus substrate, fol-
lowed by incubation in the complete reaction
medium for 2 h at 37 C. The reaction medium
consisted of 0.3 ml distilled water and 4.7 ml of
a solution containing the following: 10 ml 0.2 M
acetate buffer, pH 5; 10 ml 1.25% sodium f-gly-
cerolphosphate, pH 5; and 20 ml 0.22% lead
nitrate. The controls consisted of (a) basidio-
spores incubated in the reaction medium minus
substrate or (b) basidiospores pretreated for 60
min at 25 C with 0.01 M NaF in 0.05 M acetate
buffer before being incubated in the complete
medium also containing 0.01 M NaF. After treat-
ment spores were washed 3 times each in reac-
tion mixture buffer followed by fixative buffer,
then post-fixed in OsO4 and processed for elec-
tron microscopy, except that section staining was
omitted. Lysosomes were also localized at the
light microscopic level using the acridine orange
procedure of Wilson et al. (1978) and examined
by means of fluorescence microscopy with the
filter combination indicated above.
Cytochemical localization of catalase was at-
tempted with the DAB procedure of Novikoff
and Goldfischer (1969). Basidiospores were fixed
and washed as for acid phosphatase. After frac-
turing the wall, spores were rinsed with three
changes of 0.05 M 2-amino-2-methyl-l,2-pro-
panediol buffer (AMP), pH 9.2. Spores were
preincubated in the reaction mixture minus sub-
strate for 45 min at 25 C, followed by 60 min at
37 C in complete medium containing 3,3-di-
aminobenzidine-tetrahydrochloride. Controls
consisted of (a) medium minus hydrogen per-
oxide, (b) spores preincubated for 45 min at 25
C in AMP buffer containing 0.02 M aminotri-
azole and transferred to complete medium also
containing 0.02 M of the inhibitor, (c) a sodium
fluoride treatment as for acid phosphatase. After
sequential washes of the appropriate buffers the
material was processed for electron microscopy
as before.
Attempts to localize malate synthase were
made using the method ofTrelease et al. (1974).
Basidiospores were fixed in 0.5% glutaralde-
hyde-4% formaldahyde (paraformaldehyde) in
0.05 M cacodylate buffer, pH 6.9, at 4 C for 60-
90 min (Powell, 1976). After fixation, spores were
washed with buffer, fractured, then washed three
times, 15 min each, with the incubation medium.
The spores were resuspended in the incubation
medium and transferred to 37 C for 60 min.
Heat-killed basidiospores (20 min at 60 C) and
reaction mixture minus sodium glyoxylate and
acetyl CoA were used as controls. After incu-
bation the spores were washed in three 15 min
changes of 0.05 M cacodylate buffer, pH 6.9, and

FIGS. 1-7. Psilocybe cubensis basidiospores. 1. Basidiospore wall layers, x 20,200. 2. Nonmedian section
through the hilar appendage of a basidiospore, x 21,100. 3. Near median section through the germ pore of a
basidiospore, x 32,300.4. Apex of spore showing pore cap and subtending ectosporium, x 31,100. 5. Basidiospore
nuclei stained with mithramycin, x 1450. 6. Endoplasmic reticulum and nuclei. Note apparent continuation of
the ER and nuclear envelope (arrow), x 28,000. 7. Basidiospore showing typical binucleate condition. Compare
with FIG. 5. Arrows indicate nuclear pores, x 31,800. Abbreviations: Ec, ectosporium; En, endosporium; Ep,
episporium; ER, endoplasmic reticulum; HA, hilar appendage; L, lipid globule; M, mitochondrion; Mb, micro-
body; mt, microtubule; N, nucleus; Nu, nucleolus; P, pore cap; V, vacuole.

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390 M IYCOLOGIA

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 RUCH AND MOTTA: PSILOCYBE BASIDIOSPORES 391

