JOURNAL OF BACTERIOLOGY, June 1968, p. 2365-2373Copyright © 1968 American Society for Microbiology

Vol. 95, No. 6Printed in U.S.A.

Ultrastructural Details in Germinating Sporangiosporesof Rhizopus stolonifer and Rhizopus arrhizus

PATRICIA M. BUCKLEY, N. F. SOMMER, AND T. T. MATSUMOTO

Department of Pomology, Unziversity of California, Davis, California 95616

Received for publication 22 March 1968

Electron microscope examination of sporangiospore sections from Rhizopusstolonifer (Ehrenb. ex Fr.) Lind. and R. arrhizus Fischer revealed details on intracel-lular organization not previously reported. Aldehyde fixation followed by chrome-osmium postfixation permitted clear depiction of ribosomes hitherto unrevealed inthese cells. Mitochondria were diversiform. Spore wall structures in the two specieswere generally similar, but outer contours differed sufficiently to permit easy speciesidentification in examination of sections. The spores of both species abounded in cy-tosomes, corresponding in size, shape, and heavy-metal "stain" affinities to sphero-somes in cells of higher plants. The osmiophilic response of these spherosome-likeinclusions was intensified by treatment of sections with thiocarbohydrazide solutionand subsequent application of aqueous osmium tetroxide, which strengthens an

assumption that they are lipid-rich. The margins of the spherosome-like inclusionsin lead citrate-stained sections included dense particles, about 60 A across, whosecrystalline-like arrangements suggested that protein as well as lipid was present.Frequent and close associations between the spherosome-like inclusions andvarious cell membranes suggested that such bodies participate in membrane elabora-tion during germination.

Rhizopus species are mucoraceous fungi whichusually reproduce asexually, forming spores with-in a sporangium. The enveloping sporangialmembrane ruptures when the spores mature, thusreleasing them. Several species commonly spoilstored processed foods and fresh fruits andvegetables.

Electron micrographs of Rhizopus sporangio-spores have appeared in reports by Hawker andAbbot (10) and by Necas, Havelkovai, and Soudek(14). Those workers established that the maturesporangiospore in the two species examined, R.sexualis and R. nigricans Ehrenb. (syn. R.stolonifer Lind.), contains several nuclei, classicallyorganized mitochondria, and vesicles and granulesof various sizes and densities. A thick, ridgedouter wall encloses the protoplast. During germi-nation, an inner wall forms and extends aroundthe emerging hypha. Neither group of workersused fixation treatments which would permitthem to visualize ribosomes.The present study confirms the previous obser-

vations on R. stolonifer and extends electronmicroscope observations to spores of yet anotherspecies, R. arrhizus. The use of chrome-osmiumpostfixation made it possible to demonstrateribosomes in these spores for the first time. Newlydescribed are ultrastructural and cytochemical

features of spherosome-like inclusions, whoseassociations with membranous cell componentswere also noted.

MATERIALS AND METHODS

Collection and germination of spores. Sporangio-spores from 2-week-old cultures of R. stolonifer orR. arrhizus growing on V-8 juice-agar (17) wereharvested according to procedures previously de-scribed (1). Germination occurred in a medium (5)consisting of 10 g of glucose, 4.5 g of KH2PO4, 1.0g of NH4CI, and 0.5 g of MgSO4-7H2.O, dissolved in1 liter of distilled, deionized water, brought to pH6.0 with NaOH, and then autoclaved. When suspen-sions of 106 to 107 spores per ml were held for 6 to 7hr at 25 C in this medium, while aerated on a shaker,about 50% developed germ tubes up to several spore-diameters long, and about 40% more showed somedegree of swelling.

Preparation of spores for electron microscopy.Spores were fixed in 4% formaldehyde or 6.25%/0glutaraldehyde in 0.1 M phosphate buffer at pH 7.0to 7.2 and were postfixed in either 1% KMnO4 inphosphate buffer or in modified Dalton's fixative(3). In this modification, equal parts of aqueous 2%osmium tetroxide and aqueous 2% potassium di-chromate (pH 6.8) were mixed immediately beforeuse. No sodium chloride was added. Spores wereleft for 1 hr at room temperature in the permanganatefixative and for 12 to 18 hr at 5 to 7 C in the chrome-

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BUCKLEY, SOMMER, AND MATSUMOTO

osmium mixture. Details of the dehydrating, embed-ding, and sectioning procedures were reportedpreviously (1). Usual procedures for staining sectionsincluded application of Reynolds' lead citrate (15)for 10 min, or uranyl acetate (saturated solution inmethanol) for 2 min, followed by lead citrate for 10min.

