JOURNAL OF BACTERIOLOGY, July 1973, p. 316-322 Copyright 0 1973 American Society for Microbiology Vol. 115, No. 1 Printed in U.SA. Electron Micrograph Study of the Asci and Ascospores of Metschnikowia Kamienski L. T. TALENS,1 M. W. MILLER, AND MARY MIRANDA Department of Food Science and Technology, University of California, Davis, California 95616 Received for publication 2 April 1973 Internal and surface structures of asci and ascospores were studied by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM) to establish the character and number of ascospores within the ascus of Metschnikowia krissii. Enzyme digestion with snail gut enzymes and SEM examination suggested the presence of a single ascospore enclosed in a thick sheath of epiplasmic materials. Two closely associated ascospores without an epiplasmic sheath were clearly distinguishable from asci of M. bicuspidata var. chathamia when similarly treated. Ultramicrotomy and TEM established conclusively that M. krissii produced a single ascospore per ascus. Neither SEM nor TEM revealed any morphological detail of the ascospores of taxonomic significance. Metschnikoff (7) first described and named Monospora bicuspidata, a yeast-like fungus parasitizing the fresh-water crustacean Daphnia magna. The organism reproduced by multilateral budding and formed club-shaped asci containing single, needle-like ascospores with both ends pointed. Kamienski (1) observed a similar type of yeast in the brine shrimp Artemia salina and renamed the genus Metschnikowia. Several species and varieties of Metschnikowia are presently recognized (6, 8). These species are differentiated on the basis of physiology and manner of sexual reproduction, including shape and number of ascospores per ascus. Treatment of the ascus with enzymes obtained from snail gut demonstrated the presence of two closely appressed spores in M. bicuspidata var. chathamia when it was examined by light microscopy (8). When the same enzyme treatment was used on sporulating suspensions derived from M. krissii, some observations suggested that this species might also produce two spores per ascus. Additional evidence, by means of electron microscopy, shall be necessary to clarify these observations. MATERIALS AND METHODS The culture of M. krissii used in this study (UCD, FS&T 61-31) was the type strain (CBS 4823) isolated from sea water in La Jolla, Calif. The culture was transferred to a slant of V-8 juice-agar medium (6) 1Present address: Department of Veterinary Microbiology, School of Veterinary Medicine, University of California, Davis, Calif. 95616. and incubated at 20 C for 2 weeks. M. bicuspidata var. chathamia (UCD 67-2) was the type strain (CBS 6011) isolated from a fresh-water pond on Chatham Island, New Zealand. The culture was incubated on diluted V-8 juice-agar medium (1:20, vol/vol) for 10 days at 12 C to obtain ascospores. Light microscopy examinations of these cultures were made to ascertain at what point the yeast cells were at maximum sporulation. For scanning electron microscopy, surface growth of a sporulating culture was scraped from the agar slant and suspended in 50 mm phosphate buffer (ph 7.0) containing 1- to 2-mM mercaptoethanol. After 20 min the cells were washed twice with 20-mM phosphate buffer (ph 7.0), which was used in all subsequent washes. The cells were suspended in 0.5 ml of buffer to which was added 0.25 ml of a 1:10 dilution of a preparation from the digestive tract of the garden snail Helix pomatia. The suspension was incubated for 2 h (or until enough ascospores were released) at 37 C, at which time 0.05 ml of ribonuclease (Calbiochem, Los Angeles, Calif; 2 mg/ml) was added. Incubation was continued for 2 more h at 37 C, and then 0.05 ml of Pronase (Calbiochem; 2 mg/ml) was added. After 1 h, again at 37 C, the sample was washed three times in buffer. Subsequent specimen fixation, dehydration, and preparation for scanning electron microscopy (SEM) examination was by the methods of Talens et al. (9). Specimen preparation for transmission electron microscopy (TEM) also were by the methods of Talens et al. (9). RESULTS Digested asci of M. bicuspidata var. chathamia showed two, frequently closely appressed, but distinguishable, ascospores (Fig. 316
