Zoospore germination in the water mold, Blastocladiella emersonii

Reprinted Copyright

from DEYELOPYENTIL 0 1969 by Academic

DEVELOPMENTAL

BIOLOGY, Press, Inc.

Volume

20, No. 3, September,

PTinted

1969

in 1T.S.A.

20, 183-217 (1969)

BIOLOGY

Zoospore

Germination

in the Water Mold,

Blastocladiella I. Measurement

emersonii

of Germination

and Sequence of Subcellular

Morphological

Changes’

D. R. SOLL, R. BROMBERG,AND D. R. SONNEBORN Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706 Accepted

March

14,1969

INTRODUCTION

While much has been learned about the structure and function of different subcellular organelles, very few analyses are as yet available concerning their involvement in eukaryotic cell differentiation. The life cycle of the water mold Blastocladiella emersonii furnishes favorable material for such an analysis. The Blastocludiella zoospore contains a highly ordered array of organelles. Parts of this array include: (a) a single posteriorly situated flagellum, (b) a single nucleus containing a single nucleolus, (c) a single membrane-bound “nuclear cap” overlying the nucleus and containing most, if not all, of the cell’s ribosomes, (d) a single giant mitochondrion positioned eccentrically around the nucleus, and (e) a single membrane-bound “lipid granule”-containing body extending along the outer margin of the long arm of the mitochondrion. [See Cantino and Lovett (1964) and Fuller (1966) for reviews of zoospore structure.] An additional distinguishing feature of the zoospore is the absence of a cell wall. The zoospore is a motile cell which does not, so far as is known, grow. Through a series of abrupt changes in cell architecture, it transforms into a sessile cell capable of growth, the germling. This latter cell no longer contains a flagellum, a nuclear cap, or a single, unbranched mitochondrion but is surrounded by a cell wall. Growth ’ This work has been supported by Grant GB 6030 from the National Science Foundation, by Grant GM 1382 from the National Institutes of Health, and by institutional grants originating with the Wisconsin Alumni Research Foundation and the American Cancer Society. The senior author is supported by a predoctoral fellowship on Training Grant 5.TOl-GM 01435 from the National Institutes of Health. 183 Copyright

0 1969 by ilcademic

Press,

Inc.

184

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ROUND-CELL C.W.

ROUND-CELL

life cycle of Blastoc2adielh emersonii. FIG. 1. Sketch of the “ordinary colorless” Drawings are not necessarily drawn to scale (e.g., the sporangium at the end of growth should be much larger) but are intended to show the dispositions of organelles. C. W., cell wall; F, flagellum; G. P., gamma particle; G.T., germ tube; L.G., lipid granules; M, mitochondrion; N, nucleus; N.C., nuclear cap; NW, nucleolus; P., exit pore; Rh., rhizoids; Ribs., ribosomes.

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is accompanied by nuclear division in the absence of cell division. After the completion of growth along the “ordinary colorless” (Cantino and Lovett, 1964) or “zoosporangial” (Lovett, 1967) pathway, the multinucleate syncytium can cleave into a population of uninucleate zoospores each displaying the characteristic arrangement of internal organelles. The zoospores are then released through one or more exit pores, leaving behind the cell wall plus the anucleate rhizoid system (cf. life cycle sketch, Fig. 1). Two phases of the life cycle are thus clearly marked by dramatic examples of organelle morphogenesis-the formation and release of zoospores (sporulation), and the conversion of zoospores to germlings (germination).2 In the present paper, procedures are presented whereby the progress of germination in populations of transforming zoospores may be quantitatively evaluated. Quantitative evaluation of the progress of eukaryotic cell differentiation has been beset with technical and interpretive difficulties. Such cell differentiations are customarily accompanied by a complex, progressive sequence of functional and morphological changes rather than by a single event. Even when single events are examined, curves describing phenotypic change in cell populations are composed of two rate components: a cell velocity component and a cell heterogeneity component. The former component refers to the rate at which cells participating in a given change proceed through the change. The latter component arises because cell populations are never completely synchronous-i.e., each cell in the population does not accomplish a given phenotypic change at the same instant as every other cell. Since the cell heterogeneity component has customarily remained unknown or difficult to define with precision, the relative contribution of the two components to the curves obtained has not been resolved. To help overcome these and other difficulties, Fulton and Dingle (1967) have recently suggested and discussed in detail a quantitative methodology. Their suggestion is to dissect from the continuum of events accompanying cell differentiation single characteristics which can be monitored as all or none, or quantal, changes. This method allows direct measurement of the cell heterogeneity component of phenotypic change. In addition, the initial or average time of change ’ For a study of organ& changes accompanying (1968). Diagrammatic sketches of organelle changes found in Lovett (1968).

sporulation, accompanying

see Lessie and Lovett germination can be

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can be determined with the method and these parameters yield information concerning the velocity component. With respect to zoospore germination, we distinguish three quantal phenotypes-zoospore, round cell, and germling. Procedures are described which reproducibly allow rapid, complete, and moderately synchronous cell transformation. Attention is then directed to the sequence of subcellular morphological changes which accompany the shifts in quanta1 phenotype. On the basis of this and other information, a novel working hypothesis is presented to account for most of the changes which occur during zoospore germination. MATERIALS

and

METHODS

Organism and stock maintenance. Stocks of Blustocludiellu emersonii were generously provided by Drs. Edward Cantino and James Lovett. These stocks were all derived from the original isolate described by Cantino (Cantino, 1951; Cantino and Hyatt, 1953). From the stock received from Dr. Lovett, four clonal lines were initiated. These lines have each been subjected to periodic subcloning over the course of three years. Recently, two stocks were received from Dr. Cantino, his standard stock and his Be. var. 1. The latter stock is distinguishable from the standard stock in its growth behavior and pigmentation on standard agar growth plates (Cantino, personal communication). All these cultures yield zoospore populations which behave indistinguishably in germination assays. Stock and mass cultures were maintained on Difco Cantino PYG agar (Difco Laboratories, Detroit, Michigan). Freshly released zoospores were obtained by flooding overnight mass cultures with distilled water. All manipulations of living material were performed with sterile technique. Preparation of zoospore suspensions for germination assays. Fresh zoospore suspensionswere counted and diluted into 10 ml of defined liquid growth medium (cf. below) in 100 X 20 mm tissue culture dishes (Falcon No. 3003). The range in inoculum size for different experiments was 1.5 to 5 x lo6 zoospores per dish. Once the cells germinated, they adhered firmly to the bottom of the dish.3 Growth was terminated after 9-14 hours at 20°C by decanting the growth medium. The attached cells were rinsed four times with 3 ml of sporulation solu,’ Tissue culture, rather than bacteriological, plastics have been used for all dish cultures because the rapidity and tightness of adhesion of cells to the dish bottom are much more pronounced and regular.