Osmium tetroxide staining. Special staining forenhancement of lipid-rich components in chrome-osmium-fixed sections of spores required slight modi-fication of the OTO method described by Seligmanand co-workers (16). Sections mounted on uncoatednickel grids were immersed for 0.5 to 1 hr in warm(40 to 45 C) aqueous 0.5% thiocarbohydrazide.They were washed several times in warm 50%0 ethylalcohol and once in distilled water before beingstained by immersion for 10 to 15 min in aqueous 1%h/osmium tetroxide. The ethyl alcohol rinses freed thetreated sections from a precipitate often found whenonly distilled-water rinses were used.

Acid phosphatase reaction. A method described byEricsson and Trump (6) for demonstrating acidphosphatase activity was used on formaldehyde-treated spores. Spores were incubated with theglycerophosphate substrate for 1 hr at 32C. Theywere then treated as prescribed and postfixed withchrome-osmium. The dehydrating and other prepara-tive procedures were carried out as usual. All sectionswere examined with RCA EMU-3 microscope3.

Chilling treatment. Spores which had been incu-bated for 6 hr at 25 C in liquid medium were trans-ferred to chilled fresh medium in cotton-pluggedflasks. These were held on a shaker for 4 days in aroom maintained at 0 C i 2 C. About 10% of thechilled spores retained their ability to form normalcolonies according to plating tests done at that time.

RESULTS AND DISCUSSION

General observations. Nuclear and mitochon-drial outlines, cell wall details, and various cellinclusions were clearly seen in aldehyde-perman-ganate-fixed sections after lead citrate staining(Fig. 1, 3a, and 4). The relatively poor contrastof organelles against ground plasms in chrome-osmium-fixed material was improved by stainingsections with uranyl acetate followed by leadcitrate (Fig. 3b).The numerous nuclear outlines were circular,

oval, or sometimes very elongate, but not asirregular in shape in germinating as in nonger-minating spores. Mitochondria were dispersedthroughout the sections and often showed con-torted, lobed profiles. Cristae were closely paralleland frequently extended almost completely acrossa mitochondrion. Except for minor differences,the intracellular organization of the R. arrhizusspore closely resembled that of R. stolonifer (Fig.la and 3a). R. arrhizus spores were smaller thanthe R. stolonifer spores (about 5 , for nonger-minating spores of R. arrhizus, compared withabout 7.5 A for this strain of R. stolonifer).

Average numbers of nuclei and mitochondriawere greater in R. stolonifer spores, but the shapesassumed by those organelles were very similar inthe two species.One detail of spore ultrastructure not previ-

ously emphasized in studies of these fungi is theappearance of ribosomes. After chrome-osmiumpostfixation, it was possible to observe dense,approximately round, cytoplasmic particles whichlay apparently free throughout the cells (Fig. 2a).An average diameter for the largest ones was ap-proximately 170 A, which is in the size rangeascribed to ribosomal particles from many otherkinds of cells. Endoplasmic reticulum strandswere sparse in these spores and ribosomal associa-tions with endoplasmic reticulum were not ob-served. The great abundance of the ribosomalparticles at all stages of germination and also innongerminating spores contributed to the diffi-culty of distinguishing organelle outlines clearlyafter chrome-osmium postfixation.

Walls and plasmalemmas in both species weredelineated equally well with both fixation treat-ments (Fig. 1, 2, and 3). The wall present beforegermination (Fig. lb), referred to as the outerwall (ow), exhibited two distinctly different zonesor layers. In R. stolonifer, an electron-opaqueouter layer displayed numerous prominences(Fig. la and 2b) representing the ridges first pic-tured by Hawker and Abbott (10). Below thislayer was a light, thicker zone, strewn with elec-tron-dense areas. The wall structure in R. arrhizuswas quite similar, except that the prominences ofthe outer opaque layer were less abrupt. Thus, inR. arrhizus, the spore contours were more gentlycurved and this made it possible to distinguish thespores of the two species in sectioned material.As is shown in Fig. lb, the absence of an inner

wall was clearly evident in the ungerminatedspore. An inner wall appeared only in germinat-ing spores (Fig. la, 2a, 2b, 3, and 4) and sur-rounded the emerging tube, as Hawker andAbbott also noted. It was composed of a singlezone of moderately electron-dense material whichsometimes appeared fibrillar (Fig. 2b). Infoldingsof the plasmalemma, which lay directly below theinner wall, were seen occasionally (Fig. 2a). Atsuch places, membranous material occupied thespace between the inner wall and the plasma-lemma.