processed for electron microscopy as with other
enzyme localizations.
Catalase, isocitrate lyase, and malate synthase
were also determined in vitro according to the
methods of Liick (1963), Dixon and Kornberg
(1959), and Cooper and Beevers (1969a) respec-
tively. Basidiospores were collected by centrif-
ugation and suspended in 5 ml cold grinding
medium consisting of 1 mM EDTA; 0.15 mM
Tricine buffer [N-tris(hydroxylmethyl)meth-
ylglycine] pH 7.5; 10 mM KC1; 1 mM MgCl2; 1
mg/ml dithioerythritol; and 0.1% Triton X-100
(Cooper and Beevers, 1969a). A basidiospore/
glass bead mixture was homogenized in a Braun
Cell Homogenizer MKS for 40 s in the cold. The
homogenate was centrifuged (500 x gfor 10 min
at 4 C), lipids, if present, removed, the pellet
resuspended in the supernatant, and the homog-
enate used for analysis. As a check, a similar
preparation of endosperm from 4 day old castor
bean seedlings was assayed simultaneously (Coo-
per and Beevers, 1969a). Total protein was de-
termined by the method of Lowry et al. (1951)
with crystalline bovine albumen as a standard.
RESULTS
Basidiospores of Psilocybe cubensis have a thick
wall composed of three distinct layers (FIGS. 1,
2). The ectosporium, which is slightly electron-
opaque and appears mucilaginous, varies in
width, being widest at the apex of the spore (FIG.
3). Associated with the ectosporium and partially
covering the germ pore is an electron-opaque
pore cap (FIGS. 3, 4, 14). The pore cap is trap-
ezoidal, with the shorter of the two parallel sides
facing the germ pore. The pore cap was never
observed to be in contact with the epi- or en-
dosporium. The episporium is a thick, electron-
opaque layer of uniform thickness (600-700 nm
in median section) except for the regions of the
germ pore and hilar appendage where it thins
significantly (FIGS. 1-4, 14). The endosporium is
electron-transparent and is also of uniform thick-
ness (approximately 100 nm) but becomes thick-
er at the hilar appendage and germ pore where
it makes up the bulk of the wall material. The
germ pore, approximately 1 ,m diam, is well
defined and in thin section no depression of the
spore wall is seen at this point (FIG. 3).
Freshly discharged basidiospores of P. cu-
bensis have a distinct internal morphology (FIG.
10). The protoplasm is bound by a well defined
plasma membrane characterized by many dis-
tinct invaginations (FIG. 8). At the poles of the
spore large numbers of nonmembrane-bound
lipid-like globules occur (FIGS. 10, 13, 14). Two
nuclei, each approximately 1.5-2 Am diam, are
typically present in each spore. The nuclei are
always closely associated with each other and
generally occupy the center of the spore, lying on
a plane perpendicular to its long axis (FIGS. 5,
7). The nucleoplasm, which appears homoge-
neous except for a large, prominent nucleolus, is
surrounded by a distinct nuclear envelope. Nu-
clear pores appear along the envelope of inter-
phase nuclei. In all cases, nucleoli are laterally
positioned within the nucleus and in close prox-
imity to the nuclear envelope.
Well defined mitochondria are distributed
throughout the protoplasm (FIGs. 10, 12). In sec-
tion each mitochondrion is bound by a well de-
fined outer membrane often associated with ri-
bosomes (FIGS. 10, 12). Only a few, well
delineated plate-like cristae are observed. Mi-
tochondria are circular to ellipsoid in section,
300-700 nm diam. Branching was not observed
and it appears that many mitochondria are pres-
ent, although serial sections were not made to
confirm this.
Endoplasmic reticulum (ER) is sparsely dis-
tributed throughout the cytoplasm of dormant
basidiospores and is sometimes confluent with
the nuclear envelope (FIG. 6). No continuity be-
tween the ER and plasma membrane was ob-
served.
Each spore contains numerous single mem-
brane-bound vacuoles of various sizes (FIGS. 10-
12). These vacuoles contain an amorphous elec-
tron-dense material which may or may not fill
the entire matrix. In addition, many microbody-
like organelles can be observed. They appear as
small, less than 500 nm diam, circular structures
in cross section, bound by a single unit mem-
brane (FIGS. 7, 10, 12-14). The matrices of these

4-

FIGS. 8-10. Psilocybe cubensis basidiospores. 8. Plasma membrane of spore with distinct invaginations,
x 76,300. 9. Light micrograph of spore stained with Sudan Black B to show polar distribution of lipid, x 2780.
10. Longitudinal section of basidiospore illustrating various organelles. Nuclear pores indicated by unlabeled
arrows, x30,300.