MORPHOLOGICAL

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187

tion (lo-” M CaCl* in lo-:’ M Tris-maleate, pH 6.7) and were then incubated at 27” C in 4 ml sporulation solution. Zoospore release began after 3-3.5 hours and within 40-60 minutes thereafter virtually every sporangium had discharged its zoospore progeny. Within 1 hour after the completion of zoospore release, the zoospores were gently pipetted into uniform suspension and withdrawn for use in the germination assays. The defined liquid growth medium, DM-2. In spinner flask growth cultures (Murphy and Lovett, 1966), DM-2 liquid growth medium permits a mass doubling time approximating that of the undefined peptone-yeast extract-glucose (PYG) medium standardly used for Blastocladiella. However, the mass yield after exponential growth is less in DM-2 than PYG cultures (Gottlieb, unpublished). A difference between zoospores obtained from DM-2 as opposed to PYG cultures will be discussed in the accompanying paper (Sol1 and Sonneborn, 1969). For such reasons, we view DM-2 as a workable, but not optimal, defined growth medium. The medium is sterilized by Millipore filtration and contains (per liter): 10 ml of the amino acid mixture detailed below; 20 gm L-glutamic acid; 20 pg thiamine HCl; 0.5 gm glucose; lo-’ M MgS04 ; lo-” M CaCL ; 10m4 M Na phosphate, pH 7.0; lo-” M Tris-maleate buffer, pH 6.8 plus enough KOH to bring the medium to pH 6.8; KC1 to bring the concentration of K’ to 5 X lo- ’ M. All inorganic salts are added by dilution from concentrated stock solutions. Glass double-distilled water is used throughout. The stock solution of L-amino acids is dissolved in 350 ml 1 N HCl and then brought to 2 liters with distilled water. It contains: arginine HCl, 4.21 gm., glycine, 1.50 gm; histidine-HCl, 2.10 gm; isoleutine, 5.25 gm; leucine, 5.25 gm; lysine-HCl, 7.32 gm; methionine, 1.49 gm; phenylalanine, 3.3 gm; serine, 2.00 gm; threonine, 4.74 gm; tryptophan, 0.82 gm; tyrosine, 3.62 gm; valine, 4.68 gm. The dish assay of germination. Zoospores are diluted into germination solution (cf. below) in petri dishes. At successive time intervals, dishes are swirled so that zoospores are suspended whereas cells undergoing germination remain attached to the dishes. The attached cells are of two types: round cells, each lacking both an external flagellum and a germ tube; and germlings, each possessing at least one germ tube. The concentration of suspendable cells is determined as is the proportion of round cells and germlings on the dish bottom. The complete protocol is as follows. 60 X 15 mm tissue culture

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dishes (Falcon No. 3002), each containing 3.7 ml of germination solution (10m3 M CaCh, lo-’ M MgCb, 5 X lo-’ M KC1 in 10m3 M Tris-maleate, pH 6.7) are preincubated at 27” C (+l”C) for at least 30 minutes. Inoculation of each dish with 0.3 ml of prewarmed zoospore suspension is performed at room temperature, inoculation of the entire series of dishes being completed. within 1 minute. The dishes are swirled for 30 seconds and returned to the 27OC incubator. The completion of inoculation is taken as zero time, and at each time point a single dish is removed from the incubator. It is swirled in alternating directions for 30 seconds, after which an aliquot of the supernatant is immediately fixed with formaldehyde (9 parts cell suspension to 1 part 35% formaldehyde, CaC03 neutralized). The remaining supernatant is decanted and the dish bottom with attached round cells and germlings is washed and fixed.4 The fixed supernatants are counted after independently filling the opposing chambers of a hemacytometer. Four fields (10e4 ml/field) in each chamber are counted. The initial cell concentration is determined with the zero time supernatant and the range for different assays was 9 to 35 X lo4 cells/ml. The relative proportions of round cells and germlings on the dish bottoms are scored under an inverted microscope with phase optics at 160 x total magnification. At least five fields, chosen at random, are scored. Knowledge of the initial zoospore concentration, the concentration of zoospores at each sampled time point, and the relative proportion of round cells and germlings at that time allows the calculation of round-cell and germling concentrations. Data from a representative assay are plotted in Fig. 5A. The spinner flask assay. The zoospore inoculum is suspended in germination solution in a water-jacketed Bellco spinner flask mounted on a magnetic stirrer. The inside of the flask is siliconized (Siliclad, Clay Adams, Inc., New York, New York) to prevent excessive cell adhesion to the glass surface. Temperature is maintained at 27°C f 0.5OC throughout by means of water circulated through the water jacket from a Forma minitemp waterbath (Model No. XKE-4, Forma Scientific, Marietta, Ohio). In a typical spinner flask assay, 6 ml of prewarmed (27°C) zoospore suspension is added to 74 ml of prewarmed germination solu’ Formaldehyde fixation does not alter the scoring fixed controls. Overnight storage in formaldehyde were routinely scored within 6 hours of fixation.

of cell type as compared is permissible, although

with unsamples

MORPHOLOGICAL

CHANGES

IN

ZOOSPORE

GERMINATION

189

tion in a loo-ml capacity spinner flask (final zoospore concentration, 10 to 25 x lo4 cells/ml). Spinner flasks of greater capacity may be used for experiments demanding greater bulk. At selected time points, 0.9 ml of cell suspension is withdrawn and mixed with 0.1 ml of 35% neutralized formaldehyde. Differential counts are made of the three cell types with phase optics under 600 X total magnification using acid cleaned slides and coverslips. For each time point, 100-150 cells were scored. Data from a representative assay are plotted in Fig. 5B. Classification of cell types. The following paragraphs describe the conventions used in the different germination assays for distinguishing each of the three quanta1 phenotypes. In all assays, each cell examined is scored as one, and only one, of these phenotypes. In the routine dish assay, scoring is based on the segregation of zoospores into the supernatant and of round cells plus germlings to the dish bottom of swirled cultures. Microscopic observations of the fixed dish bottoms consistently revealed that zoospores were quantitatively removed with the supernatants. However, in the supernatants, small numbers of cells were observed which, on morphological grounds, were not zoospores (see next paragraph). To examine this feature of the dish assay, differential counts of zoospores us. round cells in the supernatant samples were performed under high magnification (500-600~) with phase optics using hemacytometer assemblies specially constructed for use with phase optics (A0 No. 1475 American Optical Co., Buffalo, New York). Such differential count assays are referred to as refined dish assays. Where zoospores and round cells are scored in the same sample, as in the spinner flask assays and in the supernatants of the refined dish assays, our convention has been to score as round cells only those cells which are nonflagellated and exhibit smooth-surfaced, perfectly round shapes at high magnification under phase optics. A minor fraction of zoospores no longer possess a visible flagellum at the time of counting (about 1% of the zoospores in the dish assay and less than 6% of the zoospores in the spinner flask assay); such zoospores nevertheless display an irregular, or rough, surface outline of variable cell shape. Without high magnification and/or without phase optics, some of these cells would be classified as round cells. The magnitude of this difference is small under our conditions, but there are other conditions where the difference may be sizable (e.g., Lovett, 1968). That our convention is operationally useful is evi-