Thus, germinating sporangiospores of R.stolonifer and R. arrhizus were found to beamenable to electron microscope examinationafter either of two fixation treatments in wideuse for both plant and animal material. It wasexpected that the spores of two closely relatedspecies would resemble each other in manyaspects of subcellular organization. The details

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FIG. la. Section through germinating sporangiospore of Rhizopus stolonifer showing nuclei (N), mitochondria(m), vacuoles (V), and dense inclusions (S), some of which appear to be membrane-associated (arrows). Outerwall (ow) has broken; new inner wall (iw) surrounds protoplast. KMnO4-postfixed, lead citrate-stained.

FIG. lb. Portion of nongerminating sporangiospore, characterized by absence of inner wall (arrow) betweenouter wall and protoplast. Note numerous dense inclusions (S), several mitochondria, and part ofa nucleus. KMnO4-postfixed, lead citrate-stainied. All line markers denote distance of I IA, unless otherwise indicated.

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2b 2c.FIG. 2a. Portion of germinating Rhizopus stolonifer sporangiospore postfixed in chrome-osmium, showing de-

tails of inner (iw) and outer wall (ow) structures, plasmalemma (P), dense inclusions (S), and mitochondria (m).Ribosomal particles (R) appear freely distributed in hyaloplasm. Uranyl acetate, lead citrate staining.

FIG. 2b. Portion of cell section that was osmium tetroxide-treated. S inclusions appear dark, unbounded. Wallprominences typical for R. stolonifer spore.

FIG. 2c. S inclusions in chrome-osmium-fixed cell, showing regularly arranged dense particles at margins.Lead citrate-stained.

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FIG. 3a. Portion of Rhizopus arrhizus germling showing mitochondria (m), nuclei (N), outer and inner walllayers (ow, iw), and S inclusions, some associated with nuclear envelopes and other membranes. Also note S asso-ciations in inset, above left. Outer-wall contour characteristic for this species. KMnO4-postfixed, lead citrate-stained.

FIG. 3b. S inclusions and nucleus in R. arrhizus spore postfixed with chrome-osmium. White arrows indicatenuclear envelope. Note similarity ofS inclusion associations with nuclei in this and Fig. 3a.

FIG. 3c. Enlarged view of S inclusion from section shown in 3b. Black arrows point to membrane segmentsat margins ofS inclusions. Uranyl acetate, lead citrate staining.

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2370 BUCKLEY, SOMMER, AND MATSUMOTO J. BACrERIOL.

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VOL. 95, 1968 SPORANGIOSPORES OF R. STOLONIFER AND R. ARRHIZUS

of wall structure and previously described organ-elles agreed with the reports on Rhizopus by otherworkers, and new information has been addedabout ribosomal particles. The evident com-plexity of the spore walls, shown by both fixations(Fig. 2b and 3a), leaves unanswered many ques-tions concerning the location and distribution ofmelanin, chitin, lipid, etc., in the several zones orlayers.

Cytosomes in Rhizopus spores. Dense inclusionsand clear vacuoles abounded in these cells. Par-ticularly noticeable in permanganate-fixed mate-rial were dense, crenulated inclusions (denoted"S," for spherosome-like), which appeared to bemembrane-bounded (Fig. la, lb, 3a, and 4). Inchrome-osmium-fixed material, in contrast,boundaries of the more nearly round bodies,representing the same inclusions, appeared to belacking (Fig. 2a, 2b, 2c). In many respects, theinclusions fit the descriptions given for sphero-somes of higher plant cells (4, 7, 8). The diametersof many of the S inclusions in Rhizopus spores fellin the range 0.2 to 0.6 ,t. This is within the sizerange reported (7, 8) for spherosomes (0.2 to1.3 ,) and also for certain plant cytosomes saidto resemble animal microbodies (12). In theirreport on plant microbodies, Mollenhauer andco-workers (12) mentioned, but did not illustrate,the presence of microbodies in Rhizopus materialwhich they had examined; whether the materialincluded spores was not stated.