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392 MYCOLOGIA

c. *

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FIGS. 11-19. Psilocybe cubensis basidiospores. 11. Thin sections illustrating typical, single membrane-bound
vacuoles, x 88,100. 12. Mitochondrion with associated ribosomes, x 31,500. 13. Microbodies located between
lipid globules and hilar appendage, x 20,900. 14. Microbodies located between lipid globule and germ pore,
x 36,300. 15. PAS-stained thick section showing distribution of insoluble polysaccharides. Note absence of
staining between the two areas of lipid, x 2010. 16. Aniline blue-black-stained thick section. Note unstained
polar regions. Arrow indicates nucleolus, x2010. 17. Non-specific staining of hilar appendage with acridine
orange, x 1770. 18. Broken cell with acridine orange staining of presumptive lysosomes, x 1770. 19. Acid
phosphatase localization with Gamori method x 2780.

--4

FIGS. 20-22. Psilocybe cubensis basidiospores. Acid phosphatase localization. 20. Acid phosphatase without
post-staining, x 21,500. 21, 22. Acid phosphatase with post-staining to illustrate unreactive microbodies, x 26,600
and x 44,000, respectively.

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 RUCH AND MOTTA: PSILOCYBE BASIDIOSPORES 393

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394 MYCOLOGIA

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FIGS. 23-25. Psilocybe cubensis basidiospores. 23. Acid phosphatase, sodium fluoride control, x 23,000. 24.
Acid phosphatase, reaction mixture minus the substrate control, x 24,100.25. Catalase localization. Note reaction
product only in mitochondria, x 26,800.

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 RUCH AND MOTTA: PSILOCYBE BASIDIOSPORES 395

TABLE I

CATALASE AND GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE 500 X G SUPERNATANT PREPARATIONS OF
PSILOCYBE CUBENSIS AND CASTOR BEAN ENDOSPERM

Catalase Malate synthase Isocitrate lyase

Unitsa SAb Unitsa SAb Unitsa SAb

P. cubensis 668 33.2 0 0 3.9 0.18

Castor bean 5280 117.6 10.8 0.24 7.7 0.17

a Units of enzyme activity per gram fresh weight. (One enzyme unit is defined as the amount of enzyme that
produced 1 Amol of product/min.)
b Specific activity equals units per mg protein.

organelles are homogeneous and no crystalline
inclusions are observed. Microbody-like organ-
elles are often associated with the lipid globules
and were the only organelles, except ribosomes,
occurring between the lipid globules and the cell
wall. No physical association between ER and
microbody-like organelles is observed and the
limiting membrane of the latter is free of ribo-
somes.
Neither dictyosomes nor rosettes of glycogen
(a-particles) were observed in basidiospores and
microtubules were seen only occasionally (FIG.
13).
Sudan Black B stains clusters of globules at the
poles of the spore (FIG. 9) corresponding to those
observed in thin sections. Thick sections of dor-
mant basidiospores stained by PAS-Schiffs ex-
hibit a strong positive reaction along the endo-
sporium and germ plug (FIG. 15). Other than a
very weak positive reaction of lipids, there ap-
pears to be a conspicuous absence of staining
within the protoplasm of the spores. When sec-
tions are counterstained with aniline blue-black
however, the internal compartment of the spore
stains uniformly blue (FIG. 16).
Unfixed basidiospores treated with acridine
orange do not exhibit internal fluorescence if the
spore wall remains intact. Only the spore wall,
especially near and including the hilar appen-
dage, fluoresces orange (FIG. 17). No fluorescence
of the spore wall is observed if the wall is frac-
tured during preparation. In such spores, there
is bright yellow-orange to red-orange fluores-
cence of numerous particulate bodies, which are
distributed throughout the protoplasm (FIG. 18).
In light microscopy, the distribution of reaction
product in spores stained by the Gamori method
for acid phosphatase is also as discrete, randomly
distributed particles (FIG. 19). Ultrastructurally,
reaction product appears as heavy deposits of
electron-opaque material primarily in single
membrane-bound vacuoles (FIGS. 20-22). How-
ever, lead deposits are also observed along the
plasma membrane, in the space between the plas-
ma membrane and the wall, in the wall itself,
especially the endosporium, and within the nu-
clei. No lead deposits are seen in nuclei of the
sodium fluoride control (FIG. 23) or in the spores
incubated in the reaction mixture minus the sub-
strate (FIG. 24).
The distribution of reaction product in the cat-
alase procedure is illustrated in FIG. 25. Only
mitochondrial cristae have a positive DAB re-
action. Reaction product is never observed in
any of the other organelles. Similarly, attempts
to visualize malate synthase activity were all neg-
ative. The in vitro experiments to detect activity
of these enzymes is summarized in TABLE I. Both
catalase (33.2 units/mg protein) and isocitrate
lyase (0.18 units/mg protein) appear to be pres-
ent, but no malate synthase activity is detected.
DISCUSSION
Psilocybe cubensis has a thick, multilayered
basidiospore wall characteristic of the spore wall
described for other Psilocybe species (Olah, 1973;
Stocks and Hess, 1970) and several Pholiota
species (Arita, 1979; Clemencon, 1974; Melen-
dez-Howell, 1967a, b). The germ pore of P. cu-
bensis is similar in structure to the germ pore of
P. mutans McKnight (Stocks and Hess, 1970),
except that there is no depression of the epi-
sporium in thin section. The germ pore of P.
cubensis is formed by the extension of the en-
dosporium into the episporial layer, which forms
a thin covering over the endosporium. Thus, the
bulk of the germ plug is formed from the inner,
electron-transparent endosporial layer. This type
of germ pore, type A of the five types described
by Melendez-Howell (1967a, b), is characteristic
of most species of Strophariaceae (Pegler and
Young, 1971), several Pholiota species (Arita,
1979; Melendez-Howell, 1967a, b), and most

