Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1964 Flagellar initiation and centrioles in Albugo Jerry Dean Berlin Iowa State University
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BERLIN, Jerry Dean, 1934- FLAGELLAR INITIATION AND CENTRIOLES IN ALBUGO.
Iowa State University of Science and Technology Ph.D„ 1964 Botany
University Microfilms, Inc., Ann Arbor, Michigan FLAGELLAR INITIATION AND CENTRIOLES IN ALBUGO
by
Jerry Dean Berlin
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY '
Major Subject: Cell Biology
Approved:
Signature was redacted for privacy.
Signature was redacted for privacy. Head of Department
Signature was redacted for privacy.
iu? of Gradate
Iowa State University Of Science and Technology Ames, Iowa
1964 il
TABLE OP CONTENTS
Page
I. INTRODUCTION 1
II. LITERATURE REVIEW 3
A. The Life Cycle of Albugo Candida 3
B. Organization of Fungal Cells 5
C. Centrioles 6
D. Basal Bodies 10
E. Flagella 11
F. Electron Microscopy of Filamentous Structures Associated with Centrioles, Basal Bodies, or Flagella 14
III. MATERIALS AND METHODS 16
A. Organism 16
B. Specimen Preparation for Electron Microscopy 16
IV. OBSERVATIONS AND DISCUSSION 20
A. General Fine Structure 20
1. Intercellular hyphae 20 2. Sporangiophores 25 3. Sporangia 26
B. Centrioles 36
C. Basal Bodies 4g
D. Flagella 54
E. Filamentous Structures Associated with Basal Bodies , 59
' 1. Spur 59 2. Rhizoplast 60 lii
Page
3. Ancillary cytoplasmic filaments 60
F. Filamentous Structure; a Review 63
V. SUMMARY 65
VI. ACKNOWLEDGEMENTS 67
VII. LITERATURE CITED 68 1
I. INTRODUCTION
Flagella are of widespread occurrence in the animal kingdom, but in plants are restricted to gametes and zoo spores of certain algae and fungi and to the spermatozoids of certain higher plants. Flagella, when present in eucaryotic cells, are formed from basal bodies. The origin of basal bodies, however, is somewhat in doubt. Basal bodies have been found to apparently arise de, novo in some organisms and from pre-existing centrioles in others. In plants, centrioles are generally assumed to be present only in the division preceding motile cell formation where they are thought to function as basal bodies in the development of flagella.
The Phycomycetes are the only fungal group having motile cells. Although amphlastral figures have been reported by light microscopists in a number of fungi, primarily in the
Ascomycetes, studies of fungal fine structure have not demonstrated centrioles or basal bodies despite their striking appearance at high magnifications. The present electron microscope investigation was undertaken to determine the origin of basal bodies in the zoospores of a Phycomycete, a group which has not been previously studied in this respect.
The electron microscope is a notoriously poor survey instrument. Therefore, when working with this tool, for reasons of efficiency, it is necessary to choose an experl- 2 mental subject in which the various stages to be investigated may be anticipated in respect to time and position. Albugo
Candida (Pers.) Kuntze, a Phycomycete which produces biflagel- late zoospores during the asexual stages of its life cycle, was selected as the organism for this investigation. This fungus possesses a chain of multinucleate sporangia and each sporangium gives rise to a number of uninucleate zoospores.
The protoplast of each sporangium of a chain is in a different stage of zoospore differentiation. Hence, this organism presents material at predictable stages of development in precisely known positions and thus is well suited for this study. II. LITERATURE REVIEW
I A. The Life Cycle of Albugo Candida
The life history of A. Candida, an obligate parasite of crucifers, has been described by de Bary (1863), Wager (1896), and others. The asexual life cycle is as follows: The
branching hyphae in the intercellular spaces of the spongy mesophyll of the host leaf form knob-like haustoria in the I host cells. The hyphae give rise to sporangiophores which form large numbers of multinucleate sporangia. Nuclear divi sion does not occur in the developing sporangia. Pour to eight nuclei enter a sporangium and each becomes the nucleus of a zoospore (Wager, 1896). The leaf epidermis is ruptured by local accumulations of sporangia. In a favorable environ ment the sporangia absorb water and, depending upon the temperature, germinate either by biflagellate zoospores or germ tubes (Alexopoulos, 1952). These zoospores have been described as possessing one "whip lash" and one "tinsel" type of flageHum. Zoospores are extruded into a sessile vesicle
(Vanterpool, 1959)• de Bary (1863) did not observe flagella until the zoospores were free from the sporangium. The zoo spores swim for a period of time before they develop a cell wall and lose their flagella. Each encysted spore germinates by producing a germ tube which enters the host plant through 4 the stoma; thus completing the asexual life cycle, which may be repeated several times during a growing season.
The sexual stages of the life cycle begin in the leaf where oogonia and antheridia are formed. The oogonium con tains approximately one hundred nuclei, the antheridium six to twelve. All these nuclei undergo a simultaneous division throughout both organs before an oosphere (a naked female gamete) is formed within the oogonium. After completion of differentiation of the oogonium into periplasm and ooplasm the nuclei of the antheridium and the ooplasm undergo a second mitotic division. The nuclei of the periplasm disintegrate at this stage. A single femaleinucleus moves from the ooplasm to the center of the oosphere while the other ooplasmic nuclei move to the periplasm. A fertilization tube, carrying a single male nucleus, penetrates the oosphere and ruptures.
The male nucleus fuses with the female nucleus forming a zygote. A wall forms around the zygote and the resulting structure is termed an oospore. The nuclei left in the antheridium and the periplasm disintegrate. The nuclei of the oospore divide several times with two of these divisions constituting meiosis. The oospore germinates, producing more than one hundred uninucleate, biflagellate zoospores which are similar to the zoospores produced in the asexual cycle. These zoospores swim, encyst, and germinate via germ tubes which invade the host leaves (Fitzpatrick, 1930). 5
Melhus (1911) found that high germination and, conse
quently, high infection rates were obtained when sporangia of
A. Candida were chilled in water prior to being placed on
susceptible host plants. Under these conditions sporangia
germinate in three to five hours liberating numerous zoospores.
This finding permits continuous greenhouse culturing of this
organism.
B. Organization of Fungal Cells
Moore and McAlear (I960, 1961a, 1961b, 1961c, 1962,
1963a, 1963b, 1963c), in the most exhaustive study of fungal fine structure to date, have pointed out the similarity of cells of several fungal species to other eucaryotic cells.
They have observed plasma membrane-endoplasmic reticulum- nuclear envelope continuity as well as mitochondria, vacuoles, lipoidal inclusions, ribosomes, and perinuclear Golgi. No structures resembling plastids have been reported. Lomasomes, complex elaborations of the plasma membrane (Peyton and Bowen,
1963), appear to be unique to the three classes of Bumycophyta.
