Orientated zoospore attachment and cyst germination in Catenaria anguillulae, a facultative endoparasite of nematodes

Mycol. Res. 101 (5) : 513–522 (1997)

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Orientated zoospore attachment and cyst germination in Catenaria anguillulae, a facultative endoparasite of nematodes

J. W. D E A C O N1 A N D G E E T A S A X E N A2 Institute of Cell and Molecular Biology, University of Edinburgh, Daniel Rutherford Building, Mayfield Road, Edinburgh EH9 3JH, U.K.

Zoospores of the nematode-parasitic Catenaria anguillulae (Chytridiomycota) were studied by videomicroscopy in sealed films of water on microscope slides in the presence or absence of freeze-inactivated nematodes (Panagrellus redivivus). Zoospores swam for more than 1 h at a mean velocity of 104 µm s−", interspersed with repeated phases (1–2 min) of amoeboid crawling on glass or nematode surfaces. They were attracted to and encysted near the mouth, excretory pore, and anus of nematodes, or eventually encysted at random on glass and nematode surfaces. The single posterior flagellum was immobile during amoeboid crawling but resumed rapid beating when the last pseudopodium was being retracted. Zoospores encysted by adhesion of the anterior of an amoeboid cell to a surface ; then the cell posterior was raised above the anterior so that the flagellum projected perpendicular to the surface, and the flagellum was retracted by rotation of the cell contents. Cysts germinated within 20–60 min by a narrow germ-tube at the site of adhesion. The germ-tube grew a short distance, then formed an intercalary vesicle into which the cyst contents emptied by expansion of a cyst vacuole. In several cases the germ-tube penetrated a nematode and formed the vesicle inside the host. Rhizoids or assimilative hyphae developed from the vesicle or by growth of the germ-tube tip. An increasing proportion of zoospores that remained motile after 1 h in water films had a globose body in contrast to the normal elongated form. This seemed to be caused by damage during repeated transitions between the amoeboid and swimming phases, because pseudopodia sometimes remained firmly attached to a glass surface. C. anguillulae showed consistent orientation (polarity) of zoospore encystment and cyst germination. This parallels the behaviour of other zoosporic fungi or fungus-like organisms (Plasmodiophora brassicae, Rozella allomycis, Pythium, Phytophthora and Saprolegnia spp.) suggesting that it is a common feature of zoosporic parasites. Surface-recognition for encystment by C. anguillulae was mediated by the zoospore soma, not the flagellum. In addition, we redefine the early development of C. anguillulae, including flagellar retraction by rotation of cell contents, non-specific adhesion of zoospores and cysts to surfaces, and evacuation of cyst contents into a vesicle from which further growth occurs.

The establishment and maintenance of growth polarity is central to the biology of fungi. Tip growth maximizes the efficiency of nutrient capture and enables fungi to penetrate surfaces such as the outer barriers of plant and insect hosts. However, the control of growth polarity is still poorly understood (Gow & Gadd, 1994 ; Gooday, 1995 ; Harold, 1995). The spores of many fungi do not have a fixed point of germination. Instead, they synthesize new wall materials over most of the cell periphery in the early stage of germination, then a germ-tube arises from some point (Bartnicki-Garcia & Lippman, 1969 ; Bartnicki-Garcia, Hegert & Gierz, 1989). The site of outgrowth can be influenced by exogenous factors such as applied electrical fields (Lever et al., 1994), oxygen gradients (Robinson, 1973) and the proximity of other cells (Robinson, 1980 ; Allan, Thorpe & Deacon, 1992). For parasitic fungi it is likely to be influenced by host factors (Deacon, 1996) but this has been demonstrated in only a few cases (Jansson et al., 1988 ; Allan et al., 1992). " Corresponding author. # Permanent address : Applied Mycology Laboratory, Botany Department,

University of Delhi, Delhi – 110 007, India.

The zoospores of lower fungi can behave differently from other spores. They locate a host or other nutrient source by taxis (Carlile, 1983) then encyst following recognition of a host or other surface (Held, 1974 ; Hinch & Clarke, 1980 ; Estrada-Garcia et al., 1990 ; Donaldson & Deacon, 1993). The biflagellate zoospores of Pythium and Phytophthora spp. (Oomycota : Peronosporales) have a pre-determined site of germination (Mitchell & Deacon, 1986 ; Paktitis, Grant & Lawrie, 1986) and the zoospores orientate during encystment so that the cysts germinate towards the host (Hardham & Gubler, 1990 ; Jones, Donaldson & Deacon, 1991). In these fungi the flagella seem to mediate encystment by surfacerecognition because encystment can be induced in vitro by monoclonal antibodies that bind to the flagella (Hardham & Suzaki, 1986 ; Estrada-Garcia et al., 1990). Other zoosporic fungi have not been examined in such detail, but there is evidence for orientation during encystment and cyst germination in Rozella allomycis (Chytridiomycota : Chytridiales) and Plasmodiophora brassicae (Plasmodiophoromycota). The posteriorly uniflagellate zoospores of R. allomycis settled with a defined lateral region of the zoospore next to the host, then the cysts germinated from the region of contact (Held, 1973).