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dented by the fact that, by employing it with populations of deflagellated zoospores (where scoring must be purely on the basis of cell appearance rather than presence or absence of a flagellum), curves of zoospore conversion to round cells were obtained which were indistinguishable from those with flagellated zoospores (cf. Sol1 and Sonneborn, 1969). The distinction between round cell and germling is based on the absence or presence of a germ tube. The first visible thin projection beyond the phase halo of round cells is considered to be a germ tube. This convention, while useful, certainly overestimates the time of actual initial appearance of the germ tube. The small, thin projections are easily distinguished from the larger, round “blebs” which appear at the time and site of flagellar retraction in a small percentage of round cells. Reproducibility of the germination assays. Adherence to the simple set of manipulations and scoring procedures detailed above is necessary in order to reproduce the germination kinetics described below. The degree of reproducibility is considered in the Results section. The influence of cellular and environmental variables on germination kinetics is discussed in the accompanying paper (Sol1 and Sonneborn, 1969). Electron microscopy. Freshly released zoospores were obtained by flooding overnight agar cultures with distilled water and were immediately fixed in a buffered glutaraldehyde solution (0.25% glutaraldehyde, 1.25% polyvinyl alcohol, 10m3 M sodium phosphate, pH 7.1). After 45-60 minutes at room temperature, the cells were pelleted and washed several times in phosphate buffer containing polyvinyl alcohol. The cells were postfixed for 45 minutes in osmium textroxide (0.1% OsOl in phosphate buffer plus polyvinyl alcohol). After washing once in phosphate buffer and two times in distilled water, the cells were treated for 2 hours with 0.5% uranyl acetate in 0.1 strength Kellenberger’s Veronal-acetate buffer (Ryter and Kellenberger, 1958). Subsequent steps included washing in Veronal-acetate buffer, dehydrating through a graded series of alcohol concentrations (50-lOOQ, embedding in Epon-Araldite (Mollenhauer, 1964), and sectioning on an LKB microtime fitted with a glass knife. Sections were transferred to 200-mesh carbon-Formvar coated grids (Watson, 1955), then stained for 2 hours at 38°C with magnesium uranyl acetate (Frasca and Parks, 1965) followed by IO-15 minutes with lead citrate (Reynolds, 1963). Micrographs were taken with an Hitachi HS75 electron microscope using Kodak lantern slides.

MORPHOLOGICAL

CHANGES

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GERMINATION

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Round cells and germlings were obtained by sampling germinating spinner flask cultures at appropriate times. The samples were immediately prefixed with glutaraldehyde (1% glutaraldehyde, lo-’ M sodium phosphate, pH 7.1). After 5 minutes, the cells were pelleted and the glutaraldehyde concentration was increased to 4%. After 2 hours, the pellets were washed with phosphate buffer and then incubated in buffer overnight at 4°C. The pellets were postfixed with osmium tetroxide (1% 0~0~ in lo-’ A4 phosphate buffer) ‘for 1 hour. The subsequent steps were carried out in the same manner as described for the zoospores. RESULTS

Population Analyses of Phenotypic Change .Qualitative description of the population. Immediately upon inoculation of zoospores into germination solution, the, cells undergo a momentary, but dramatic, reduction in motility and assume a characteristic oblong shape. Within the first few minutes, zoospore motility reappears. As the period of transformation to round cells approaches, two changes in the population are striking: (a) zoospore cell shape, especially in fixed preparations, may vary from oblong to amoeboid to irregularly ovoid. Despite this variability, zoospores are clearly distinguishable from round cells (cf. Materials and Methods); (b) in dish cultures, zoospores become n,onrandomly distributed near the dish bottom. Loose attachments, sometimes visible in the form of amoeboid pseudopodia, may be made and broken. These attachments are broken by the swirling procedure used to obtain the supernatant for the zoospore count. Once the transformation to round cells occurs, the vast majority of such cells are firmly attached to the dish bottom. In spinner flask cultures, this transformation is accompanied by a noticeable decrease in the turbidity of the suspension and, at cell densities higher than those employed here, severe clumping occurs. The transformation of round cell to germling is accompanied (and scored) by the appearance of a localized germ tube from which the rhizoids of the growing plant originate. Quantitative Assays of Germination The routine dish assay. It is apparent from the results of a typical dish assay (Fig. 5A) that virtually every zoospore transforms to a round cell and then to a germling. Population synchrony is suffi-

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ciently high that essentially pure populations of the three phenotypes can be obtained by sampling at the appropriate time intervals. At zero time, the population consists entirely of zoospores; at 21 minutes, over 90% of the population consists of round cells; and

FIGS. 2-4. Photographs of formaldehyde-fixed cells taken under phase optics with Nikon inverted microscope (original magnification X 125; final magnification X 625). FIG. 2. Zoospores fixed 1 minute after inoculation into germination solution. f, flagellum. FIG 3. Cells attached to dish bottom at 20 minutes after germination initiation; 20 minutes was the peak of round cell formation in this assay. FIG. 4. Cells attached to dish bottom at 35 minutes after germination initiation: gt, germ tube. The cells are nearly all germlings (every cell containing a germ tube cannot be shown in one photograph, since the germ tubes occur in more than one focal plane).

MORPHOLOGICAL

E + J i 5 y a :

CHANGES

IN

ZOOSPORE

100

100

90

90

so

SO

70

70

60

60

50

50

40

40

30

30

PO

20

193

GERMINATION

10

10

0

0 0

10

20 TIME

30 IN

MINUTES

‘lo

50

0

10

20 TIME

IN

30

‘lo

MINUTES

FIG. 5. Population kinetics of germination in the refined dish assay (A) and the spinzoospore; 0 = round cell; 9 = germling. ner Aask assay (B) ./=

beyond 40 minutes, over 90% of the population consists of germlings. Figures 2-4 are photographs of cultures taken at appropriate times to demonstrate this separation of phenotypes. Figure 5A shows that, after an initial lag period of about 5 minutes, zoospores begin to disappear from the population. Between 5 and 20 minutes, the proportion of the population which is zoospores decreases from 100% to 10%. The last 5% of the zoospores disappear at a decreased rate. The curve describing the percent round cells us. time is composed of two major components which have slight overlaps; (a) the ascending portion of the curve, the slope of which is the reciprocal of the zoospore disappearance curve, and (b) the descending portion of the curve, the slope of which is the reciprocal of the germling appearance curve. Germlings begin to appear between 17 and 20 minutes and then accumulate so that by 37 minutes they constitute 80% of the population. Thereafter, the synchrony of germling formation decreases in a fashion corresponding to the decrease in synchrony of round cell disappearance. The refined dish assay. In routine dish assays, all cells in the supernatants are scored as zoospores. When careful differential counts are made on such supernatants, round cells can be observed.

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The percentage of the total cells which are suspendable round cells is never high (Fig. 6). Nevertheless, the following trends have emerged from data such as those presented in Fig. 6: (a) the magnitude of the contribution of round cells to the supernatant tends to be greater as the synchrony of zoospore disappearance is increased (where synchrony is varied as a function of growth density; cf. Sol1 and Sonneborn, 1969); (b) when figured on the basis of either total cells or supernatant cells, the percentage of suspendable round cells varies significantly and predictably during the course of the zoospore disappearance curve. Peak values are reached during the latter portions of such curves (cf. tabulated data in Fig. 6).