These observations are in general agreement with the studies of Phycomycetes by Koch (1956), Blondel and Turian (i960),
Hawker and Abbott (1963a, 1963b), and Peyton and Bowen (1963); of Ascomycetes by Contl and Naylor (1959, 1960a, 1960b),
Shatkln and latum (1959), Hashimoto, Gerhardt, Contl and Naylor 6
(I960), Thyagarajan and Naylor (1962), and Ehrlich and Ehrlich
(1963b); and of Basldiomycetes by Girbardt (1958, 1961) and
Ehrlich and Ehrlich (1963a).
The work of Berlin and Bowen (1964) on A. Candida is the only electron microscopic study of this organism published to date. They studied the organization of the host-parasite interface and described the haustoria in detail. A. Candida was shown to possess a cell organized much like that of
Peronospora manshurlca (Naom. ) Syd. ex Gaum (Peyton and Bowen,
1963).
0. Centrioles
The centriole literature is confusing because of the lack of a standard terminology. Wilson (1928) gives at least 15 synonyms used by various workers for centrioles or centro- somes, or both. Undoubtedly this confusion is due to the number of independent workers in the field, the minuteness of centrioles, and the resolution limits of the light microscope.
The electron microscopists, Bemhard and de Harven (i960), described a centriole as "une structure cylindrique composée de neuf tubules parallèles ou de neuf groups de tubules."
Several other terms are useful in discussing centrioles.
The "centrosome", according to most workers, includes the centriole and the peri cent riolar zone which Bemhard and de
Harven (i960) describe as having a "diffuse density" under the 7 electron microscope. 11 Diplosome" is generally used to designate a pair of centrioles. The "proximal", as opposed to the 11 distal", end of a centriole is that end lying nearest the other centriole of the diplosome.
Centrosomes were first described by Flemming (1875) in the egg of the fresh water mussel Anodonta. According to
Wilson (1928) and Lepper (1956), centrioles had been described by 1900 as occurring in animal and lower plant cells, but had not been found in seed plants. It is still generally assumed that centrioles are present only in plants possessing motile male gametes and then only in the division preceding spermato- zoid differentiation. Since their discovery, centrioles have been controversial objects, and there was even a period when they were considered to be artifacts by many cytologists.
Huettner (1933) followed the continuity of centrioles in the fruit fly Drosophilla and showed that centrioles were self- perpetuating and continuous from one cell generation to the next. The magnificent light micrographs presented in
Heuttner's paper, most of which were made by E. B. Wilson, dispel any doubts about the reality of centrioles.
The fine structure of centrioles was first described by de Harven and Bemhard (1956), and this description has been subsequently confirmed by Bessis and Breton-Gorius (1957),
Amano (1957) and many others. Berkaloff (1963), working with the brown alga Hlmanthalia Lorea, appears to have been the 8 first electron microscopist to describe centioles in vegetative plant cells. Centrioles described to date have certain fea tures in common. They are about 150 mP- in diameter, 400 to 500 mU long, and are composed of nine triplets arranged in a cylinder. Bach triplet is composed of a row of three appressed filaments and the individual filament is 15 to 25 my- in diameter, appearing tubular. The nine triplets are organized in a "pin-wheel" array such that each row of fila ments makes an angle of about 4o* with the tangent to the cylinder. Bemhard and de Harven (i960) reported 70 mP- diameter pericentriolar structures, which they termed "satel lites", attached to the sides of centrioles. Gall (1961) occasionally observed a central filament and suggested that it was present at the proximal end of centrioles.
Centriole formation has been the subject of several investigations. The possibility that centromeres may give rise to centrioles was suggested by the work of Pollister and
Pollister (19*3). Cleveland (i960) states that centrioles are autonomous organelles and describes elaborate growth stages for centrioles. However, the structure he is dealing with is more than a centriole in the sense used here.
Bemhard and de Harven (I960) theorized that centrioles reduplicate themselves by a process of lateral budding.
Mazia, Harris and Bibring (I960) present evidence for the
"generative" replication of cell centers involving the 9 formation of a pro-body of smaller size than the parent structure. Gall (1961) reported procentrioles of the same diameter as the mature centriole, but shorter in length, arising at right angles to the mature centriole. The pro centriole was generally found about 70 mU from the mature centriole and was never observed in actual contact with it.
In the water fern Marsilea Gall (1963) found a cluster of procentrioles prior to sperm development. In Marsllea the procentrioles did not appear to arise from a pre-existing centriole and their origin is not clear. Based primarily on morphological evidence, the method of centriole replication is presently thought to be composed of at least two processes: the formation of a short procentriole and its growth into a centriole (Roth, 1964). I The precise function of centrioles remains unknown, but they are apparently associated with several different kinds of filamentous structures. According to Sleigh (1962) they are either functionally concerned with filament production or they may act as organizing centers for filamentous structures.
There appears to be little doubt that centrioles occur in the Ascomycetes (Wager, 1894; Bagchee, 1925; Sharp, 1934).
No observations of centrioles in the Phycomycetes are reported in the literature, but Davis (1900) suggested that centrioles were present in the sexual stages of A. Candida but were too small to be seen. Centrioles have not been observed in any 10 fungus by electron microscopy. i
D. Basal Bodies
The structure found at the base of flagella and cilia in eucaryotic cells has been given various names. This termi nology is reviewed by Fawcett (1961) and, following this author, the descriptive term "basal body" will be used exclu sively in this study. The "proximal" end of the basal body is that end nearest the nucleus.
Basal bodies are very similar in structure to centrioles.
Their fine structure has been reported by Rhodin and Dalhamn
(1956), Afzelius (1959), Gibbons and Grimstone (I960),
Gibbons (1961), and others. They are cylindrical, composed of nine triplets, and have a 150 mil diameter and a variable length. They are closed distally by a basal plate and Gibbons and Grimstone (I960) have described a central filament in the proximal end of basal bodies of two protozoans. The lumens of basal bodies show structural variation in different organisms
(Fawcett, I96I).
That basal bodies may be centriolar in origin was dis cussed independently by Henneguy and Lenhossek in 1898 and is today termed the Henneguy-Lenhossek theory. Fawcett and
Porter (1954), de Harven and Bemhard (1956), Burgos and
Fawcett (1956), Rouiller and Faurl-Fremiet (1958), Rhodin 11
(1963), and others have noted structural similarities between
centrioles and basal bodies at the electron microscope level.
Sotelo and Trujillo-Cen6z (1958a, 1958b) and Gall (1961)
observed flagella originating from pre-existing centrioles.
Conversely, Ehret and Powers (1959) and Ehret and de Haller
(1963) feel that the Paramecium basal bodies arise de novo
by development from more elementary structures.
1 E. Flagella
Fawcett (1961) calls motile organs "cilia" when they are short and there are many per cell and "flagella" when they are long and few per cell. Sleigh (1962) objects to this separation and separates the two according to their mode of use or movement. 1 Pitelka (1963) believes cilia are merely one of several varieties of flagella. Mycologists have uniformly called the motile organs of fungal zoospores
"flagella" and this term will be used in this study.
