J. Cell Set. 83, 135-140 (1986) 135Printed in Great Britain © The Company of Biologists Limited 1986

BASAL BODY LOSS DURING FUNGAL ZOOSPOREENCYSTMENT: EVIDENCE AGAINST CENTRIOLEAUTONOMY

I. BRENT HEATH, SUSAN G. W. KAMINSKYJBiology Department, York University, 4700 Keele Street, North York, Ontario, M3J 1P3,Canada

AND TOM BAUCHOPDepartment of Biochemistry, Microbiology and Nutrition, The University of NewEngland, Armidale, NSW 2351, Australia

SUMMARYThe 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, remainsunresolved. All previous reports on shedding or withdrawal of cilia and flagella showed that theirbasal bodies (= centrioles) were retained in the cells where they may, or may not, subsequentlydisassemble. We show that in the fungusNeocallimastix sp. the basal bodies are discarded with theflagella when zoospores encyst. This shedding of basal bodies argues against centriolar persistencein any form and thus against their autonomy and endosymbiotic origin.

INTRODUCTION

Eukaryotic flagellar axonemes develop from basal bodies, which are in at leastsome cases derived from centrioles. Centrioles persist throughout the life of manycells and organisms, and may contain nucleic acids (reviewed by Fulton, 1971;Margulis, 1970; Wheatley, 1982). Consequently, they have been described as semi-autonomous organelles that may have an endosymbiotic origin from a free-livingprokaryotic progenitor (Margulis, 1970). Fulton (1971) has fully discussed theuncertainties of the autonomy hypothesis and more recent data have still notprovided unequivocal support for or against the hypothesis (Wheatley, 1982).Persistence of centrioles in non-flagella-bearing cells is an argument for their essentialcontinuity and autonomy, but their undoubted absence at certain life-cycle stagesand their de novo synthesis before flagellogenesis in a number of species argue againstautonomy (Fulton, 1971; Margulis, 1970; Wheatley, 1982; Pickett-Heaps, 1971).The latter arguments are not compelling, because a hypothetical informationalmolecule or structure could remain after centriole disassembly and persist andreplicate, either in a different form such as a nucleus-associated organelle (Girbardt& Hadrich, 1975) or, unrecognized, in the cell lineage until used for the nextcentriole-synthetic event. The life cycles of many species include the transientproduction of flagellate cells such as sperm or zoospores. Because most of these

Key words: basal bodies, centrioles, zoospores, flagella, fungi, organelle evolution.

136 /. B. Heath, S. G. W. Kaminskyj and T. Bauchop

flagellate cells retain either the whole axonemes, or just the basal bodies, inside thecells when they cease to be flagellate (e.g. at encystment) (Bloodgood, 1974),essential continuity and autonomy of the centrioles seems likely, even when en-cystment is followed by disassembly of the basal bodies. Clear evidence for sheddingof the entire axonemes, including basal bodies, into the environment at encystmentwould be a strong argument against continuity and autonomy. We report suchevidence for the chytridiomycete rumen fungus, Neocallimastix sp.

MATERIALS AND METHODS

Isolate PN2 of a species of Neocallimastix was grown and prepared for serial-section-basedtransmission electron microscopy of individually selected zoosporangia as previously described(Heath et al. 1983; Heath & Bauchop, 1985). Briefly, cultures were grown in a liquid nutrientmedium, fixed by addition of phosphate-buffered glutaraldehyde, mixed with added molten agarand poured into a thin layer to gel in a Petri dish. Areas of gel containing samples of fungus wereexcised, fixed further in glutaraldehyde followed by osmium tetroxide and flat-embedded in epoxyresin. Individual zoosporangia or clusters of cysts were selected from the polymerized blocks andtrimmed 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). Untilwe 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 ofNeocallimastix sp. is outlined in Fig. 1. The organism is maintained in axenicculture by continuous cycles of this proliferative sequence. In addition to the normalsequence of zoospore release and encystment, some apparently normal sporangiafailed to release their zoospores. Instead, the zoospores encysted and began togerminate within the sporangium (Fig. 2). These sporangia were ideal for analysis ofthe fate of flagella at encystment because everything was contained within thesporangium. We have examined three such sporangia, one containing only germi-nated cysts and the others containing a mixture of cysts and unencysted spores thatwere presumably about to undergo encystment at the time of fixation. We have alsoexamined a cluster of germinating cysts that had encysted in a group outside asporangium. In all four samples the cysts were accompanied by bundles of flagellathat were no longer attached to the cells. We could not obtain accurate counts butsubjective observations of essentially random sections suggested an approximatelyequal number of cysts and bundles. We have not traced all shed axonemes to theirproximal ends, but all ends that we have seen terminated in basal bodies, whichcontained triplet microtubules and central cartwheels (Figs 4—7). Each basal bodywas accompanied by the 'skirt' and 'spur' (Heath et al. 1983) components of theflagellar root system (Figs 4-7), but the spurs bore no microtubules comparable tothose found attached to them in the adjacent unencysted zoospores. The shed flagellaseemed to begin disintegration from their proximal ends at the time of sheddingbecause, whilst many bundles were coherent and similar to attached bundles alongmost of their lengths, they were always frayed at the basal body ends. Frayinginvolved both separation of individual flagella and dissociation of the flagellar