FIG. 6. Corrections for suspendable round cells in dish assays. Synchrony of round cell formation was modified by varying the population density of the growth cultures from which zoospores were obtained (cf. Sol1 and Sonneborn, 1969)‘; (A) 1.5 x lo6 cells/dish; (B) = 2.5 x lo6 cells/dish, (C) 5 x lo6 cells/dish. 0 - 0 are disappearance curves for total suspendable cells (routine dish assay). 0 - 0 are disappearance curves for zoospores only (refined dish assay). “Percent supernatant” refers to percent of the suspendable cells which are round cells. “Percent total” refers to percent of the total population which are suspendable round cells. Apparent 7’50 and 7’~ are the times for 50% and 90% of the suspendable cells to disappear; actual T,, and Tso are the times for 50% and 90% of the zoospores to disappear. FIGS. 7-11. Electron micrographs of sequence of morphological changes during zoospore germination. Scale lines in all figures except Fig. 9C represent 1~. Lines in Fig. 9C represent 0.5 p. Abbreviations as follows: cm, cytomembrane; cw, cell wall; g, gamma particle; gl, glycogen particles; gt, germ tube; km, inner nuclear cap membrane; if, internal flagellar axoneme; inm, inner nuclear membrane; 1, lipid granules; m, mitochondrion; mu, multivesicular bodies; n, nucleus, nc, nuclear cap; nu, nucleolus; om, outer nuclear membrane; 00, open vesicle; p, side body particles.

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195

While these trends are reproducible, the refined dish assay is needed only for special purposes. The contribution of suspendable round cells is sufficiently small and regular that, for most purposes, the routine dish assay suffices. The spinner flask assay. This assay differs from the dish assay in that germination occurs while cells are kept in suspension rather than while they attach to a plastic surface. Correspondingly, the methods of scoring the progress of germination in the two assays differ in some details (cf. Materials and Methods). Nevertheless, the spinner flask assay yields results (Fig. 5B) which agree closely with those obtained with the dish assay (Fig. 5A). These results are considered in greater detail in succeeding sections. Quantitative Assays

Analysis

and

Reproducibility

of

the

Germination

Ration&e. The germination assays are potentially useful in at least two respects: (a) to provide methods for quantitating the effects of environmental and physiological variables on zoospore germination, and (b) to provide means of assessingthe degree of cellular heterogeneity at any time point in the germination process. The precision with which the assays may be used depends, in part, upon the reproducibility of the results under standard conditions. In order to assessreproducibility, several quantitative measures were obtained from germination curves such as those shown in Fig. 5. Routine measures include: (a) zoospore T,,-the time necessary for 50”; of the zoospores to become round cells. Since no germlings have appeared at this time, it also represents the TjO for round cell appearance; (b) round-cell peak-the time necessary for the germinating population to reach the greatest proportion of round cells; (c) germling Tso-the time necessary for 50:~ of the population to become germlings. Since virtually all zoospores have disappeared by this time, it also represents the T50 for round cell disappearance. For comparing results obtained under different experimental conditions (Sol1 and Sonneborn, 1969) either additional quantitative measures or entire transformation curves must be used in order to evaluate the relative roles of the heterogeneity and velocity components of such curves. Since these curves measure the rates at which the population undergoes quanta& rather than continuous, change, the slopes of the curves furnish measures of populational heterogeneity and in no sense can be used to deduce the velocity of

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transformation [e.g., the slope of the zoospore disappearance curve is in terms of percent zoospores per minute and is a measure of heterogeneity in time (asynchrony) of zoospore disappearance rather than a measure of the velocity with which a zoospore becomes a round ce11].5 Departures from the major slope are observed in later portions of transformation curves (Fig. 5). It is often useful to have comparative indices of such departures (cf. for example, Fig. 6), and, for this purpose, we have chosen zoospore TgO and germling !I’,,. With respect to the measurement of the velocity component of transformation curves, T50 provides a measure of the mean velocity of transformation-i.e., TSO is the summed time for all events leading to the measured quanta1 change in the average cell. TSO can be used to measure the relative velocity of transformation under certain experimental conditions-namely, under conditions where population heterogeneity is similar. Where heterogeneity is dissimilar-i.e., where the distributions of times of transformation are different, and particularly where the distributions are overlappinginterpretation of T50 in terms of relative velocity is not straightforward. An alternative measure of the velocity of transformation is the time at which the first cells undergo a quanta1 change.6 Reproducibility of quantitative measures. Table 1 presents a summary of the data from a number of germination assays. The assays included were performed over a two-year period and were selected solely on the basis that they were performed under the standard conditions outlined in Materials and Methods and that enough time points were scored relative to the appropriate quantitative measure. Attention is called to the following general features of these data. (1) Reproducibility of both dish and spinner flask assays is quite 5 The experimental points along the major rise (or fall) portions of transformation curves can be fitted to straight lines (cf. Fig. 5A), and it is from such lines that major slopes have been determined (Table 1). However, analyses of transformation curves by means of the probit technique (Finney, 1962; cf. Fulton and Dingle, 1967) suggest that the different times of cell transformation in the population are distributed normally around the mean time rather than linearly. While this distinction is crucial to considerations of the biology of population heterogeneity, the slopes approximated by straight line fits furnish simple, convenient indices for comparing heterogeneity under different experimental conditions. It is in this sense that “major slope” is used throughout this and the accompanying paper (Sol1 and Sonneborn, 1968). ’ We present this time in terms of the “initial lag period” prior to change in the first cells (Table 1). Ideally, this time should be independent of the slope of the curve. ln practice, however, it is difficult to determine accurately and the determination is influenced by the initial slope of the curve.

MORPHOLOGICAL

CHANGES

IN

TABLE REPRODUCIBILITY

AND

QUANTITATIVE

Zwspore

ZOOSPORE

197

GERMINATION

1 EVALUATION

OF

Assays

GERMINATION Germling

--~

Round

l-80 (min)

Dish assay Mean SD’ No. of expts. Spinner flask assay Mean SD No. of expts. Refined dish assay Mean SD No. of expts.