Instead of triplets, as found in centrioles and basal bodies, flagella have nine peripheral doublets (two appressed filaments). These doublets are arranged around two single central filaments. All of the filaments have a diameter of
15 to 25 mV>. The doublets are oriented 30e to 50* to the tangent of the flagellar cylinder. As the flagellum tapers toward a point, various of the filaments terminate, the 12 sequence of termination depending upon the organism. At the I base of flagella, the outer nine doublets are assumed to merge with the basal body triplets. The two central filaments do not reach the basal body, but end in the "transition region" between flagellum and basal body. Finally, a membrane, which is continuous with the plasma membrane, surrounds the flagellum. The structure, general biology, and present knowledge of the chemistry and function of flagella is reviewed by Fawcett (1961) and Sleigh (1962). i
Other workers had previously observed 11 filaments in flagella, but Manton and Clarke (1951), studying flagella in fern spermatozoïde, were the first to suggest that a pair of centrally positioned filaments were surrounded by nine others.
This nine plus two pattern was first confirmed with thin- section electron microscopy by Fawcett and Porter (1954) on ciliated spithelia. Since these early studies in flagellar fine structure, the nine plus two pattern has been shown to occur in widely diverse organisms and cell types, e.g.,
Protozoa (Roth, 1958; Gibbons and Grimstone, I960; Lang,
1963a; Pitelka, 1963), Mollusca (Gibbons, 1961), rat trachea
(Rhodin and Dalhamn, 1956), sperm (Fawcett, 1958; Afzelius,
1959; Telkka, Fawcett and Christensen, 1961), Algae (Manton,
Clarke and Greenwood, 1955; Gibbs, Lewin, and Philpott, 1958;
Hoffman and Manton, 1962), Bryophyta (Manton and Clarke, 1952) and Fungi (Koch, 1956; Blondel and Turian, I960). This 13 pattern is so consistent that variations are now subjects for research papers (Afzelius, 1963).
Pease (1963) reported the walls of each individual filament in negative stained rat sperm tails could be broken down into ten sub-units which are also longitudinally oriented.
Whether the ten sub-units are as consistent in flagellar filaments as the nine plus two pattern is in flagella is a question which will be answered only by further investigations.
The biflagellate zoospores found in the Phycomycetes appear to have one flagellum of the tinsel type and one of the whip lash type (Alexopoulos, 1952). The whip lash type is apparently a typical flagellum while the tinsel type has hair like projections along its entire length.
In the fungi, shadowed preparations of flagella in
Saprolegnia, Allomyces, and Olpidlum have been published by
Manton, Clarke, Greenwood, and Flint (1952); in Phytophthora by Ferris (1954) and Kole and Horstra (1959); in chytrids i>y
Koch (1956); and in Plasmopara by Santilli (1958). Using thin-section techniques, Blondel and Turian (I960) observed the nine plus two pattern in flagella of differentiating gametes in gametangia of Allomyces. 14
P. Electron Microscopy of Filamentous Structures
Associated with Centrioles, Basal Bodies, or Flagella I
The literature abounds with such terms as filaments, fibrils, fibers, microtubules, etc. describing essentially similar structures found in flagella, basal bodies, centrioles, spindles, rootlet systems, etc. These structures appear to be tubular in nature, possessing a circular wall with a less electron-dense lumen, are 15 to 30 mU in diameter, and their precise function is unknown, as is their chemistry. Although these structures may have a bimodal diameter distribution
(Slautterback, 1963; Roth, 1964) the only clear basis we have of distinguishing the above structures is by their location within a cell. It is convenient to attach a name to these structures and the term "filament", accompanied by an adjec tive indicating location, will be used throughout this study.
Filamentous structures associated with centrioles represent a part of the mitotic apparatus and were reviewed by Mazia (1961). Spindle structure has been described by
Amano (1957), Ruthmann (1959), Bernhard and de Harven (i960),
Roth, Obetz, and Daniels (I960), Harris (1961, 1962), Roth and Daniels (1962), and Roth and Shigenaka (1964). Astral filaments have been reported by Gall (I96I) and Harris (1961,
1962).
Rootlet structures are assumed to serve flagella in an 15 anchoring capacity and are discussed by Fawcett (1961) and
Sleigh (1962). Schuster (1963) reported filamentous struc tures in the flagellate stage of the amoebo-flagellate
Maeglerla which are of special interest to the present inves tigation. Two sets of basal body-associated filaments were described. The first, described by Schuster (1963) as being unique to Saegleria, is termed a 11 spur" and is composed of a linear array of 14-mP diameter filaments which attaches broadly to the proximal region of the basal body. The second set of filaments consist of isolated 22-mU diameter elements which appear to radiate from the basal body complex.' Similar isolated structures are found associated with basal bodies in molluscs (Gibbons, 1961) and in other protozoa (Roth, 1958)•
Finally, it should be recalled that the filaments com posing the centrioles, basal bodies, and flagella have pre viously been described as being 15 to 25 mil in diameter.
Slautterback (1963) extensively reviews the literature of cytoplasmic filaments in this size range. There appears to be great similarity of structure, and probably homology, between the various filamentous structures of cells (Roth,
1964). 16
III. MATERIALS AND METHODS
A. Organism
Initially leaves of the common garden radish, Raphanus
satlvus L., infected with Albugo Candida (Pers.) Kuntze were
collected and identified by Dr. J. 0. Gilman. Sporangia from
mature pustules were suspended in glass-distilled water and
immediately sprayed on young radish plants in the greenhouse.
The plants were enclosed in plastic bags and placed in a 10°C
cold box. Condensation resulted in the formation of a film
of water on the plants. After chilling for five hours the
plastic was removed and the plants were returned to the green
house where pustules containing sporangia appeared on leaves
in one to two weeks.
B. Specimen Preparation for Electron Microscopy
Discs 0.8 mm in diameter were punched out of pustules
with a hypodermic needle as described by Peyton and Bowen
(1963). These discs of infected leaf tissue were immediately
placed in vials containing fixative.
Most of the material studied was fixed in glutaraldehyde
followed by a post-fixation in OSO4 (modified slightly from
Sabatini, Bensch, and Barrnett, 1963). One part of 0.067 17
M KHgHPO^ was added to four parts of 0.067 M EagHPO^ giving
a pH of 7.3. This buffer was used to dilute 25# glutaralde- hyde to a final mixture of 6.5$ glutaraldehyde. The tissue
was fixed 12 to 16 hours at 0 to 4°C, rinsed three times (1 to
3 minutes each) in cold phosphate buffer prepared as above, and then placed in 1% OSO4 buffered with veronal-acetate to pH 7.3 (Palade, 1952), at 4°C for 60 minutes.