Basal body loss during zoospore encystment 137

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

membranes from the axonemes, with concomitant coiling of the axonemes within thedilated membranes. Because of this fraying and relatively short series of sections itwas 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. Wehave also looked inside the recently encysted cells in the samples and have not seenany withdrawn axonemes, basal bodies or centrioles. We have examined enoughseries of sections of fresh cysts to have revealed many basal bodies if the cysts hadbeen flagellated zoospores. This assertion is supported by the frequent detection ofbasal bodies in the cytoplasm of the flagellate zoospores (e.g. see Fig. 3), which werestill present in two of the sporangia examined.

DISCUSSION

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

138 /. B. Heath, S. G. W. Kaminskyj and T. Bauchop

Basal body loss during zoospore encystment 139

flagella, it is unlikely that shedding is restricted to the unusual sporangia with slowzoospore release. Munn et al. (1981) have also shown flagellar shedding by freezoospores, but they did not determine whether the basal bodies were shed with theflagella, because they used relatively low-resolution scanning electron microscopy.Because shed bundles of flagella seem to be as abundant as cysts and because we haveseen no withdrawn axonemes, it is likely that shedding is the typical and probably theonly behaviour. Shedding of basal bodies is also consistent with the absence ofcentrioles 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 lossof basal bodies. This physical discarding of basal bodies shows that if any part ofthem, including undetected material carrying morphogenetic information essentialto the perpetuation of the centrioles, is retained by the cell, then it must be separatedbefore they are shed from the cell. We cannot exclude this possibility but thenecessary extra control system renders it less likely. Because the zoospores arepolyflagellate, one basal body may be retained while the rest are shed. Our inabilityto 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 ensuredifferential behaviour among the variable numbers of flagella on each zoospore. Sucha control system seems error-prone and is unlikely, on the principle of accepting thesimplest hypothesis.

The present demonstration of an encystment system that leads to the discarding ofthe cellular complement of basal bodies and centrioles adds to the weight of evidenceagainst a semi-autonomous existence for the centriole and indirectly argues against anendosymbiotic origin for this organelle. Such conclusions illustrate the unexpectedresults that can come from investigation of these little-known fungi, which clearlydeserve greater attention.

Fig. 2. Survey of sporangium with a persistent wall (open arrow) showing approximateequivalence 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 theencircled flagella were inserted into the zoospore in adjacent sections. X4850.

Fig. 3. Unencysted zoospore from one of the sporangia examined. Note that fixation didnot cause loss of flagella (f) or basal bodies (6), two of which are still clearly attached tothe cell. X32500.

Fig. 4. Longitudinal tangential view of the base of a discarded axoneme showing thetermination 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 discardedaxoneme showing triplet microtubules (white shaded arrows), a cartwheel hub (smallblack arrows) with associated spokes, and the adjoining skirt (s) and spur (p) of theflagellar root system. X99000.

Fig. 6. Transverse section of the basal body of a discarded axoneme showing tripletmicrotubules and the associated skirt (s). X124200.

Fig. 7. Transition zone of a discarded axoneme showing the osmiophilic plate charac-teristic of this zone in this organism. X110 500.

140 /. B. Heath, S. G. W. Kaminskyj and T. Bauchop

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

REFERENCESBLOODGOOD, 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 Kern-

assoziierten 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 itspolyflagellate zoospore ultrastucture. Can.J. Bot. 61, 295-307.

HOLLOWAY, S. A. & HEATH, I. B. (1977). Morphogenesis and the role of microtubules insynchronous populations of Saprolegnia zoospores. Expl Mycol. 1, 9-19.

MARGULIS, L. (1970). Origin of Eukaryotic Cells, pp. 211-237. New Haven: Yale UniversityPress.

MUNN, E. A., ORPIN, C. G. & HALL, F. J. (1981). Ultrastructural studies of the free zoospore ofthe 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 theNeocallimasticaceae 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)