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396 MYCOLOGIA
species in the Cortinariaceae (Hawker and Mad-
elin, 1976).
McLaughlin (1977) described the develop-
ment of a pore cap in Coprinus cinereus (Schaef-
fer:Fr.) Fr. The immature basidiospore of C.
cinereus had a six-layered wall. Pore cap for-
mation was initiated by the disruption of the
third wall layer at the spore apex. As the spore
matured, a well developed pore cap appeared to
form by the irregular interdigitation of wall ma-
terials continuous with layers three and four. Like
C. cinereus but unlike P. mutans (Stocks and
Hess, 1970), P. cubensis has a distinct pore cap
embedded in the ectosporial layer over the germ
pore. Although its origin was not directly deter-
mined in this study, it appears that the pore cap
of P. cubensis may originate from the epispo-
rium. Since the electron-opacity of the epispo-
rium and the pore cap are similar, and the char-
acteristic trapezoidal pore cap matches the break
seen in the episporium, the latter hypothesis
seems reasonable and consistent with Mc-
Laughlin's (1977) interpretation.
Mitochondria of dormant basidiospores have
been described as poorly defined with only a few
cristae (Arita, 1979; Hyde and Walkinshaw, 1966;
Voelz and Niederpruem, 1964). However, the
freeze-fractured basidiospores of P. mutans ex-
hibited well defined mitochondria (Stocks and
Hess, 1970). Similar mitochondria, having well
delineated cristae, were seen in P. cubensis. The
number of cristae visible in any single mito-
chondrion is highly dependent on the section
through that mitochondrion.
The positive reaction of material distributed
at the poles of the spore stained with Sudan Black
B is consistent with the hypothesis that the sim-
ilarly distributed, nonmembrane-bound globules
visible in the electron microscope are primarily
lipid in character. The relative abundance of this
material in the spore, coupled with the apparent
lack of an insoluble polysaccharide such as gly-
cogen, as demonstrated by the negative PAS re-
action and the absence of particles in the cyto-
plasm, is significant. This and the observation of
the relative abundance of microbody-like organ-
elles in association with the presumptive lipid
bodies led us to the attempts to localize enzymes
typically identified with microbodies, specifical-
ly, those of glyoxysomes.
Glyoxysomes, as described from higher plants,
contain an array of enzymes, among them cat-
alase, all of the enzymes of the glyoxylate cycle,
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and the enzymes for oxidation of fatty acids
(Breidenbach et al., 1968; Cooper and Beevers,
1969a, b). Basidiospores incubated in the DAB
medium for the localization ofcatalase, however,
gave a negative reaction. Similarly, attempts to
localize malate synthetase in the spores were un-
successful. Since it has been established that the
conditions for in situ demonstration of these en-
zymes might vary with the species (see Maxwell
et al., 1977), we attempted in vitro demonstration
of catalase, malate synthetase and isocitrate lyase.
The presence of catalase and isocitrate lyase in
basidiospore homogenates (TABLE I) lends sup-
port to Maxwell et al.'s (1977) hypotheses that
the appropriate conditions for in situ demon-
stration of these enzymes were not met in these
experiments. The inability to demonstrate ma-
late synthetase activity, neither in situ nor in
vitro, was unexpected. However, several expla-
nations are possible in addition to those already
alluded to, including the hypothesis that this par-
ticular enzyme may simply not be present until
conditions for germination exist. Clearly, many