VOL. 115, 1973 ELECTRON MICROGRAPH STUDY OF METSCHNIKOWIA 317 1). SEM examination of digested asci of M. krissii suggested the presence of two spores as indicated by a bifurcate shape frequently observed at the terminus of the ascospore (Fig. 2a). However, the spore(s) was virtually enclosed in a relatively thick sheath of epiplasmic material that easily could conceal two closely adjacent spores (Fig. 2c). Comparison of digested spore preparations indicated a considerable breakdown of epiplasmic materials surrounding the ascospores in M. bicuspidata var. chathamia, whereas in M. krissii the sheath was apparently resistant to the treatments. Various aspects of the digested asci and ascospores of M. krissii are shown in Fig. 3. Rupture of the ascus wall and subsequent release of the ascospore in M. krissii did not appear to be as extensive as in M. bicuspidata var. chathamia. TEM observations of ultrathin cross sections of over 30 asci from several different specimen preparations showed that the ascus of M. krissii contained only one ascospore. The morphological appearance of the spore varied, depending upon the angle and location of the section in reference to the ascus (Fig. 4 and 5). A perpendicular section of the narrow, pedunculate portion of the ascus and an oblique section obtained in the elliptical end of another ascus are shown in Fig. 4 and 5, respectively. The thick, epiplasmic sheath containing vestigial organelles was conspicuous; cytoplasmic details were lacking within the heavy-walled ascospore. In undigested preparations the ascus wall was very thick (90 nm), completely intact, apparently two-layered, and virtually indistinguishable from the wall of a vegetative cell (Fig. 4, 5, and 6). The indistinct appearance of the ascospore as viewed by light microscopy may be attributed to the epiplasmic sheath. The spore wall was thick and electron dense, but it did not appear to have a two-layered composition similar to that reported by Kreger-van Rij (5; Ph.D. dissertation, Univ. of Leiden, Netherlands) for ascospores of other yeasts. Certain areas of the spores tend to pick up more stain than others, but little detail of the internal structures of the spore is discernible in the micrographs. The section through the enlarged portion of the ascus indicates that the interior of the ascospore was virtually devoid of cytoplasm (Fig. 5), whereas in the peduncle region the interior of FIG. 1. Scanning electron micrograph of sporulating cells of M. bicuspidata var. chathamia digested with snail gut enzymes. Note the presence of two ascospores (arrows) in juxtaposition. Some ascospores were separated completely by the enzyme treatment.
318 TALENS, MILLER, AND MIRANDA J. BACTERIOL. FIG. 2. Scanning electron micrograph of sporulating cells of M. krissii digested with snail gut enzymes. Note (a) the bifurcate end of the ascospore, (b) remnants of the ascus structure, and (c) the epiplasmic sheath immediately surrounding the ascospores. the spore was filled with cytoplasmic material (Fig. 4). Figure 6 shows a rarely observed, longitudinal section through the ascus and ascospore. The major portion of the enlarged end of the ascus appears to be less electron dense than does the area surrounding the ascospore, which can be attributed to the epiplasmic sheath. The spore is somewhat curved and the visible terminus appears to be blunt. The obscured terminus appears to taper gradually which would result in a single-pointed terminus as is observed by light microscopy. DISCUSSION Little attention was given to the submicroscopic structure of yeast until Kawakami (2) and Kawakami and Nehira (3, 4) published electron micrographs of the various ascospores of yeast with some reference to their possible taxonomic usefulness. Although it has been known for many years that spores of yeast exhibit considerable variation in shape, surface as well as internal structures could reveal details which could lead to further characterization and conceivably to speciation, and such structural details would be valuable in studies of yeast species with similar ascospore shapes as observed by light microscopy. The investigations by Kreger-van Rij (5; Ph.D. dissertation, Univ. of Leiden, Netherlands) have been a significant step in the direction of the possible use of spore ultrastructure in yeast taxonomy. Particular attention has been given to the spore wall, which shows considerable variation as compared with the internal structures, which have disclosed too little detail to be useful. The ascospore wall of M. krissii that was examined by TEM did exhibit varying electron density. Whether this represents the presence of more than one wall layer, as reported for many yeast spores by Kreger-van Rij, or whether the preparatory treatments are responsible for the differential electron stain uptake is not certain. No distinguishing surface details can be recognized because the spore wall is smooth, presumably through its entire length. Demonstration of the internal details of the ascospore has been unsuccessful. Due to the length and narrow diameter of the ascospore, the ideal section of the spore through its entire length is virtually impossible to obtain.