Zoospore attachment and cyst germination in Catenaria anguillulae The biflagellate zoospores of P. brassicae settled with the flagella projecting away from the host, and again the cysts germinated from the region of contact (Aist & Williams, 1971). Thus, unlike Pythium and Phytophthora, in both the chytrid and the plasmodiophorid the flagella were not directly involved in recognition of the host surface. Our aim was to extend these studies to a fungus of a different taxon, in order to determine whether there are common patterns of behaviour. For this we chose Catenaria anguillulae Sorokı, n (Chytridiomycota : Blastocladiales), a facultative endoparasite of nematodes. Here we report that the zoospores of C. anguillulae show consistent polarity of zoospore encystment and cyst germination. We also describe several new aspects of the behaviour of this fungus. MATERIALS AND METHODS Nematodes naturally infected by C. anguillulae were extracted from soil at King’s Buildings, University of Edinburgh, by the Baermann funnel technique (Cairns, 1960). A pure culture was obtained by allowing diseased nematodes to release zoospores then transferring the spores to dense suspensions of the saprotrophic nematode Panagrellus redivivus L. in distilled water. Zoospores released from infected Panagrellus were streaked onto YpSs medium (20 g soluble starch, 1 g yeast extract, 1 g K HPO , 0±5 g MgSO \7H O, 15 g agar in 1 l # % % # distilled water) containing streptomycin (50 µm ml−") and chlortetracycline (50 µm ml−"). The fungus was maintained at 27 °C on either YpSs or PYG agar (1±25 g Difco bacteriological peptone, 1±25 g yeast extract, 3 g glucose, 15 g agar in 1 l distilled water). All experiments were done with pure suspensions of zoospores, obtained by immersing blocks of colonized agar in an autoclaved mixture (1 : 1) of lake water and distilled water and incubating overnight at 27°. Panagrellus cultures were maintained on moistened, unsterile oat meal that supported large bacterial populations (mainly Bacillus spp.) as a food source. Nematodes were washed from this medium with distilled water and most of the microbial cells were removed by 3 or 4 cycles of centrifugation and resuspension in sterile water. Because zoospores could not settle on rapidly moving nematodes, observations were made with freeze-inactivated nematodes. For this, nematodes were added to drops of water and frozen for 1–3 min on the freezing block of a microtome. After thawing, they were transferred with a needle to a 20 µl drop of zoospore suspension on a microscope slide. A cover-slip was placed on the drop and its edges were sealed with microscope immersion oil to prevent water loss. Similar slides were prepared with zoospore suspension alone. All observations were made at room temperature (ca 20°) with a Leitz Orthoplan microscope, using a 70¬ phasecontrast objective. The microscope was equipped with a Panasonic S-VHS F15 colour video camera, connected to a Panasonic S-VHS AG-6720 video recorder and BT-M1420PY colour video monitor. Recordings were made on Master Broadcast S-VHS videotapes. Photographs were taken from the screen of the video monitor, using a tripod-mounted camera with Ilford black and white film ISO 65, or ISO 400 for motile cells.

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For detection of nuclei, cysts were stained with the fluorochrome DAPI (1 µg ml−" distilled water) and observed with a 70¬ objective and epifluorescence. RESULTS Zoospore behaviour Motile zoospores were elongated, 6±1–6±8 µm long, 3±7 µm wide, with a posterior flagellum 19–20 µm long. The terminal 2 µm of the flagellum was narrower than the rest of the flagellum. The spores contained a conspicuous nuclear cap, a posteriorly located nucleus, a microbody-lipid globule complex (MLC) to one side of the nucleus, and a large mitochondrion near the base of the flagellum. The arrangement of these organelles was best seen in abnormal, globose zoospores (Fig. 1) described later. The anterior of zoospores was sac-like and contained few identifiable organelles except for occasional small, phase-bright vacuoles and phase-dark particles (probably gamma particles). All these observations were consistent with published electron micrographs of zoospores of C. anguillulae (Chong & Barr, 1974). Under sealed cover-slips, zoospores swam in straight paths at 88–124 µm s−" (mean 104³5±2 ..., n ¯ 20) measured over periods of 0±5 s from successive video frames (50 s−"). In the absence of nematodes, the spores remained motile for up to 2 h, but many began to encyst after about 60 min, and most of those that were still motile after 90 min had a globose body form. Swimming was interrupted periodically by phases of amoeboid crawling on the surface of the slides (never on the overlying cover-slip). Amoeboid crawling was initiated by contact of the spore anterior with the glass. The flagellum then ceased its rapid beating and projected more or less immobile from the cell posterior, with only weak, intermittent waving (Figs 2–4). A broad, rounded pseudopodium developed from the anterior, sac-like region of the spore, creating a large area of contact with the glass. The pseudopodium produced lobes in a wave-like manner (Figs 2, 3) and sometimes also produced tapering, finger-like projections (Fig. 4). During amoeboid crawling, the major visible

Fig. 1. Globose zoospore of C. anguillulae prior to encystment, showing a nuclear cap (NC), nucleus (N), microbody-lipid globule complex (L), basal mitochondrion (M) and flagellum (F). Bar ¯ 10 µm.