Major SlOPt? F70/min)e

4.8 2.8 37

12.7 1.8 77

25.3 4.8 46

6.0 1.6 11

20.5 2.6 10

20.9 2.4 10

29.0 3.6 12

36.7 4.3 12

6.7 1.0

6.8 1.7 15

12.6 2.0 30

20.7 3.0 30

6.5 1.5 22

21.3 3.2 15

22.4 2.7 19

31.0 4.4 19

39.0 5.5 17

6.2 2.0 15

6.2 1.4 10

12.1 1.1 10

22.2 3.3 10

7.5 2.0 10

a Determined by extrapolation of major slope to 100% zoospores. fi Determined by best fit to a straight line of point,s on curve between 90y0 and 10% zoospores. c Determined by extrapolation of major slopes of appearance and disappearance portions of round-cell curve. d Det,ermined by extrapolation of major slope to 0% germlings. e Determined by best fit to a straight line of points on curve between 20% and 80% germlings. 1 SD = standard deviation.

high; i.e., the standard deviations of the quantitative measures are reasonably low. These standard deviations set limits for determining whether environmental or physiological variables have significant effects on the progress of germination. Zoospore TSo displays the smallest standard deviation, and this is the measure we have relied upon most extensively in the study of environmental and physiological variables. (2) The major slopes of the zoospore disappearance curves and of the germling appearance curves are quite similar. In other words, for the majority of cells, there is no detectable altera-

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tion in population synchrony between zoospore disappearance and germling appearance. However, the decrease in synchrony with respect to the last cells to transform (cf. Fig. 5) typically involves a greater proportion of cells in germling appearance curves (lo-30%) than in zoospore disappearance curves (510%). (3) The dish and spinner flask assays yield similar quantitative measures. As a consequence it can be concluded that the acquisition of cellular adhesiveness must be a very early event in round cell formation. In the dish assay, but not in the spinner flask assay, measurement of round cell appearance depends on cell adhesion; yet the population kinetics of round cell formation in the two assays are in close agreement. By a similar argument, it can be concluded that the population kinetics of flagellar retraction and of germ tube formation are not significantly altered by whether cells are allowed to adhere to a dish surface or are kept in suspension. Reproducibility of zoospore populations. As set forth in Materials and Methods, six independently maintained stocks yield zoospores which behave indistinguishably in germination assays. Four of these stocks have been repeatedly subcloned; the other two stocks have been cultured separately from the rest (by Cantino) for many years. One of the stocks is known to differ from the others by criteria other than germination performance. Within these limits, therefore, performance in germination assays constitutes a heritably stable trait. Sequence of Morphological Electron Microscopy

Events Accompanying

Germination-

With the spinner flask assay procedures described above, samples have been taken at different time points during germination and examined in the electron microscope. Our ultrastructural studies to date have led us to classify germinating cells into four distinct groups. Group 1. Zoospore. Figure 7 contains two electron micrographs of sectioned zoospores. Each section shows single profiles of nuclear cap, nucleus, nucleolus, and mitochondrion. The single mitochondrion is placed eccentrically around the posterior periphery of the nucleus [plus cap]; one mitochondrial arm extends much farther anteriorly than the other. The flagellar basal body plus accessory rootlet system extends through membrane-bound gaps in the single mitochondrion (Fig. 7a; cf. figures in Cantino et al., 1963; Reichle and Fuller, 1967; Lessie and Lovett, 1968). The single flagellum is continuous with the longer of the two basal structures shown in Fig. 7a. The second, shorter basal structure or “associated centriole” is often seen,

MORPHOLOGICAL

FIG. 7. Zoospore. centriole.” x 16,700.

(a) Long (b) Arrow,

CHANGES

arrow, portion

IN

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flagellar basal body; short of accessory rootlet system.

arrow,

“accessory

X 15,700.

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and both structures appear to terminate near the outer nuclear membrane (cf. also Reichle and Fuller, 1967; and Lessie and Lovett, 1968). Numerous membrane pores are seen where nuclear and nuclear cap membranes are juxtaposed (shown more clearly in micrographs of the next stage, esp. Fig. 9b; cf. also Reichle and Fuller, 1967). Beyond this area of juxtaposition, we interpret the membrane relationships of nucleus and cap to be as follows (cf. Fig. 7b): the nucleus and the cap share a common outer membrane, but each possesses its own inner membrane. Cantino et al. (1968) apparently concur in this interpretation (cf. their diagram, p. 127); in addition, Fig. 54 of Lessie and Lovett (1968) can be so interpreted. However, Reichle and Fuller (1967) have presented a different interpretation of these membrane relationships. Other structural features of the zoospore include the presence of: (1) a single nucleolus typically located in the posterior portion of the nucleus directly above the flagellar basal body (Fig. 7); (2) a membrane-bound organelle adjacent to the outer margin of the long arm of the mitochondrion, termed a “lipid sac” by Cantino et al. (1968), and containing large “lipid granules” interspersed with “sb particles” of unknown function (Fig. 7a) (Lessie and Lovett, 1968; Reichle and Fuller, 1967); (3) cytoplasmic glycogen granules (seen more clearly in micrographs of the next stage, Figs. 8 and 9b; cf. also Lessie and Lovett, 1968); and (4) various morphological types of cytoplasmic vesicles-the large vesicles with electron-dense diskshaped elements within them (Fig. 7; also recognized by all the authors cited above and tentatively identified as “gamma particles” by Cantino et al., 1963), electron transparent vesicles of various sizes (Figs. 7-ll), and large vesicles with internal vesicular and/or tubular elements (referred to below as “multivesicular bodies”; cf. Figs. 7, 8, 9, 11. Noteworthy by its absence is a second outer cell envelope beyond the plasma membrane. Group 2. Round cell I. When sections of round cells are examined in samples taken during the rise portion of the round cell curve (15 minutes), the only ultrastructural changes that we have observed are: (1) flagellar axoneme lacking a surrounding membrane is found inside the cell proper (Fig. 8). Other workers have also observed internal flagellar axonemes (cf. Discussion). It is highly probable that the entire length of the flagellar axoneme is drawn intact into the cell proper since (a) the vast majority of round cell I sections

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show at least some segment of the flagellar axoneme; (b) some sections display more than one obliquely cut axonemal section, and these oblique sections are often 180 degrees apart around the inner periphery of the cell; and (c) a few sections display one long, continuous longitudinal profile of the internal axoneme. Supporting evidence for this latter observation comes from phase microscopy of intact cells (Cantino et al., 1968; Koch, 1968). (2) A second cell envelope, which we tentatively identify as the initial cell wall, can be seen (Figs. 8 and 9). (3) Some of the cytoplasmic vesicles possess an open channel to the plasma membrane (Figs. 8 and 9). Cantino et al. (1968, and personal communication) have also observed open vesicles and, in a preliminary communication, commented on “ . . . the formation of ‘vacuole’-like bodies . . . which originate near the nuclear cap, migrate to the cell surface, and there seem to ‘break through’ it, apparently release their contents, and then disappear. We have the impression that some of these migrating ‘vacuoles’ arise from gamma particles” (Cantino et al., 1968, p. 141). With respect to these comments, we have as yet not seen any of the presumed “gamma particle” vesicles with openings to the plasma membrane. In fact, as contrasted to the zoospore, such “gamma particles” are impressive by their scarcity in sections of this and succeeding stages. Open vesicles of the other two types have been seen (Figs. 8 and 9). Open “multivesicular bodies” often appear to contain internal elements in the “neck” region of the opening (Figs. 8a, 9c). Despite these findings, it may well be that the morphological classes of vesicles are of common origin. Possible intermediate forms, apparently containing both electron dense disks and tubular elements, have occasionally been observed. (4) Whether the mitochondrion has begun to undergo structural alteration at this point is still an open question. Many sections display a single extensive mitochondrial profile, but, in other sections, more than one profile is seen (cf. further comments below). Group 3. Round cell II. When samples are taken during the descending portion of the round cell curve (beyond 20 minutes), round cell sections display the following structural changes: (1) No evidence of the flagellar axoneme can be found in the great majority of sections. In a minority of sections, a small portion of what is presumably flagellar axoneme may be observed (Fig. 9a). (2) Ribosomes are no longer confined within the nuclear cap, but are liberally scattered throughout the cytoplasm. In most sections, the nuclear

