basal body loss during fungal zoospore encystment: evidence against ...

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autonomous organelles that may have an endosymbiotic origin from a free-living prokaryotic progenitor (Margulis, 1970). Fulton (1971) has fully discussed the.
J. Cell Set. 83, 135-140 (1986)

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BASAL BODY LOSS DURING FUNGAL ZOOSPORE ENCYSTMENT: EVIDENCE AGAINST CENTRIOLE AUTONOMY I. BRENT HEATH, SUSAN G. W. KAMINSKYJ Biology Department, York University, 4700 Keele Street, North York, Ontario, M3J 1P3, Canada

AND TOM BAUCHOP Department of Biochemistry, Microbiology and Nutrition, The University of New England, Armidale, NSW 2351, Australia

SUMMARY The controversial question of the possible autonomy of centrioles, as shown by the persistence of all or part of them in the generative cell line throughout the life cycle of organisms, remains unresolved. All previous reports on shedding or withdrawal of cilia and flagella showed that their basal bodies (= centrioles) were retained in the cells where they may, or may not, subsequently disassemble. We show that in the fungusNeocallimastix sp. the basal bodies are discarded with the flagella when zoospores encyst. This shedding of basal bodies argues against centriolar persistence in any form and thus against their autonomy and endosymbiotic origin.

INTRODUCTION

Eukaryotic flagellar axonemes develop from basal bodies, which are in at least some cases derived from centrioles. Centrioles persist throughout the life of many cells and organisms, and may contain nucleic acids (reviewed by Fulton, 1971; Margulis, 1970; Wheatley, 1982). Consequently, they have been described as semiautonomous organelles that may have an endosymbiotic origin from a free-living prokaryotic progenitor (Margulis, 1970). Fulton (1971) has fully discussed the uncertainties of the autonomy hypothesis and more recent data have still not provided unequivocal support for or against the hypothesis (Wheatley, 1982). Persistence of centrioles in non-flagella-bearing cells is an argument for their essential continuity and autonomy, but their undoubted absence at certain life-cycle stages and their de novo synthesis beforeflagellogenesisin a number of species argue against autonomy (Fulton, 1971; Margulis, 1970; Wheatley, 1982; Pickett-Heaps, 1971). The latter arguments are not compelling, because a hypothetical informational molecule or structure could remain after centriole disassembly and persist and replicate, either in a different form such as a nucleus-associated organelle (Girbardt & Hadrich, 1975) or, unrecognized, in the cell lineage until used for the next centriole-synthetic event. The life cycles of many species include the transient production of flagellate cells such as sperm or zoospores. Because most of these Key words: basal bodies, centrioles, zoospores,flagella,fungi, organelle evolution.

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flagellate cells retain either the whole axonemes, or just the basal bodies, inside the cells when they cease to be flagellate (e.g. at encystment) (Bloodgood, 1974), essential continuity and autonomy of the centrioles seems likely, even when encystment is followed by disassembly of the basal bodies. Clear evidence for shedding of the entire axonemes, including basal bodies, into the environment at encystment would be a strong argument against continuity and autonomy. We report such evidence for the chytridiomycete rumen fungus, Neocallimastix sp. MATERIALS AND METHODS Isolate PN2 of a species of Neocallimastix was grown and prepared for serial-section-based transmission electron microscopy of individually selected zoosporangia as previously described (Heath et al. 1983; Heath & Bauchop, 1985). Briefly, cultures were grown in a liquid nutrient medium, fixed by addition of phosphate-buffered glutaraldehyde, mixed with added molten agar and poured into a thin layer to gel in a Petri dish. Areas of gel containing samples of fungus were excised, fixed further in glutaraldehyde followed by osmium tetroxide and flat-embedded in epoxy resin. Individual zoosporangia or clusters of cysts were selected from the polymerized blocks and trimmed for serial sectioning. The specific epithet for isolate PN2 is uncertain (Heath & Bauchop, 1985); it may be a variant oifmntalis (Heath et al. 1983) ovpatriciae (Orpin & Munn, 1986). Until we have resolved this uncertainty we shall refer to it as Neocallimastix sp.