Other fixatives used included: unbuffered 4# KMnO^ at room temperature for 10 to 15 minutes (Luft, 1956; Mollenhauer,
1959), 1# OsO^ buffered with veronal-acetate to pH 7*3 (Palade,
I952) both at room temperature and at 4°0 for 60 minutes, and unbuffered 2# OSO4. for 60 minutes at room temperature
(Malhotra, 1962). Of the fixatives used in this study only giutaraldehyde-osmium stabilizes filaments of the mitotic apparatus (Roth, Jenkins, Johnson and Robinson, 1963).
After OSO4 fixation or post-fixation, the fixative was diluted with an equal volume of 70# ethanol for 15 to 30 I seconds, i.e., long enough to homogenize the mixture by gentle rotation of the fixing vial. Dehydration was then completed in five minute intervals of 70#, 95#, and three changes of
100# ethanol. After KMnO^ fixation, specimens were rinsed
/ briefly in distilled water and dehydrated five minutes in each of the following: 50#, 70#, 95# and three changes of 100#.
Dehydration and infiltration was accomplished at room tempera ture. 18
Dehydrated tissue was carried through three changes of propylene oxide at five minute intervals and infiltrated by soaking in Epon-propylene oxide mixtures as follows: 15 minutes in a 1:3 mixture, 30 to 45 minutes in a 1:1 mixture, three to four hours in a 3:1 mixture and 12 hours in 100#
Epon. Infiltration was facilitated by placing the specimen vials on a tilted, slowly rotating wheel until just prior to embedding. The tissue was then embedded in flat, aluminum foil "boats".
The Epon was prepared just before using as described by
Luft (1961). Three volumes of dodecenyl succinic anhydride plus Epon 812 were mixed with two volumes of methyl nadic anhydride plus Epon 812. To this mixture DMP-30 (2,4,6- dimethylaminomethyl-phenol) was added to give a final con centration of 2# v/v.
After embedding, the plastic was polymerized with heat according to the following schedule: 8 to 18 hours at 35*0;
8 to 18 hours at 45*0; and three to seven days at 60®C. The specimens were then ready for sectioning.
By observing the specimens under a dissecting microscope it was possible to trim the blocks so that selected portions of the fungus, namely, intercellular hyphae, sp orangi opho re s, or sporangia, could be sectioned for electron microscopy.
Sections were obtained using duPont diamond knives and an LKB
Dltrotome. Sections showing interference colors in the gray- 19
silver-pale gold (30 to 90 mU) range were selected for elec tron microscopy and picked up on either 400-mesh unsupported or 150-mesh Formvar coated copper grids.
Sections were stained with lead (Karnovsky, 1961;
Millonig, 1961; Reynolds, 1963) or 2% unbuffered, aqueous uranyl acetate (modified from Watson, 1958). With the latter stain a temperature of 45® to 50®0, rather than room tempera ture, was used to obtain increased contrast in shorter times
(Behnke, 1963).
Specimens were observed with an ROA EMU 3F electron microscope equipped with 30 to 4o P objective apertures and operated at 50 kv. Micrographs were taken at magnifications of 2,000 to 17,000 diameters on Kodak contrast plates. 20
IV. OBSERVATIONS AND DISCUSSION
Observations in this study are confined to a description
- of the intercellular hyphae, sporangiophores and developing
sporangia of Albugo Candida. Berlin and Bowen (1964) have
described the fine structure of haustoria of this fungus in
detail, but electron microscope study of the sexual stages
remains to be done. Although this study primarily concerns
itself with centrioles, basal bodies, and flagella, incidental
observations of cytoplasmic organization will be reported.
A. General Fine Structure
1. Intercellular hyphae
The cell is bounded by a cell wall varying in thickness
from 0.25 to 0.8 The cell wall is relatively electron
transparent except for a 10 mP dense external layer (Figure
1). The plasma membrane is a typical unit membrane - approxi
mately 7 mil thick and consisting of two outer electron dense
layers enclosing a less dense layer. As shown by Berlin and
Bowen (1964), mitochondria, ribosomes, lipoidal inclusions,
lomasomes, elements of the endoplasmic reticulum, vacuoles,
and perinuclear Golgi complexes are present (Figures 1, 2, and
3). No septa were observed in the coencytlc fungal vegetative
body. These observations agree with those made by Peyton and Figure 1. Survey view of intercellular hypha. The fungal cell wall (FW) and its external dense layer (DL) are apparent. A perinuclear Golgi complex (G), mitochondria (M), vacuoles (V), a lomasome (LO), endoplasmic reticulum (BR), rîbosomes (R), a nucleus (N), and a centriole (0), in an indenta tion of the nuclear envelope (NB), are present. Note the chloroplasts (CL) in the host cell above the hypha. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 2. Intercellular hypha. Numerous mitochondria (M), ribosomes (R), vacuoles (V), and a spindle- shaped nucleus (H) containing ribosome-like particles (R) interspersed between spindle filaments (S) are observed. Nuclear pores (HP) are present in the nuclear envelope (NB). Glutaraldehyde post-osmium fixation. Section stained 20 minutes with lead (Hillonig)
Figure 3. Survey view of intercellular hypha and spor anglophore. A constriction of the protoplast (arrows) occurs between the intercellular hypha on the left and the club-shaped sporanglophore on the right. Many lipoidal inclusions (L) are found in the intercellular hypha. A thick wall (SPW) is observed in longitudinal and cross section around sporanglophore bases. Spherical nuclei (N), mitochondria (M), and vacuoles (V) are present in the sporanglophore. Glutaralde- hyde post-osmium fixation. Sections stained two hours in uranyl acetate
Figure 4. Survey view of sporanglophore and young spor angia. The thick sporanglophore cell wall (SPW) is evident as are numerous nuclei (H), mito chondria (M), vacuoles (V), and lipoidal inclu sions (1). Note the continuity of the protoplast between the sporanglophore and the second sporangium. Glutaraldehyde post-osmium fixation. Sections stained two hours in uranyl acetate 24 25 I
Bo wen (1963) on the intercellular hyphae of the closely related Phycomycete, Peronospora manshurica (Naom.) Syd. ex
Gaum.
2. Sporangiophores
The sporangiophores are specialized branches of the intercellular hyphae located between the host mesophyll cells and the lower epidermis. The cytoplasm of the intercellular hyphae is continuous with that of the club-shaped sporangio phores and is constricted at the base of the sporanglophore
(Figure 3). The wall of the sporanglophore is very thick (up to two micra) except at its tip where sporangia are produced
(Figures 3 and 4). The organization of the protoplasts in the sporangiophores and the intercellular hyphae is essentially similar.