FIG. 8. Round cell I. (a) Note tubular elements in neck region of open vesicles. x 15,700. (b) Later section of same cell as in (a). Note cell wall covering open vesicle. x 15,200. 202

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FIG. 9. (a) Round cell II. Arrow points to origin of cytomembrane at nuclear membrane. X 21,200. (b) Round cell I. Arrows point to pores between nucleus and nuclear cap. X 24,000. (cl Open vesicles. Arrows point to open neck regions showing membrane continuity between vesicle and plasma membrane. Upper figure, ~42,400. Lower figure, enlargement of open vesicle in Fig. 9a. X, 48,700.

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cap double membrane is no longer intact. However, long lengths of cytomembrane are frequently visible among the dispersed ribosomes (Fig. 9a). Some of these cytomembranes can be traced back to origins near, if not adjoining, the nuclear double membrane. Marked align-

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ment of groups of ribosomes along both these membranes and the nuclear membrane can be observed (Fig. lOa). (3) Multiple, rather than single, mitochondrial profiles are regularly observed by this time. However, we certainly do not claim that this indicates that the mitochondrion has fragmented. Certain sections (e.g., Figs. lob and c) suggest that the mitochondrion is undergoing complex shape changes, including branching. Other sections of cells at a later stage suggest that the single mitochondrion, or some portion of it, can undergo considerable elongation (cf. below). The question of shape

FIG. 10. Round cell II. (a) Long arrows point to ribosome “alignment” or “clustering” along cytomembrane. Short arrows point to ribosome clustering along nuclear membrane. X 33,800. (b) “Branching” mitochondrion. x 26,500. (c) Four adjacent mitochondrial profiles. The middle two profiles display an area of contunuity. x 27,500.

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change us. fragmentation may be resolved by methods which allow visualization of three dimensional structure. Our own preliminary results with serial sections suggest that “the mitochondrion” at this stage can possess more than two ends (Bromberg, unpublished). Group 4. Germlilzg. Little change in ultrastructure has as yet been observed between the round cell II and germling phenotypes. The distinguishing feature in the electron microscope as in the light microscope is the germ tube (Fig. 11). When electron micrographs are taken at higher magnifications than those of Fig. 9a and Fig. 11, the thickness of the layer(s) external to the plasma membrane appears to have increased between round cell II and germling; this increase appears to continue as the cells enter the growth phase

FIG. 11. Germling.

(a)

x

207

15,800.

(b) x 15,300.

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(Bromberg, unpublished). However, since much of this increase appears as a relatively electron transparent area between an outer electron dense area and the plasma membrane, differential shrinkage of the cytoplasm away from the outer limiting layer during f?xation cannot at the moment be ruled out. Cytoplasmic elements (mitochondria, vesicles, ribosomes, etc.) are routinely observed in the germ tube, but no evidence of nuclear entrance into the germ tube has been found. Often, a quite long, single, mitochondrial profile is seen in the germ tube (Fig. lla). Occasionally, these long, slender profiles are seen to extend from the tip of the growing germ tube up to and around the inner periphery of the cell proper. The longest single profile that we have observed to date measures some 35 ,L (Bromberg, unpublished). This length must be considerably greater than that of the original zoospore mitochondrion (the latter extends less than halfway around the inner periphery of a cell about 7 p wide and 9 P long). Sequences of Events Accompanying Assays with the Light Microscope

Germination-Supplemental

The acquisition of resistance to lytic agents. Zoospores, which lack a cell wall, are quite sensitive to lysis by a variety of agents, e.g., HCl, KOH, and detergents (both anionic and nonionic). At appropriate concentrations, these agents do not lyse cells which have progressed to the round cell stage or beyond. The population kinetics of acquisition of resistance to lysis during germination is compared in Fig. 12 to the kinetics of cell attachment in untreated companion cultures (dish assay). The two curves are in very close agreement. This indicates that cell surface change(s) monitored by two different techniques-resistance to lytic agents and adhesion to the dish bottom -become effective at closely similar, if not identical, times and with similar, if not identical, population heterogeneities. The disintegration of the nuclear cap-visualization aceto-carmine staining. The basic dye aceto-carmine

by means of

may be used to nuclear cap is intact from cells are not confined to the nuclear stained over the nuclear cap an intense stain dispersed over between the two extremes can

discriminate cells within which the within which the bulk of ribosomes cap. The former cells are intensely region whereas the latter cells exhibit the entire cytoplasm. Intermediates also be visualized and scored as such. Figure 13 shows the results of a spinner flask assay in which cells

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209

0

FIG. 12. Resistance of germinating cells to lysis by detergent. Dish assays were scored at successive time points for relative number of cells attached to the dish bottom. 0 - 0, population kinetics of round cell (and germling) formation (control). 0 -0, population kinetics of appearance of resistant cells (experimental). At each point in the latter curve, cultures were treated for 1 minute with 0.5% 7~ detergent (Linbro Chemical Co., New Haven, Connecticut) before swirling, washing, and fixing. Zoospores lysed instantaneously in the 7 x solution.

were scored for intact, disintegrating, or fully disintegrated nuclear caps. The curve describing the appearance of fully disintegrated caps furnishes a preliminary characterization of the appearance of round cell II in the population. This curve is clearly distinguishable from the curve describing the appearance of round cell I (the ascending portion of the round cell curve) and the curve describing the appearance of germlings. The curve describing the appearance of partially disintegrated caps is obviously dependent on the ability to resolve whether or not the stain is confined within the cap (in this case, scoring is done under 1000 X total magnification). However, both this curve and our inability so far to observe any cap disintegra-

210

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FIG. 13. Disintegration of nuclear caps during germination. In conjunction with a normal spinner flask assay, l-ml samples were removed at different time points and were immediately mixed with 0.3 ml of aceto-carmine stain. Intact, partially disintegrated, and totally disintegrated nuclear caps were then scored under 1000x magnification. A - A represents the percent disintegrating and disintegrated caps. V -V represents the percent disintegrated caps only.

tion in electron micrographs of early round cells suggest that there is a short time interval after flagellar retraction and initial cell wall formation before cap disintegration begins. Furthermore, the displacement in time between the partial and complete disintegration curves reenforces the notion gained from electron microscopic examination that release of ribosomes from the nuclear cap is not an instantaneous event but rather is a gradual event, occupying several minutes for completion. DISCUSSION

Two different kinds of analytical tools have been provided in this paper: (a) a methodology has been developed which permits quantitative evaluation of the progress of zoospore germination, and (b)