It is believed that spherosomes contain bothlipid and protein (7). S inclusions respondedsimilarly to fixations and were assumed to besimilar in composition. To test for the presumedhigh lipid content of S inclusions, we used a modi-fication of a method (16) which required treat-ment of osmium-fixed sections with thiocarbo-hydrazide and subsequent staining with aqueousosmium tetroxide. Figure 2b shows a typicalresult. The osmiophilic reaction of the S inclusionswas intensified by the osmium tetroxide staining,although the details of other cell components wereless clear. The intensified osmiophilic responsewas considered added evidence that the bodies inquestion are rich in lipid.An interesting feature of S inclusion ultrastruc-

ture was brought out by lead citrate staining.Lead citrate in alkaline solution is commonlyused in electron microscopy of biological sub-jects to enhance contrast. The effect is attributedto the higher affinity of certain cell componentsfor the heavy metal ions. Figure 2c shows, atrelatively high magnification (125,000 X), theappearance of two S inclusions in a lead citrate-stained, osmium-fixed spore section. Against abackground of ribosomal particles, the bodiesappear moderately dense and homogeneous,

except for some small (50 to 60 A) particles onand near their margins. These particles, arrangedin crystalline-like patterns, lie in the size rangepossible for globular protein particles havingmolecular weights from 20,000 to 80,000 (8). Thepresence of these particles, visible after the leadcitrate treatment, implies that S inclusions have aprotein component, possibly enzymatic. Whatseem to be surface coatings or marginal accumu-lations of these particles on the S inclusions inosmium-fixed cells were likely responsible fortheir membrane-bounded appearance in the per-manganate-fixed cells. Such an effect, apparentbounding by a membrane, has been demonstratedat interfaces of oil-gelatin mixtures after KMnO4treatment (8). Carasso and Favard (2), in theirstudy of the "vitelline plaquettes" (yolk deposits)in Planorbis corneus eggs, observed crystalloidstructures which they interpreted as being com-posed of protein granules, about 60 A in diameterand with 80 A intergranular spacing. AlthoughMollenhauer and co-workers (12) saw densematerial in the cytosomes which they designatedplant microbodies, they found no crystallineinclusions.The indication that S inclusions contain pro-

tein, coupled with their other similarities tospherosomes and microbodies, raised the questionof whether S inclusions also are sites of enzymeactivity. A modification of Ericsson and Trump'smethod (6) was used to test for acid phosphataseactivity. Fine granulation accumulated on themargins of S inclusions and was considered pre-sumptive evidence of activity. However, thedemonstration that regularly spaced, dense gran-ules occupied those locations after application oflead citrate alone raises some doubt about thereputed specificity of a response to the lead-con-taining glycerophosphate substrate in the Gomorireaction. Tests for other enzymes have not beenmade.

Mollenhauer and co-workers (12) noted thatmicrobodies were resistant to damaging treat-ments given cells. In our experience, S inclusionsalso remained apparently unaltered when ger-minating spores were damaged by -y radiation(unpublished data) or chilling. As an illustrationof the effect of chilling, Fig. 4 shows S inclusions,some associated with nuclei and vacuoles, in agerminating spore left 4 days in liquid medium at0 C. Mitochondria are aggregated and deformed,and the nuclei show degenerative changes, butthe dense inclusions appear unaffected.The above comparisons, based on both mor-

phological and cytochemical data, make it clearthat S inclusions are cytosomes having propertiesin common with both microbodies and sphero-somes, but corresponding exactly with neither.

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BUCKLEY, SOMMER, AND MATSUMOTO

Many workers have noted inclusions presentin fungal cells (9, 10, 13, 19, 20), but little hasbeen said about their probable uses. Also unclearare the functions of microbodies and spherosomes(7, 8), although it has been argued that sphero-somes may function catabolically because theypossess acid phosphatase and other lytic enzymes(18, 21). In germinating Rhizopus spores examinedin the present study, S inclusions were associatedfrequently with nuclear and vacuolar membranes(Fig. la, 3, and 4) and less often with the plasma-lemma or mitochondrial membranes. Many sec-tions contained S inclusions surrounded by, andin contact with, loops or strands of unidentifiedmembranes (Fig. 3). No associations betweenmembranes and dense inclusions were observedin sections of ungerminated spores, althoughmany dense inclusions were present in them (Fig.lb). Possibly the bodies first exhibit functionalactivity during germination. The numerous in-stances in which S inclusions in the spores of bothRhizopus species were found to be associated withnuclear and other membranes make it temptingto propose for them a functional role in mem-brane formation in growing cells with rapidlyexpanding membrane requirements. Hyde andWalkinshaw (11), speculating on the significanceof large osmiophilic bodies which were alwaysassociated with a large vacuole and vesicularmembrane system in germlings of Lenzitessaepiaria, suggested that "this entire complex mayrepresent the mobilization of nutrients for theemerging germ tube." In the Rhizopus spore,vacuoles enlarge as germination proceeds. Also,during germination, the nuclei replicate, the sporebecomes larger, and the mitochondria increase insize and number. Thus, even before the germ tubeappears, the spore must have among its growthrequirements an urgent need for structural unitsfor membrane assembly. The information aboutthe sequence of events during germination is stilltoo fragmentary to tell whether the membrane-associated inclusions will be used up, or will per-sist, acting only as templates or sites for theassembly. It might be alternatively proposed thatS inclusions are being produced by the mem-branous components with which they are associ-ated. However, this would require an explanationfor their formation at diverse sites. The reverseproposal recognizes that membrane elaborationshould require the same materials, protein andlipid, to be brought together, whatever the site.The relative abundance of the inclusions in thespores should encourage attempts to isolate andfurther characterize them.