Sporangia are continuously produced, forming a chain at the tips of the sporangiophores (Figure 4). The nearer the sporangium is to the sporanglophore the younger it is in relation to the other sporangia of the same chain. The protoplast of the sporanglophore is continuous with that of the first sporangium and is frequently continuous with the protoplast of the second sporangium (Figure 4). 26
3. Sporangia
Sporangia of A. Candida have been estimated to contain between four and eight nuclei (Wager, 1896; Endo and Linn,
I960). Although a complete set of serial sections through a sporangium was not obtained, these numbers would seem con servative in that five profiles of nuclei were seen in a single section of a sporangium (Figure 5). The sporangia contain many mitochondria and ribosomes (Figures 5 and 6).
In addition, endoplasmic reticulum (Figures 6 and 7) and perinuclear Golgi (Figure 7) appear to be better organized than in the intercellular hyphae. lomasomes are fewer in number and less complex than those found in the intercellular hyphae and sporangiophores. It is likely that differences observed in organelle morphology between intercellular hyphae and sporangia are results of varying functional requirements in the different structures.
Lipoidal inclusions are found in all parts of the fungal protoplast, but are particularly abundant in sporangia where they are found both in the ground cytoplasm and free in the vacuoles (Figures 5 to 10). After permanganate fixation, lipoidal inclusions appear as electron transparent regions surrounded by thin, irregularly-shaped electron dense zones
(Figure 7 and 8). Osmium-fixed lipoidal inclusions appear as electron dense masses (Figure 9). Lipids are poorly fixed by the aldehyde and post-osmium procedure and, after this
I Figure 5. Sporangium. Five nuclear profiles (N), numerous lipoidal inclusions (L), and mitochondria (M) are present. Glutaraldehyde post-osmium fixation. Sections stained two hours in uranyl acetate
Figure 6. Sporangium. Note lipoidal inclusions (L), mitochondria (M), and the endoplasmic reticulum (BR) with attached ribosomes (R). Glutaralde- hyde post-osmium fixation. Sections stained 20 minutes in lead (Millonig)
Figure 7. Sporangium. A nucleus (N), a perinuclear Golgi complex (G), mitochondria (M), and endoplasmic reticulum (BR) are observed. Lipoidal inclusions (L) appear electron transparent. Permanganate fixation 28 Figure 8. Sporangium. Electron transparent lipoidal inclusions (L) are found both in vacuoles (V) and free in the ground cytoplasm. Permanganate fixation
Figure 9. Sporangium. Electron dense lipoidal inclusions (L) are observed in vacuoles (V) and in the ground cytoplasm. A spindle-shaped nucleus (N) is present. This shape indicates that the nucleus is undergoing mitosis, however, spindle filaments are not preserved by this fixation, A centriole (0) is located in an indentation of the nuclear envelope (NE). Veronal acetate buffered osmium fixation. Section stained 20 minutes in lead (Reynolds) 30 Figure 10. Survey view of a sporangium. An old sporangium with several eccentroids (E), two flagella (FG), lipoidal inclusions (L), and vacuoles (V) is shown. The vacuoles appear to line up and separate each nucleus (N) and its surrounding cytoplasm from adjacent nuclear-cytoplasmic areas. Unidentified bodies (U) of moderate electron density are found near the sporangial wall (Stf). Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 11. Sporangium. Unidentified bodies (U) are observed lying near the sporangial wall (SW). A basal body (B) is found between the nucleus (N) and the sporangial wall and an eccentroid (E), perinuclear Golgi complex (G), several vacuoles (V), and cross-sectioned filaments (F) are evident. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
I 32 33
treatment, lipoidal inclusions are difficult to identify
(Figures 5, 6, and 10).
In the oldest sporangia a vacuolate zone roughly
separates each nucleus and a surrounding region of cytoplasm
from the cytoplasm associated with adjacent nuclei (Figure
10). Presumably these vacuolate zones mark the future planes
of cytokinesis, although differentiation of zoospores was not
followed.
In glutaraldehyde-po st-o smium-fixed sporangia there are
many unidentified bodies of moderate electron density which
range from 200 to 350 mî* in diameter and appear to be bounded
by a single membrane. These bodies were not observed by any
other fixation and it is possible that they are either arti
facts of glutaraldehyde fixation or that they are not fixed
by osmium or permanganate. In older sporangia they are
primarily located adjacent to the sporangial wall (Figures 10
and 11) and they may possibly be acrosomal in function.
Previously undescribed spherical bodies with a character
istic internal arrangement are found in the oldest sporangia
(Figures 10 to 14). These structures have an eccentric
organization and will be called "eccentroids". Eccentroids
are relatively uniform in size, about 250 m# in diameter, and
are membrane limited. In osmium or glutaraldehyde post-osmium
fixation they contain an eccentrically-placed granular mass
roughly 100 to 150 mP in diameter. The remaining cap-shaped Figure 12. Sporangium. Eccentroids (E) with moderately electron dense eccentric regions and electron transparent caps. Veronal acetate buffered osmium fixation. Section stained 20 minutes in lead (Karnovsky)
Figure 13. Sporangium. Eccentroids (E) showing lamella like organization of the eccentric region and the electron transparent cap. Unbuffered osmium fixation. Section stained 20 minutes in lead (Millonig)
Figure 14. Sporangium. Eccentroids (E) electron transparent eccentric region and a moderately electron dense cap. Permanganate fixation 35
j*K5Ï 36
portion of the eccentrold is electron-transparent (Figures 10
to 12). In unbuffered osmium-fixed material the eccentrically-
placed internal sphere appears to have a lamellar organization
(Figure 13). Eccentroids are not well fixed by permanganate
but it appears that the eccentric mass is electron transparent
while the cap is moderately electron-dense (Figure 14). The
possibility exists that the internal structure of eccentroids is an artifact of fixation. However, all of the fixatives used gave uniform results in regard to the eccentric pattern.
Eccentroids bear at least a superficial resemblance to poorly resolved 90 by 160 mP "dense bodies" described by Schuster
(1963) in all stages of the life cycle of the protozoan
Haegleria. Schuster (1963) suggests that the dense bodies in
Naeglerla may be viral particles. Since eccentroids were found only in the oldest sporangia it seems unlikely that they could be viral in nature.