VOL. 115, 1973 ELECTRON MICROGRAPH STUDY OF METSCHNIKOWIA 319 FIG. 3. Scanning electron micrograph of sporulating cells of M. krissii digested with snail gut enzymes, showing the effect of the enzyme treatment on ascal walls. Epiplasmic sheath (a) and ascus wall (b) remain virtually intact (arrows).
320 TALENS, MILLER, AND MIRANDA J. BACTERIOL. o.- FIG. 4. Thin section of an ascus of M. krissii containing a single ascospore. The cross section is from the pedunculate portion of the ascus. Vestigial organelles are observable in the sheath, but little detail can be noted in the ascospore. FIG. 5. Thin section of an ascus of M. krissii showing a single ascospore. The section is through the enlarged end of the ascus and inclined at an angle towards the peduncle. Vestigial organelles are concentrated in the epiplasmic sheath. Interior of the ascospore appears primarily as an electron light void.
VOL. 115, 1973 ELECTRON MICROGRAPH STUDY OF METSCHNIKOWIA 321 'V. sl Ṣ-8 j S j~~~~!.s S;t Lllm~~~~~~~~~- l.o.1r'> FIG. 6. Thin section of an ascus of M. krissii containing an ascospore. The section is longitudinal and includes nearly the entire length of the ascus. Note the concentration of epiplasmic materials around the ascospore and the empty appearance of the enlarged end of the ascus. The ascospore of M. krissii is conspicuously needle-like in shape, with one terminus distinctly pointed. Ascospores of M. bicuspidata var. chathamia, on the other hand, are clearly pointed at both ends. This investigation has revealed conclusively that the bifurcated terminus as seen by light and SEM microscopy was not due to two closely appressed ascospores, but rather to a single spore enclosed in a substantial epiplasmic sheath. The distinct longitudinal line of weakness and figure 8-shaped cross section observed by Pitt and Miller (8) in heavily squashed preparations also can be explained by the presence of the surrounding sheath. ACKNOWLEDGMENTS We are grateful for the facilities and technology offered by Y. Zee of the Department of Veterinary Microbiology. We also thank J. Pangborn and the staff of the Facility for Advanced Instrumentation for advice and technical assistance.
322 TALENS, MILLER, AND MIRANDA J. BACTERIOL. LITERATURE CITED 1. Kamienski, T. 1889. Notice preliminaire sur la nouvelle espece de Metschnikowia (Monospora Metschn.). Trav. Soc. Imp. Natural. (St. Petersburg) 30:363-364. 2. Kawakami, N. 1958. Electron microscopy of fungi. V. The morphological study of the spore of non-pellicle group in the genus Debaryomyces and the classification of the genus Debaryomyces. J. Electronmicrosc. 7:35-38. 3. Kawakami, N., and T. Nehira. 1958. Electron-microscopy of fungi. IV. The morphological study of the spore of pellicle group in the genus Debaryomyces. J. Electronmicrosc. 7:33-34. 4. Kawakami, N., and T. Nehira. 1959. Electron-microscopy of fungi. IX. Intracellular structures of Rhodotorula glutinis and Nadsonia fulvescens and their relation to the physiological characters and taxonomic affinity. J. Ferment. Technol. 37:125-132. 5. Kreger-van Rij, N. J. W. 1966. Some features of yeast ascospores observed under the electron microscope. In Yeasts, Proc. Sec. Symp. Yeasts. Vydavatel'stvo Slovenskej Akademe Vied, Bratislava, 1969. 6. Lodder, J. 1970. The yeasts, a taxonomic study. North- Holland Publishing Co., Amsterdam. 7. Metschnikoff, E. 1884. Uber eine Sprosspilzkrankeit der Daphnien. Beitrag zur Lehre uber den Kampf der Phagocyten gegen Krankheitserreger. Arch. Pathol. Anat. Physiol. Klin. Med. Virshows 96:177-195. 8. Pitt, J. I., and M. W. Miller. 1970. Speciation in the yeast genus Metschnikowia. Antonie van Leeuwenhoek J. Microbiol. Serol. 36:357-381. 9. Talens, L. T., M. Miranda, and M. W. Miller. 1973. Electron micrographic study of bud formation in Metschnikowia krissii. J. Bacteriol. 114:413-423.