ACKNOWLEDGMENTS

We are grateful to Jack Pangborn for makingavailable to us the excellent facilities of the ElectronMicroscope Laboratory of the University of Cali-fornia at Davis.

This investigation was supported by Atomic EnergyCommission contract AT(11-1)-34, Project 73. Thisis report number UCD-34P73-21.

LITERATURE CITED

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2. Carasso, N., and P. Favard. 1958. L'origine desplaquettes vitellines de l'oeuf de Planorbe.Compt. Rend. 246:1594-1597.

3. Dalton, A. J. 1955. A chrome-osmium fixativefor electron microscopy. Anat. Record 121:281.

4. Drawert, H., and M. Mix. 1962. Die Spharosomenim elektronenmikroskopischen Bild. Ber.Deut. Botan. Ges. 75:128-134.

5. Ekundayo, J. A., and M. J. Carlile. 1964. Thegermination of sporangiospores of Rhizopusarrhizus; spore swelling and germ-tube emer-gence. J. Gen. Microbiol. 35:261-269.

6. Ericsson, J. L. E., and B. F. Trump. 1964. Elec-tron microscopic studies of the epithelium ofthe proximal tubule of the rat kidney. I. Theintracellular localization of acid phosphatase.Lab. Invest. 13:1427-1456.

7. Frey-Wissling, A., A. E. Grieshaber, and K.Mtihlethaler. 1963. Origin of sphaerosomesin plant cells. J. Ultrastruct. Res. 8:506-516.

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9. Hawker, L. E. 1965. Fine structure of fungi asrevealed by electron microscopy. Biol. Rev.Cambridge Phil. Soc. 40:52-92.

10. Hawker, L. E., and P. McV. Abbott. 1963. Anelectron microscope study of maturation andgermination of sporangiospores of two speciesof Rhizopus. J. Gen. Microbiol. 32:295-298.

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12. Mollenhauer, H. H., D. J. Morre, and A. G.Kelley. 1966. The widespread occurrence ofplant cytosomes resembling animal microbodies.Protoplasma 62:44-52.

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14. Necas, O., M. Havelkova, and D. Soudek. 1963.Submicroscopic morphology of Rhizopusnigricans. Folia Microbiol. (Prague) 8:290-292.

15. Reynolds, E. S. 1963. The use of lead citrate athigh pH as an electron-opaque stain in electronmicroscopy. J. Cell Biol. 17:208-212.

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16. Seligman, A. M., H. L. Wasserkrug, and J. S.Hanker. 1966. A new staining method (OTO)for enhancing contrast of lipid-containingmembranes and droplets in osmium tetroxide-fixed tissue with osmiophilic thiocarbohydraz-ide (TCH). J. Cell Biol. 30:424-432.

17. Sommer, N. F., M. Creasy, R. J. Romani, andE. C. Maxie. 1964. An oxygen-dependent post-irradiation restoration of Rhizopus stolonifersporangiospores. Radiation Res. 22:21-28.

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19. Tanaka, K., and T. Yanagita. 1963. Electronmicroscopy on ultrathin sections of Aspergillusniger. I. Fine structure of hyphal cells. J. Gen.Appl. Microbiol. (Tokyo) 9:101-118.

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21. Walek-Czernecka, A. 1965. Histochemical demon-stration of some hydrolytic enzymes in thespherosomes of plant cells. Acta Soc. Botan.Polon. 34:573-598.

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