B. Centrloles
Paired centrloles were observed near both interphase and mitotic nuclei in intercellular hyphae » sporangiophores, and young sporangia (Figures 1, 9, and 15 to 20). They are usually adjacent to indentations of the nuclear envelope
(Figures 1, 9, and 16 to 19). Each centriole is about 200 m# in diameter and length and consists of a cylinder composed of I
Figure 15. Intercellular hypha. Paired centrloles (G) located near a nucleus (N) are shown in near median longitudinal section. Note the close end-to-end association between the two centrloles. The proximal end of the centrloles contain a central filament and a dense zone (DZ) is present in the distal ends. Radiating astral filaments (A) are present. Glutaralde hyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 16. Sporanglophore. A centriole (0) is shown in cross -section near the nucleus (N). Note the central filament and the radial elements which give the appearance of a hub and spoke arrangement. An astral filament (A) is present. Glutaraldehyde post-osmium fixation. Sections stained two hours in uranyl acetate 38 /XV'
Figure 17. Survey view of a sporangium. Note the centriole (0) located in an Indentation of the nuclear envelope (NE). Spindle filaments (S) are present in the nucleus (N) and a perinuclear Golgi complex (G) is evident. An elaborate nuclear bleb of unknown significance is shown. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 18. Enlarged view of a section serial to that shown in figure 17. Note the spindle filaments (S) oriented with respect to the obliquely- sectioned centriole (0) even though separated from it by the nuclear envelope (NE). An astral filament (A) is also present. Glutar aldehyde post-osmium fixation. Section stained two hours in uranyl acetate 4o Figure 19. A prophase nucleus in a sporanglophore. Note the centrloles (0) at both poles where they are located in indentations of the nuclear envelope (NE). A perinuclear Golgi complex (G), a lomasome (L0), and numerous astral filaments (A) and mitochondria (M) are present. Nucleolar material (NU) is present in the spindle-shaped nucleus (N). Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate I
42 43 nine peripheral triplets. A single axial filament was noted in the proximal two-thirds to three-fourths of the centriole
(Figures 15 and 16) and an ill-defined dense zone, approxi mately 80 mV> in diameter, was found in the center of the cylinder at its distal end (Figure 15). Each filament appears tubular and the individual peripheral filaments average 26 mP in diameter while the single central filament has a diameter of about 22 mU. The three filaments of each triplet are closely appressed and arranged in a row that is inclined at an angle of about 45° to a tangent to the circumference of the cylinder. The triplets appear to be connected to the central filament by radial elements. Thus, the centrloles in cross section usually have a cart-wheel appearance with a hub and spokes (Figure 16).
The centrloles described herein represent the first fine structural description of centrloles in fungi. The structure of centrloles in A. Candida varies only in detail from that reported for centrloles in other plants (Berkaloff, 1963;
Gall, 1963) and in animals (de Harven and Bemhard, 1956;
Amano, 1957; Bessis and Breton-Gorius, 1957; Bemhard and de
Harven, I960; Gall, 1961). Typically centrloles in other organisms are about 150 mU in diameter and 400 to 500 mP long, hence, it appears that centrloles in A. Candida are shorter, but larger in diameter than most other centrloles. However, centrloles in A. Candida are longer than the 70 mP long Figure 20. A metaphase nucleus in a sporanglophore. Numerous spindle filaments (S) and astral filaments (A) are shown. Nucleolar material (NU) is concentrated near the poles and ribosome-like particles (R) are interspersed between the spindles. Chromosomes are poorly preserved but are present in the broad por tion of the spindle-shaped nucleus (N). A pair of end-to-end centrloles (0) with radiating astral filaments (A) are associated with a nucleus out of the plane of this picture. Glutaraldehyde post-osmium fixation. Section stained two hours with uranyl acetate 45
I 46 procentrioles of Vlviparus and Marsilea (Gall, 1961, 1963).
Even though the structures observed in the vegetative cells of A. Candida are short in relation to centrloles of other organisms they apparently function in mitosis as centrloles and probably should not be termed procentrioles.
The centrloles of a diplosome tend to lie end-to-end
•with little or no angle between their axis and this angle never approximates 90° as reported for the majority of other organisms. Centrloles in near-median longitudinal section were observed in very close end-to-end association, with a possible connection of central filaments (Figure 15). As suggested by Gall (1961) the proximal end of centrloles, and perhaps the central filament, may play a role in centriolar replication. It has been suggested that the perpendicular arrangement of centrloles in other organisms is related to their mode of duplication (Bemhard and de Harven, I960).
The finding of procentrioles perpendicular to mature centrl oles (Gall, 1961) lends considerable support to this theory.
However, on the grounds of end-to-end organization of centrl oles in A. Candida, it seems that centriole replication in this organism may occur end-to-end rather than in a 90° arrangement.
Centrloles of mitotic nuclei appear to be located at the foci of filamentous structures which by definition are spindle filaments when they occur in nuclei (Figures 2, 17, 18, and 47
20) and astral filaments when they occur in the cytoplasm
(Figures 15, 16, 18, 19, and 20). The filaments of spindles appear to be 15 to 20 mU in diameter while astral filaments are about 22 mP in diameter. A clear zone of approximately
11 mU characteristically surrounds both spindle and astral filaments (Figures 15, 16, 19, and 20). These filaments are oriented toward the centriole, but were never observed to make actual physical contact with it. As will be discussed later there is no breakdown of the nuclear envelope during mitosis.
In mitosis, centrioles were found at both poles outside of the nuclear envelope (Figure 19). It is interesting to note that the nuclear envelope always appeared intact between the intranuclear spindle filaments and centrioles even though the spindle filaments were rather precisely oriented with respect to the centriole (Figure 18). These observations support
Sleigh's (1962) statement that the function of centrioles appears to be either the formation or organization of fila ments.
Wager (1896) reported that nuclear division does not occur in sporangia, but spindle filaments were observed in a
sporangial nucleus in this study (Figures 17 and 18). This nucleus also has a highly irregular bleb and there is a pos
sibility that this particular nucleus is undergoing an abortive mitosis.
Interphase nuclei of A. Candida are generally spherical 48 in shape (Figures 3 to 5 and 10), average three to four miera in diameter, and are bounded by a typical porous nuclear envelope. The several mitotic nuclei observed had intact nuclear envelopes and were spindle-shaped with a long axis of about six miera (Figures 2, 19, and 20). Chromosomes were difficult to identify. Nucleolar material was clearly evident, concentrating in the polar regions at metaphase
(Figure 20). Ribosome-like particles were observed between the spindle filaments (Figures 2 and 20).
Davis (1900), studying the sex organs of A. Candida, reported an intranuclear mitosis with a persistent nuclear envelope occurred in the developing oosphere. The above observations suggest that mitosis is intranuclear in the vegetative cells. An intranuclear spindle is found in many fungi and protozoans (Darlington, 1937; Roth, 1964) and may be suggestive of a phylogenetic link between these two groups.
C. Basal Bodies
In developing sporangia, centrioles elongate forming basal bodies approximately 600 mP- long (Figures 21 and 22).