RESULTS

Because it is crucial to the comprehension of the results, the asexual life cycle of Neocallimastix sp. is outlined in Fig. 1. The organism is maintained in axenic culture by continuous cycles of this proliferative sequence. In addition to the normal sequence of zoospore release and encystment, some apparently normal sporangia failed to release their zoospores. Instead, the zoospores encysted and began to germinate within the sporangium (Fig. 2). These sporangia were ideal for analysis of the fate of flagella at encystment because everything was contained within the sporangium. We have examined three such sporangia, one containing only germinated cysts and the others containing a mixture of cysts and unencysted spores that were presumably about to undergo encystment at the time of fixation. We have also examined a cluster of germinating cysts that had encysted in a group outside a sporangium. In all four samples the cysts were accompanied by bundles of flagella that were no longer attached to the cells. We could not obtain accurate counts but subjective observations of essentially random sections suggested an approximately equal number of cysts and bundles. We have not traced all shed axonemes to their proximal ends, but all ends that we have seen terminated in basal bodies, which contained triplet microtubules and central cartwheels (Figs 4—7). Each basal body was accompanied by the 'skirt' and 'spur' (Heath et al. 1983) components of the flagellar root system (Figs 4-7), but the spurs bore no microtubules comparable to those found attached to them in the adjacent unencysted zoospores. The shed flagella seemed to begin disintegration from their proximal ends at the time of shedding because, whilst many bundles were coherent and similar to attached bundles along most of their lengths, they were always frayed at the basal body ends. Fraying involved both separation of individual flagella and dissociation of the flagellar

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Fig. 1. Summary of the asexual life cycle of Neocallimastix sp. Free-swimming uninucleate zoospores shed their flagella (A) and secrete a cell wall to become cysts. Each cyst germinates to produce a highly branched rhizoid system (stippling), which attaches the thallus to its substrate and the nucleus (filled circles) undergoes mitosis in the body of the thallus (B). The young thallus grows by enlargement of the body and successive rounds of mitosis (C,D). Ultimately the body of the thallus is separated from the rhizoid system by insertion of a cross wall and develops into a sporangium. In the sporangium, the cytoplasm cleaves to produce uninucleate zoospores, only two of which are illustrated in E. Each zoospore bears approximately 10 flagella, which beat together as a single posterior propulsive unit after the zoospores are released from the sporangium (A).

membranes from the axonemes, with concomitant coiling of the axonemes within the dilated membranes. Because of this fraying and relatively short series of sections it was not possible to prove that all flagella in each bundle terminated in a basal body, but such seemed likely; we saw no proximal ends that did not have a basal body. We have also looked inside the recently encysted cells in the samples and have not seen any withdrawn axonemes, basal bodies or centrioles. We have examined enough series of sections of fresh cysts to have revealed many basal bodies if the cysts had been flagellated zoospores. This assertion is supported by the frequent detection of basal bodies in the cytoplasm of theflagellatezoospores (e.g. see Fig. 3), which were still present in two of the sporangia examined. DISCUSSION

The simplest interpretation of our results is that the zoospores of this fungus shed their flagella, complete with basal bodies and part of their root system, when they encyst. Because encysted cells in the sporangia containing shed flagella went on to germinate, in at least one case, it is unlikely that shedding is a pathological phenomenon. Likewise, because cysts outside sporangia were accompanied by shed