more definitive experiments must be carried out
in order to answer this question.
The demonstration of acid phosphatase and
lysosome-like organelles in the basidiospores of
P. cubensis is less ambiguous. Acridine orange,
commonly used by cytologists to stain lysosomes
in living animal cells (Koenig, 1969; Dingle and
Barrett, 1969), has also been used as a marker
for fungal lysosomes (Wilson et al., 1978). Ac-
ridine orange is a cationic metachromatic dye,
whose selective staining of lysosomes and other
important cellular organelles is dependent both
upon the pH and the concentration of the dye in
the staining medium (Koenig, 1969; Motta, 1971).
The thickened walls of P. cubensis basidiospores
apparently prevented uptake of acridine orange
by cellular components. That the dye bound to
the wall and fluoresced a bright orange indicated
the presence of a polyionic charge on the wall,
particularly in the region of the hilar appendage.
Ultrastructurally, acid phosphatase activity as-
sociated with this area could not be demonstrat-
ed, however. Although this result does not pre-
clude the occurrence ofhydrolytic enzymes from
this region, it does suggest that the binding of
acridine orange to the wall was non-specific and
likely due to electrostatic forces. Fractured walls
apparently permitted penetration of the dye, and
the particles stained within the cell correspond
in relative size and distribution to the acid phos-
 RUCH AND MOTTA: PSILOCYBE BASIDIOSPORES
phatase positive vacuoles observed ultrastruc-
turally.
The presence of acid phosphatase activity in
the basidiospores of P. cubensis is consistent with
the biochemical demonstration of this enzyme
in the spores of Schizophyllum commune Fr.
(Wilson, 1972). The localization of reaction
product in vacuoles and its general distribution
in the region of cell walls and in nuclei is also
characteristic of the distribution of the enzyme
in many other fungi (Armentrout et al., 1976;
Hanssler et al., 1975; Hislop et al., 1974; Mason
and Wilson, 1979; Wilson et al., 1978).
LITERATURE CITED
Arita, I. 1979. Cytological studies on Pholiota. Rept.
Tottori Mycol. Inst. (Japan) 17: 1-118.
Armentrout, V. N., G. Hanssler, and D. P. Maxwell.
1976. Acid phosphatase localization in the fungus
Whetzelina sclerotiorum. Arch. Microbiol. 107: 7-
14.
Breidenbach, R. W., A. Kahn, and H. Beevers. 1968.
Characterization ofglyoxysomes from castor bean
endosperm. Plant Physiol. 43: 705-713.
Clemenqon, H. 1974. Die Wandstrukturen der Ba-
sidiospores. V. Pholiota und Kuehneromyces ver-
glichen mit Galerina und Gymnopilus. Z. Pilz-
kunde 40: 105-126.
Cooper, T. G., and H. Beevers. 1969a. Mitochondria
and glyoxysomes from castor bean endosperm. J.
Biol. Chem. 244: 3507-3513.
, and . 1969b. d-oxidation in glyoxy-
somes from castor bean endosperm. J. Biol. Chem.
244: 3514-3520.
Dingle, J. T., and A. J. Barrett. 1969. Some special
methods for the investigation of the lysosomal sys-
tem. Pp. 555-556. In: Lysosomes in biology and
pathology. Vol. 2. Eds., J. T. Dingle and H: B. Fell.
North-Holland Publishing Co., Amsterdam.
Dixon, G. H., and H. L. Kornberg. 1959. Assay meth-
ods for key enzymes of the glyoxylate cycle. Bio-
chem. J. 72: 3 p.
Hanssler, G., D. P. Maxwell, and M. D. Maxwell.
1975. Demonstration of acid phosphatase-con-
taining vacuoles in hyphal tip cells of Sclerotium
rolfsii. J. Bact. 124: 997-1006.
Hawker, L. W., and M. F. Madelin. 1976. The dor-
mant spore. Pp. 1-72. In: Thefungal spore. Eds.,
D. J. Weber and W. M. Hess. John Wiley and
Sons, New York.
Heath, I. B. 1980. Fungal mitosis, the significance of
variations on a theme. Mycologia 72: 229-250.
Hislop, E. C., V. M. Barnaby, C. Shellis, and F. La-