Basal bodies are found in pairs between the nucleus and the sporangia! wall (Figures 11, 22, and 23) and each basal body gives rise to a flagellum which projects into a vacuole
(Figure 22). As previously described, little or no angle is Figure 21. Sporangium. A pair of basal bodies (B) which have not initiated flagella are shown in longitudinal section. A central filament (OF) is present in the proximal end of one of the basal bodies and both basal bodies have a dense zone (DZ) in their distal ends. G-lutar- aldehyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 22. Sporangium. A longitudinal section of two flagella (FG) projecting into vacuoles (V) located between the nucleus (N) and the sporangial wall (SW). Note the spur filaments (SR), basal bodies (B), and the basal plates (BP), which separate the basal bodies and the flagella. A dense zone (DZ) is indicated in the distal end of one of the basal bodies. There appears to be an abundance of cyto plasm (CY) between the flagellar membrane (FM) and the peripheral filaments of the flagella. Glutaraldehyde post-osmium fixa tion. Section stained two hours in uranyl acetate 50 Figure 23. Sporangium. A pair of basal bodies (B) are located between the nucleus (N) and the sporangial wall (SV7). The central filament and the radiating elements present in the proximal end of basal bodies is shown. Unidentified bodies (U), vacuoles (V), and filaments (F), appressed to the cytoplasmic side of the nuclear envelope (NE), are present. Glutaraldehyde post-osmium fixa tion. Section stained two hours in uranyl acetate
Figure 24. Sporangium. A cross-section of the transition region on the flagellar side of the basal plate is shown. Note the flagellar membrane (FM) with nine undulations and the surrounding vacuole (V). The dense zone (DZ) of the transition region is seen in the middle of the flagellum. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 25. Sporangium. A slightly oblique section of the transition region near the basal plate. A triplet, characteristic of basal bodies, and doublets, of flagella, are present. Note the undulating flagellar membrane (FM) and the cross-sectioned spur (SR) which is adjacent to a vacuole (V). Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate 52 53
present between paired centrioles. However, the paired basal
bodies rather than lying end-to-end are at an angle of 120°
to 165° with each other. The individual basal bodies thus
tend to lie at angles of from 20° to 30° to a plane tangential
to the nucleus (Figure 22). Basal bodies of A. Candida have
the same diameter as centrioles and generally resemble
centrioles in structure. The central filament is present only in the proximal third of the basal body, having elongated little, if any, during the transition from a centriole. A
dense zone, similar in size and probably homologous to that
found in centrioles, occurs in the center of the basal body at its distal end (Figures 21 and 22). The distal end of the
centriole becomes the distal end of the basal body and it is
at this end that flagella are initiated. Since the central filament at the proximal end of the centriole and the central
dense zone at the distal end show little change in dimension or position during basal body formation, the suggestion that the centriole elongates interstitially is very reasonable.
However, other patterns of elongation are not necessarily ruled out.
The elongation of a procentriole into a centriole is prob ably analogous to centrioles elongating to form basal bodies.
Gall (1961) was unable to observe intermediate structures in the process of centriole development and the investigation reported here has not revealed intermediate structures in the 54
course of basal body development. These two negative observa
tions suggest that the elongation of both these structures
occurs very rapidly.
Although basal bodies in A. Candida appear to be derived from centrioles, this may not be a universal phenomenon.
Ehret and Powers (1959) and Ehret and de Haller (1963) dispute the Henneguy-Lenhossek theory and report that basal bodies
arise de novo in Paramecium.
D. Flagella
>
Only a few basal bodies (so-called because of their
length) were observed which had not started flagellar initia
tion (Figure 21). The basal plate (Figure 22) separating a
basal body from its flagellum is the most characteristic
feature of the transition region. This plate appears as two O extremely electron dense 20 A layers separated by a less dense 0 1 30 A layer. The basal plate is in a plane approximately
tangential to the membrane of the vacuole into which the
flagellum protrudes.
In most organisms, it is observed that the membrane which
encloses the flagellum is continuous with the plasma membrane
of the cell (Sleigh, 1962). Therefore, it is probable that as
the flagellum of A. Candida protrudes into a vacuole, the
vacuolar membrane becomes the flagellum membrane. On this 55 evidence, it is possible to theorize that during the final stages of zoospore differentiation (not observed in this study), the vacuoles, which roughly divide the sporangia into uninucleate portions, coalesce and their membranes become the plasma membrane of the new zoospores. Such a process would be similar to the coalescence of phragmosomes to form a cell plate reported by Porter and Machado (i960). Near the base of the flagellum the flagellar membrane lies close to the filamentous component and bulges over each doublet forming a highly regular pattern of undulations (Figures 24 and 25).
The transition region contains a central electron dense zone about 80 mP> in diameter extending approximately 100 mP above and below the basal, plate (Figures 22 and 24). This zone appears to be homologous with the similarly-positioned dense zone of the basal body and centriole.
In cross section each of the paired flagella has the expected nine peripheral doublets and two central filaments characteristic of the flagella of eucaryotic cells (Figure
26). Bach of the filaments in the doublets are about 26 ml* in diameter and each of the central filaments average 22 mP> in diameter. The doublets, like the triplets of centrioles and basal bodies, are at an angle of approximately 40° to the tangent to the flagellar cylinder. The flagellar doublets appear to be continuous with the basal body triplets, but the two central filaments terminate before reaching the basal Figure 26. Sporangium. A cross section of a flagellum (FG) is seen in a vacuole (V). Cytoplasmic material (CY) is observed between the flagellar membrane and the doublets. Glutar aldehyde post-osmium fixation. Sections stained two hours in uranyl acetate
Figure 27. Sporangium. A pair of obliquely-sectioned basal bodies (B), each of which is associated with its own longitudinally-sectioned spur (SR). The vacuoles (V) into which the flagella project are evident. Glutaraldehyde post-osmium fixation. Sections stained two hours in uranyl acetate
Figure 28. Sporangium. A spur (SR) is shown in cross section with electron dense bars above and below the flat plane of the spur. An obliquely-sectioned basal body (B) is present between the nucleus (N) and the sporangial wall (SW). Filaments (F) are seen both appressed to the cytoplasmic side of the nuclear envelope (NE) and randomly distributed in the cytoplasm. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate 57 58
plate (Figures 22, 24, and 25).
Structural elaborations such as arms, radial elements
and extra filaments occurring within flagella have been
reported by Afzelius (1959), Gibbons (1961), and Gibbons and
Grimstone (I960) in animals and by Lang (1963a, 1963b) and
Manton (1957) in plants. Similar complexities were not
observed in flagella of A. Candida. The absence of these
special structures may be due to the fact that the flagella of A. Candida observed in this investigation were rudimentary and, as yet, non-functional.
The presence of flagella in sporangia of A. Candida is in contrast with de Bary's (1863) report that flagella are not found until after zoospore dispersion. It is probable that flagellar initiation represents one of the early steps in differentiation of multinucleate sporangia into uninucleate,
biflagellate zoospores.
Flagellar formation in A. Candida cannot be completely
described until zoospore differentiation has been followed to its conclusion. The longest flagellum observed in a sporan
gium was one micron while flagella of mature zoospores are
about 25 P in length. All rudimentary flagella in the most
mature sporangia studied appeared similar and no clearcut
differences which might later distinguish 11 whiplash" from
"tinsel" flagella were observed. Flagella are generally
described as compact structures, however, in the developing 59
flagella of A. Candida a large amount of cytoplasm was fre
quently found between the flagellar membrane and the fila
mentous component (Figures 22 and 26). This material may have
a role in flagellar formation.