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a set of well-defined, simple conditions has been chosen which allows rapid, complete, and moderately synchronous germination. With the methodology, it is possible to assess the degree of cellular heterogeneity at any time point during germination. The two assays developed-the dish assay and the spinner flask assay-yield reproducible population kinetics of germination. The dish assay is preferable for the rapid examination of many variables. With the spinner flask assay, much greater numbers of cells can be routinely handled, and therefore analyses demanding bulk quantities of material become practicable. The close agreement in results obtained with the two assays is reassuring and has allowed us to conclude that acquisition of cellular adhesiveness must be an early event in round cell formation. Performance in the germination assays constitutes a heritably stable trait, and virtually every cell in the initial zoospore population transforms to a round cell and then to a germling. Population synchrony is such that a nearly complete separation of zoospores, round cells, and germlings can be effected by sampling at the appropriate time points. Furthermore, synchrony can be significantly improved by lowering the growth density in the liquid DM-2 cultures which provide the zoospores for the germination assays (Soil and Sonneborn, 1969). The analytical tools permit further analyses of the events of germination and of their control. Under otherwise well-controlled conditions, the influence of single environmental or cellular variables on the progress of germination can be quantitated (Sol1 and Sonneborn, 1969). In addition, the tools should help to delimit single shortlived events of germination and to permit bulk analyses of such events. As an example, consider the round cell phenotype which exists for only some 2% of the normal life cycle of an individual cell. Previously, its existence could be routinely observed by watching single cells under the microscope, but separation of the round cell phenotype in populations of germinating cells had not been convincingly demonstrated. Under the conditions reported here, populations of over 90% round cells, with a small proportion of zoospores, can be isolated. Furthermore, at any time during the ascending portion of the round cell curve, pure populations of round cells may be obtained by differential lysis of the zoospores (Fig. 12 and unpublished). During the same time period, populations greatly enriched for zoospores can be recovered in the supernatants of the dish assay.

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That the analytical tools permit further subdivision of the round cell phenotype into separate short-lived phenotypes is attested to by the results of both the electron microscopic examination and the supplemental assays with the light microscope. Subdivision of the zoospore and germling phenotypes may also be possible, although we have no firm evidence as yet that this is so [cf., however, Cantino et al. (1968) for suggestions of this with respect to the zoospore phenotype]. With respect to the sequence of subcellular morphological events accompanying germination, most previously reported ultrastructural descriptions have been confined to the zoospore. Our description is in general agreement with those of other authors (Cantino et al., 1963; Reichle and Fuller, 1965; Fuller, 1966; Lessie and Lovett, 1968; Lovett, 1968). Previously unemphasized details have been provided here (e.g., the presence of morphologically distinct classes of cytoplasmic vesicles), and additional details, not provided here, have been reported by the above authors. These authors, as well as Koch (1968) and ourselves, are in agreement that one of the first detectable signs of zoospore germination is the appearance of the naked flagellar axoneme within the cell proper. However, evidence is presented in the accompanying paper (Sol1 and Sonneborn, 1969; cf. also Cantino et al., 1968) that flagellar retraction is not a necessary step for the succeeding events of germination. Concerning other early events of germination, evidence presented above indicates that cellular adhesiveness is acquired early and that adhesiveness and resistance to lytic agents appear at similar, if not identical, times. A second cellular envelope (in addition to the plasma membrane) is found in electron micrographs of early round cells. These results, taken together, suggest that: (a) rapid formation of an initial cell wall accompanies the change in cell shape from zoospore to round cell; and (b) cell wall formation may be involved in the appearance of both cellular adhesiveness and cellular resistance to lytic agents7 Observations on single transforming cells (Cantino et al., 1968; Koch, 1968; Soll, unpublished) indicate that cell shape changes as the flagellar axoneme enters the cell and that the process of flagellar retraction is of short duration-within 1 minute under our conditions. ’ Cantino et al. (1968) have interpreted the results of a “flocculation test” with dense cell populations to indicate that a change in cell adhesiveness occurs prior to flagellar retraction.

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GERMINATION

There is general agreement that by the time germination is completed, cells no longer exhibit internal flagellar axonemes or nuclear caps, do exhibit multiple mitochondrial profiles in micrographic cross sections and possess both a cell wall and a germ tube. However, the sequence of intermediate morphological events accompanying germination, while previously commented upon, has heretofore received very little documentation. Our own current understanding of this sequence, based on observations and results presented above, is summarized in Table 2. Rather than presenting a detailed discussion of this summary in relation to the comments of others, we emphasize that a more rigorous, detailed description of these events is now feasible. A quantitative evaluation can be made of the percentage of cells which exhibit each of the proposed morphological changes at each of a series of time points for which the degree of cellular heterogeneity (percent zoospores us. round cells us. germlings) is known (Bromberg, unpublished). A beautifully executed example of a similar approach is available-Dingle and Fulton’s (1966) ultrastructural analysis of amoebo-flagellate transformation in Naegleria. It is of interest that, just as the progress of germination at the cellular level may be quantitatively evaluated by means of a series of quanta1 changes, the sequence of subcellular events may be similarly quantitated (e.g., external vs. internal vs. no flagellar axoneme; absence vs. presence of cell wall; intact vs. disintegrating nuclear cap; single us. multiple mitochondrial profiles). The data reported above, as well as other research in progress, TABLE

SUMMARY

OF THE SEQUENCIC Feature

External flagellmn Internal flagellum Cell wall Cell adhesion (to dish bottom) Resistance to lytic agents Vesicle “opening” Intact nuclear cap Single “unbranched” mitochondrion Germ tube

OF

EVENTS

Zoospore

f -

+ +

2

DURING Round

ZOOSPORE cell

I

+ + + + + + +?

GERMINATION

Round cell II

+ + + +

Germling

+ + +

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have led us to a working hypothesis concerning the morphological sequence of events accompanying germination, namely, that many of the changes are the result of local membrane alterations requiring rearrangements of preexisting structures. It is probable that flagellar retraction, initial cell wall formation, nuclear cap disintegration, and the appearance of multiple mitochondrial profiles do not require concomitant de nouo protein synthesis (Sol1 and Sonneborn, in preparation; cf. Lovett, 1968). In addition, flagellar retraction, the appearance of localized channels from vesicles to the plasma membrane, the release of ribosomes from the nuclear cap, the complex sequence of structural events in the mitochondrion, and the outgrowth of the germ tube all involve morphological changes at membrane-containing sites. The idea that these changes result from local membrane alterations is more speculative. Other considerations of membrane alterations during early events of germination have been discussed by Cantino et al. (1968). The mode of construction of the chitinous cell wall may furnish a particularly instructive test of the hypothesis. Of the organelle modifications we have considered, this is the only one which obviously involves de nouo formation. Previous work (Camargo et al., 1967) has indicated that chitin synthetase activity is present in both zoospores and growing cells and that, in crudely purified fractions, enzyme specific activity in zoospores is at least half that of growing cells. It is of some interest that these crude fractions, when examined in the electron microscope, appear to contain predominantly vesicular elements. Of further interest is the finding that enzyme activity in the zoospore fraction is virtually abolished by Ca2+ ion. Under our conditions, Ca++ is the sole cationic requirement for the maintenance of swimming zoospores (Sol1 and Sonneborn, 1969, and unpublished). If there is merit in the above hypothesis, it becomes of paramount importance to discover the nature of the local membrane alterations and to understand the control(s) over the temporal and spatial sequence of these alterations. Experimental control of ZOOspore germination offers a basic tool for such studies. SUMMARY

The zoospore of Blastocladiella emersonii contains a highly ordered array of intracellular organelles. Germination of the zoospore is accompanied by a series of abrupt changes in this array. Procedures have been developed whereby the progress of germination can be measured in cell populations.