borda. 1974. Localization of a-L-arabino-furan-
osidase and acid phosphatase in mycelium of
Sclerotinia fructigena. J. Gen. Microbiol. 81: 79-
99.
Hyde, J. M., and C. H. Walkinshaw. 1966. Ultra-
structure of basidiospores and mycelium of Len-
zites saepiaria. J. Bacteriol. 92: 1218-1227.
This content downloaded from 139.184.14.159 on Wed, 22 Jun 2016 13:21:15 UTC
All use subject to http://about.jstor.org/terms
397
Jensen, W. A. 1962. Botanical histochemistry. W. H.
Freeman and Co., San Francisco. 408 p.
Koenig, H. 1969. Lysosomes in the nervous system.
Pp. 111-162. In: Lysosomes in biology and pa-
thology. Vol. 2. Eds., J. T. Dingle and H. B. Fell.
North-Holland Publishing Co., Amsterdam.
Lowry, O. H., H. J. Rosebrough, A. L. Farr, and R. J.
Randall. 1951. Protein measurement with the
folin phenol reagent. J. Biol. Chem. 193: 265-275.
Luck, H. 1963. Catalase. Pp. 885-894. In: Methods
of enzymatic analysis. Ed., H. U. Bergmeyer. Ac-
ademic Press, New York.
Mason, D. L., and C. L. Wilson. 1979. Cytochemical
and biochemical identification of lysosomes in
Cryptococcus neoformans. Mycopathologia 68:
183-190.
Maxwell, D. P., V. N. Armentrout, and L. B. Graves,
Jr. 1977. Microbodies in plant pathogenic fungi.
Ann. Rev. Phytopath. 15: 119-134.
McKnight, K. H. 1971. Cultural studies on Psilocybe.
I. Variation in a new species of the P. coprophila
group. Bull. Torrey Bot. Club 98: 4-16.
McLaughlin, D. J. 1977. Basidiospore initiation and
early development in Coprinus cinereus. Amer. J.
Bot. 64: 1-16.
Melendez-Howell, L. M. 1967a. Recherches sur le
pore germinatif des basidiospores. Ann. Sci. Nat.
Bot. Paris, S6r. 8: 487-638.
1967b. Les rapports entre le pore germinatif
sporal et la germination chez les Basidiomycetes
en microscopie electronique. Compt. Rend. Hebd.
Seances Acad. Sci. 264: 1266-1269.
Motta, J.J. 1971. Histochemistry of the rhizomorph
meristem of Armillaria mellea. Amer. J. Bot. 48:
80-87.
Novikoff, A. B., and S. Goldfischer. 1969. Visualiza-
tion of peroxisomes (microbodies) and mitochon-
dria with diaminobenzidine. J. Histochem. Cy-
tochem. 17: 675-680.
Olah, G. M. 1973. The fine structure of Psilocybe
quebecensis. Mycopath. Mycol. Appl. 49: 321-338.
Olson, L. W. 1978. Preparation of"difficult" spores,
cells and sporangia for electron microscopy. Pp.
187-188. In: Lower fungi in the laboratory. Ed.,
M. S. Fuller. University of Georgia, Athens.
Oss, O. T., and 0. N. Oeric. 1976. Psilocybin: magic
mushroom grower's guide. And/Or Press, Berke-
ley, California. 63 p.
Pegler, D. N., and T. W. K. Young. 1971. Basidio-
spore morphology in the Agaricales. Beih. Nova
Hedwigia 35: 1-24, 117-123.
Powell, M. J. 1976. Ultrastructure and isolation of
glyoxysomes (microbodies) in zoospores of the
fungus Entophlyctis sp. Protoplasma 89: 1-27.
Slater, M. L. 1976. Rapid nuclear staining method
for Saccharomyces cerevisiae. J. Bacteriol. 126:
1339-1341.
Stocks, D. L., and W. M. Hess. 1970. Ultrastructure
of dormant and germinated basidiospores of a
species of Psilocybe. Mycologia 62: 176-191.
Trelease, R. N., W. M. Becker, and J. J. Burke. 1974.
Cytochemical localization of malate synthase in
glyoxysomes. J. Cell Biol. 60: 483-495.
Voelz, H., and D. J. Niederpruem. 1964. Fine struc-
398 MYCOLOGIA

ture of basidiospores of Schizophyllum commune.
J. Bacteriol. 88: 1497-1502.
Wilson, C. L., G. A. Jumper, and D. L. Mason. 1978.
Acridine orange as a lysosome marker in fungal
spores. Phytopathology 68: 1564-1567.
Wilson, R. W. 1972. Acid and alkaline phosphatase
in Schizophyllum commune. Canad. J. Microbiol.
18: 694-695.
Accepted for publication November 17, 1986

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