B. Filamentous Structures Associated
with Basal Bodies
1. Spur
A structure closely resembling the spur described by
Schuster (1963) in the protozoan Naegleria was found in A.
Candida. It consists of a linear arrangement of four to nine filaments originating near the proximal end of each basal
body. It makes an angle of about 45° with the basal body and is located on the side of the basal body away from the nucleus
(Figures 22, 27, and 28). The individual spur filaments are about 28 mil in diameter and the longest spur observed was 750 mil. Adjacent filaments are about 4 mH apart. Two electron dense bars, each 12 mU thick, are occasionally seen to lie above and below the flat plane of the spur (Figure 28). At a distance from their points of origin near the basal body, spur profiles characteristically were found adjacent to vacuoles (Figure 25). 6o
2. Rhlzoplast
Between the basal body and the spur an electron dense amorphous mass (Figure 22) was observed. An assemblege of
26 mil diameter filamentous elements appear to emanate from this mass (Figure 29). This filamentous component is apparently similar to the rootlet system or rhlzoplast described in a similar position in flagellated cells of a variety of organisms (Manton and Leedale, 1961; Anderson and
Beams, 1961; Anderson, 1962; Schuster, 1963). The rhlzoplast of A. Candida varies in width from 50 to 100 mil and the maxi mum length observed was a little over one micron. Unlike the precise striated organization of rhizoplasts of other organ isms, only occasionally was there any suggestion of striation in the rhlzoplast of this fungus. Again this may be due to the rudimentary, non-functional nature of the structures at stages observed in the present investigation.
3. Ancillary cytoplasmic filaments
Filaments about 22 mil in diameter originate in the vicinity of the proximal ends of basal bodies (Figures 23,
28, 29, and 31). The majority of these filaments are appressed to the cytoplasmic side of the nuclear envelope (Figures 22,
23, 28, 29, and 30) while others randomly radiate into the cytoplasmic region between the nucleus and the sporangial wall Figure 29. Sporangium. A longitudinally-sectioned rhlzoplast (RH) appears to originate on the side of a basal body opposite the nucleus (N). Two eccentroids (E), two basal bodies (B), one in cross section and one obliquely- sectioned, and numerous filaments (F) are shown. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
l
Figure 30. Sporangium. Two basal bodies (B), one of which exhibits a hub and spoke arrangement, and numerous filaments (F) are observed. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate
Figure 31. Sporangium. Filaments (F) radiating from the proximal ends of basal bodies have an appear ance similar to an aster. A basal body-basal plate-flagellum (B-BP-FG) is seen in oblique section. Glutaraldehyde post-osmium fixation. Section stained two hours in uranyl acetate 62 63
(Figures 28 and 30). Similar aster-like configurations accompanying basal bodies have previously been described by
Gibbons (1961) and Harris (1962).
These filaments resemble those which have been reported in a variety of non-mitosing cells, e.g., the filaments con necting the basal foot of the kinetosome of Anodonta (Gibbons,
1961); filaments noted in protozoa (Roth, 1958; Schuster,
1963); and microtubules in Hydra (Slautterback, 1963) and higher plants (Ledbetter and Porter, 1963; Berlin and Bowen,
1964).
F. Filamentous Structure; a Review
The diameters of various filamentous structures observed fall into two ranges. Included in the range of 15 to 22 mP are the spindle, astral, and ancillary cytoplasmic filaments, as well as the central filaments of centrioles, basal bodies, and flagella. The individual elements in the perpheral doublets and triplets of centrioles, basal bodies, and flagel la, as well as the individual filaments of the spur and rhlzoplast are in the 26 to 28 mP> range.
A bimodal size distribution similar to that noted above has been reported by Slautterback (1963), Ledbetter and
Porter (1963), Roth (1964), and Roth and Shigenaka (1964).
Slautterback (1963) extensively reviews the literature of 64
"cytoplasmic microtubules" and reports diameters of either
27 mP- or from 12 to 20 mP. He suggests that the smaller
filaments are involved in synthetic or metabolic cellular
activities while the larger filaments appear to function as
elastic bodies. Roth (1964) and Roth and Shlgenaka (1964) find a bimodal distribution of 15 and 21 ml*. These authors
suggest there may be transitions from one size to the other and that such transitions may be functionally important.
Until the mode of operation and functions of the structures involved is understood, it seems premature to attach any significance to the variation in filament diameter observed in this study. 65
V. SUMMARY
1. Selected asexual stages of the fungus A. Candida were
studied using the electron microscope. Various cell organel les are described, particularly in relation to specific stages of the asexual life cycle.
2. The fine structure of the intercellular hyphae, sporangiophores, and sporangia is described. Previously uncharacterlzed structures of unusual morphological organiza tion were found in older sporangia and have been termed
"eccentroids".
3. Paired centrioles occurring end-to-end, or nearly so, have been found and described in the intercellular hyphae, sporangiophores, and young sporangia of this fungus. The fine I structure of these centrioles is similar to that of centrioles found in other organisms. It is suggested that these centri oles may reproduce themselves by an end-to-end process rather than by the production of a lateral procentriole.
4. With present day techniques of electron microscopy it is impossible to actually observe a centriole being trans formed into a basal body. However, evidence accumulated in this investigation strongly suggests that centrioles elongate to become basal bodies, thus confirming the Henn eguy-Lenho s s ek theory of centriole-basal body homology, at least, when applied to this organism. 66
5. Both a morphological and functional polarity of centrioles and basal bodies was found in A. Candida. The lumen of the proximal ends of centrioles and basal bodies possesses a central filament while a dense zone is present in their distal ends. It is suggested that the proximal end of the centriole is involved in centriole replication. The proximal ends of basal bodies are associated with the forma tion or organization of a variety of filamentous structures while the distal end of basal bodies is the end from which flagella grow.
6. The various filamentous structures observed were found to have a diameter of either 15 to 22 ml& or 26 to 28 mP>. 67
VI. ACKNOWLEDGEMENTS
The author wishes to take this opportunity to thank
the many persons who inspired him throughout the course of
this investigation. First and foremost, thanks are due to
Dr. C. C. Bowen, under whose supervision this study was
carried out, for guidance and helpful and constructive criticism. The writer appreciates the interest and advice of Dr. J. 0. Gilman, who identified and provided the material studied, and is grateful to Drs. L. Tiffany and L. B. Roth for their counsel and suggestions, especially during the preparation of the thesis. The author is indebted to Dr.
J. E. Peterson, University of Missouri, for providing the original stimulus to undertake electron microscopy, and to
Mr. H. S. Pankratz for his many suggestions during this investigation.
The writer appreciates the patience and understanding of his wife, Ellen, throughout the study.
This investigation was supported in part by research
grant 0=3982 from the National Institutes of Health, United
States Public Health Service. 68
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