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Zoospores are obtained after growth in a defined nutrient medium followed by zoospore formation in buffered CaCL. The population is allowed to germinate in a defined inorganic salts medium. Three quanta1 phenotypes are distinguished: zoospore, round cell, and germling. Virtually every zoospore transforms to a round cell and then to a germling. Under the simple set of conditions adopted as standard, the sequence is rapid and population synchrony is such that nearly complete separations of the three phenotypes can be effected by sampling germinating populations at appropriate times. The beginning population is 100% zoospores; round cells begin to appear after about 5 minutes and constitute 50% and greater than 9OCO of the population at about 12.5 and 20 minutes, respectively; germlings begin to appear between 17 and 20 minutes and constitute 50% and greater than 90% of the population at about 30 and 40 minutes, respectively. Two different conditions of assay have been developed: in one, germination occurs in stationary liquid culture (dish assay); in the other, germination occurs in stirred liquid culture (spinner flask assay). In the dish assay, only zoospores remain suspendable; round cells and germlings adhere firmly to the dish surface. In the spinner flask assay, all cells remain in suspension. The two assays yield population kinetics of germination that are in close agreement. Moreover, with either assay, germination kinetics are highly reproducible, and performance in either assay constitutes a heritably stable trait. By utilizing the electron microscope and other techniques in conjunction with the population assays of germination, the order of subcellular events accompanying germination has been partially characterized. Early events of round cell formation include: retraction of the flagellar axoneme, appearance of localized channels from intracellular vesicles to the plasma membrane, construction of the initial cell wall, acquisition of cellular adhesiveness, and acquisition of resistance to lytic agents. Later events include breakdown of the nuclear cap and release of ribosomes from it into the cytoplasm, and branching or fragmentation of the single mitochondrion. Finally, germling formation is accompanied by the formation of a germ tube. We propose and discuss the hypothesis that these subcellular events are the result of local membrane alterations requiring rearrangements of preexisting structures. Dr. E. Plessmann Camargo contributed importantly to the initial stages of development of the germination assays. He was initially responsible for the construction of the defined growth medium, the sporulation solution, and the germination solution. We

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are indebted to Dr. Hans Ris, for his expert counsel and for the use of his superb laboratory facilities for electron microscopic studies; to Dr. W. H. McShan, for the use of his electron microscope; and especially to Dr. Chandler Fulton, for his painstaking, masterly critique of the manuscript. All his suggestions were considered and most of them have been incorporated. REFERENCES CAMARGO, E. P., DIETRICH, C. P., SONNEBORN, D. R., and STROMINGER, J. L. (1967). Biosynthesis of chitin in spores and growing cells of Blastocladiella emersonii. J. Biol. Chem. 242,3121-3128. CANTINO, E. C. (1951). Metabolism and morphogenesis in a new Blastoclndiella. Antonie Van Leeuwenhoek J. Microbial. Serol. 17,325-362. CANTINO, E. C., and HYATT, M. T. (1953). Phenotypic “sex” determination in the life history of a new species of Blastocladiella, B. emersonii. Antonie Van Leeuwenhoek J. Microbial. Serol. 19, 25-70. CANTINO, E. C., and LOVETT, J. S. (1964). Non-filamentous aquatic fungi: model systems for biochemical studies of morphological differentiation. Adoan. Morphol. 3, 33-93. CANTINO, E. C., LOVETT, J. S., LEAK, L. V., and LYTHGOE, J. (1963). The single mitochondrion, fine structure, and germination of the spore of Blastocladiella emersonii. J. Gen. Microbial. 31,393-404. CANTINO, E. C., TRUESDELL, L. C., and SHAW, D. S. (1968). Life history of the motile spore of Blastoclndiella emersonii: a study in cell differentiation. J. Elisha Mitchell Sci. Sot. 84, 125-146. DINGLE, A. D., and FULTON, C. (1966). Development of the flagellar apparatus of Naegleria. J. Cell Biol. 31,43-54. FINNEY, D. J. (1962). “Probit Analysis,” 2nd ed. Cambridge Univ. Press, London and New York. FRASCA, J. M., and PARKS, V. R. (1965). A routine technique for double staining ultrathin sections using uranyl and lead salts. J. Cell Biol. 25, 157-161. FULLER, M. S. (1966). Structure of the uniflagellate zoospores of aquatic Phycomycetes. PFOC. Symp. CoLston Res. Sot. 18, 67-84. FULTON, C. M., and DINGLE, A. D. (1967). Appearance of the flagellate phenotype in populations of Naegleria amebae. Deuelop. Biol. 15,165-191. KOCH, W. J. (1968). Studies of the motile cells of chytrids. V. Flagellar retraction in posteriorlyuniflagellate fungi. Am. J. Botany 55,84-859. LESSIE, P. E., and LOVETT, J. S. (1968). Ultrastructural changes during sporangium formation and zoospore differentiation in Blastocladiella emersonii. Am. J. Botany 55,220-236. LOVETT, J. S. (1967). In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.). “Aquatic Fungi” pp. 341-358. Crowell, New York. LOVE~T, J. S. (1968). Reactivation of ribonucleic acid and protein synthesis during germination of Blastocladiella zoospores and the role of the ribosomal nuclear cap. J. Bacterial., 962-969. MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114.

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MURPHY, Sr. M. N., and LOVETT, J. S. (1966). RNA and protein zoospore differentiation in synchronized cultures of Blastocladiella.

217 synthesis during Deuelop. Biol.

14,68-95.

REICHLE, R. E., and FULLER, M. S. (1967). The fine structure of Blastochdielh emersonii zoospores. Am. J. Botutzy 54,81-92. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17,20,8-212. RYTER, A., and KELLENBERGER, E. (1958). Etude au microscope electronique de plasmas contenant de l’acide desoxyribonucleique. I. Les nucleoides des batteries en croissance active. Z. Naturforsch. 136, 597-605. SOLL, D. R., and SONNEBORN, D. R. (1969). Zoospore germination in the water mold Bhstocladielh emersonii. II. Influence of cellular and environmental variables on germination. Deuelop. Viol. 20, 218-235. WATSON, M. L. (1955). The use of carbon films to support tissue sections for electron microscopy. J. Biophys. Biochem. Cytol. 1, 183-184.