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flagella, it is unlikely that shedding is restricted to the unusual sporangia with slow zoospore release. Munn et al. (1981) have also shown flagellar shedding by free zoospores, but they did not determine whether the basal bodies were shed with the flagella, because they used relatively low-resolution scanning electron microscopy. Because shed bundles of flagella seem to be as abundant as cysts and because we have seen no withdrawn axonemes, it is likely that shedding is the typical and probably the only behaviour. Shedding of basal bodies is also consistent with the absence of centrioles at the mitotic spindle poles in young thalli (Heath & Bauchop, 1985). Shedding of flagellar axonemes is not uncommon (Bloodgood, 1974; Holloway & Heath, 1977), but as far as we know this is the only report showing concomitant loss of basal bodies. This physical discarding of basal bodies shows that if any part of them, including undetected material carrying morphogenetic information essential to the perpetuation of the centrioles, is retained by the cell, then it must be separated before they are shed from the cell. We cannot exclude this possibility but the necessary extra control system renders it less likely. Because the zoospores are polyflagellate, one basal body may be retained while the rest are shed. Our inability to find any basal bodies inside cysts, or shed flagella lacking attached basal bodies, argues against this possibility, but negative evidence is not conclusive. However, retention of one basal body would require a reliable control system to ensure differential behaviour among the variable numbers offlagellaon each zoospore. Such a control system seems error-prone and is unlikely, on the principle of accepting the simplest hypothesis. The present demonstration of an encystment system that leads to the discarding of the cellular complement of basal bodies and centrioles adds to the weight of evidence against a semi-autonomous existence for the centriole and indirectly argues against an endosymbiotic origin for this organelle. Such conclusions illustrate the unexpected results that can come from investigation of these little-known fungi, which clearly deserve greater attention. Fig. 2. Survey of sporangium with a persistent wall (open arrow) showing approximate equivalence between the number of bundles of flagella (e.g. arrows) and encysted spores. The zoospore marked with an asterisk had not encysted at the time of fixation and the encircled flagella were inserted into the zoospore in adjacent sections. X4850. Fig. 3. Unencysted zoospore from one of the sporangia examined. Note that fixation did not cause loss of flagella (f) or basal bodies (6), two of which are still clearly attached to the cell. X32500. Fig. 4. Longitudinal tangential view of the base of a discarded axoneme showing the termination of the C tubule of one of the basal triplets (arrow). X90300. Fig. 5. Serial transverse sections (A and B) through the basal body of a discarded axoneme showing triplet microtubules (white shaded arrows), a cartwheel hub (small black arrows) with associated spokes, and the adjoining skirt (s) and spur (p) of the flagellar root system. X99000. Fig. 6. Transverse section of the basal body of a discarded axoneme showing triplet microtubules and the associated skirt (s). X124200. Fig. 7. Transition zone of a discarded axoneme showing the osmiophilic plate characteristic of this zone in this organism. X110 500.

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We acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada.

REFERENCES BLOODGOOD, R. A. (1974). Resorption of organelles containing microtubules. Cytobios 9, 143-161. FULTON, C. (1971). Centrioles. In Origin and Continuity of Cell Organelles (ed. J. Reinert & H. Ursprung), pp. 170-221. New York, Heidelberg: Springer-Verlag. GIRBARDT, M. & HADRICH, H. (1975). Ultrastruktur des Pilzkernes III. Genese des Kernassoziierten Organells (NAO = "KCE"). Z. allg. Mikrobiol. 15, 157-173. HEATH, I. B. & BAUCHOP, T. (1985). Mitosis and the phytogeny of the genusNeocallimastix. Can. J. Bot. 63, 1595-1604. HEATH, I. B., BAUCHOP, T. & SKIPP, R. A. (1983). Assignment of the rumen anaerobe

Neocallimastix frontalis to the Spizellomycetales (Chytridiomycetes) on the basis of its polyflagellate zoospore ultrastucture. Can.J. Bot. 61, 295-307. HOLLOWAY, S. A. & HEATH, I. B. (1977). Morphogenesis and the role of microtubules in synchronous populations of Saprolegnia zoospores. Expl Mycol. 1, 9-19. MARGULIS, L. (1970). Origin of Eukaryotic Cells, pp. 211-237. New Haven: Yale University Press. MUNN, E. A., ORPIN, C. G. & HALL, F. J. (1981). Ultrastructural studies of the free zoospore of the rumen phycomycete Neocallimastix frontalis. J. gen. Microbiol. 125, 311-323. ORPIN, C. G. & MUNN, E. A. (1986). Neocallimastix patriciae sp. nov., a new member of the Neocallimasticaceae inhabiting the rumen of sheep. Trans. Br. mycol. Soc. (in press). PICKETT-HEAPS, J. D. (1971). The autonomy of the centriole: fact or fallacy? Cytobios 3, 205-214. WHEATLEY, D. N. (1982). The Centriole; A Central Enigma of Cell Biology, pp. 1-232. Amsterdam: Elsevier.

{Received 25 November 1985 -Accepted 23 January 1986)