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Figs 2–4. Zoospores of C. anguillulae showing amoeboid crawling on a glass surface, with immobile flagella projecting upwards from the plane of focus. The nuclear cap (NC) and microbody-lipid globule complex (L) are shown. Fig. 2. Normal elongated zoospore just before it regained swimming. Fig. 3. Globose zoospore with broad pseudopodial lobes. Fig. 4. Normal elongated zoospore adpressed to the glass surface and with a finger-like projection of the pseudopodium. Bar ¯ 10 µm.

Fig. 5. Zoospore of C. anguillulae showing amoeboid crawling on the surface of a nematode, P. redivivus. The nuclear cap (NC) and microbody-lipid globule complex (L) are shown. The immobile flagellum is held above the nematode surface. Bar ¯ 10 µm.

organelles (nuclear cap, MLC, mitochondrion and flagellum) retained their normal spatial relationship to one another near the posterior of the cell. Crawling lasted for usually 1–2 min and terminated in a characteristic way : as the last pseudopodial projection was being retracted the flagellum started to beat strongly and the spore detached from the glass before the pseudopodium was fully retracted. The spores resumed their

normal shape during swimming. In some cases, however, a pseudopodium remained firmly anchored to the glass and was pulled into a long, fine thread as the spore struggled to swim away. Some of these spores were damaged permanently because they swam off with a persistent trailing filamentous appendage that resembled a second flagellum. The responses of zoospores to nematodes depended on whether these had been killed by freezing or only immobilized (but still alive) by freezing ; in immobilized nematodes the pharyngial bulb was seen to pulsate weakly for up to an hour. Zoospores were not attracted to killed, intact nematodes but seemed to encounter them at random and then crawl on the nematode surface (Fig. 5) before swimming away. This behaviour was similar to that on the surrounding glass slide. Zoospores were not attracted strongly to killed, disrupted nematodes (with released body contents) but the spores sometimes encysted in the spilled contents. In contrast, zoospores were strongly attracted to the natural body openings of living, immobilized nematodes – the mouth (Fig. 6), anus (Fig. 7) and excretory pore located near the bulb (Fig. 8). Zoospores swam directly to these regions, indicative of chemotaxis, and accumulated there in large numbers. They showed amoeboid crawling before encysting near the body openings. In all cases of amoeboid crawling the zoospores made broad lateral contact with a glass or nematode surface, but the flagellum was always held away from the surface (Figs 5, 8). Encystment Encystment was preceded by a phase of amoeboid crawling and it occurred in a consistent way. The cell stopped crawling and remained attached to a glass or nematode surface at its anterior, before the cell posterior was raised up to form a dome-shaped cell with the flagellum projecting more or less

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Figs 6–7. Zoospores of C. anguillulae accumulating and encysting in the mouth (Fig. 6) and anal regions (Fig. 7) of nematodes. Flagella (F) are shown. Bar ¯ 10 µm.

Fig. 8. Zoospores of C. anguillulae accumulating and encysting near the excretory pore, close to the pharyngial bulb (B) of a nematode. (a) One zoospore (with flagellum) is about to encyst at the end of an amoeboid crawling phase ; the cell posterior and flagellum are being drawn over the cell anterior which has adhered to the nematode. (b) After 69 s, the flagellum is held perpendicular to the nematode surface. (c) After 97 s, the flagellum has formed an arc (arrows) and is being retracted into the cell. Bar ¯ 10 µm.

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517 perpendicular to the surface. This was most clearly seen on nematodes when the spore was viewed in profile (Fig. 8) but it occurred also on glass surfaces. The flagellum then developed a kink near its point of insertion (Figs 9, 10), accompanied by a small rotational movement of the cell contents. Then the rest of the flagellum formed an arc and was retracted into the cell by rotation of the cell contents (Figs 9, 10). During this process the cell periphery seemed to remain stationary : the point where the flagellum was retracted into the cell did not change, and the cell shape (incompletely globose) also did not change. Owing to the small size of the cells and early changes in their phase-optical properties, it was difficult to follow precisely the changes in arrangement of internal organelles. However, the more conspicuous organelles (nuclear cap and MLC) were seen to rotate within the cells and retained their positions relative to one another during much of the time. The rotation of cellular contents occurred in more than one plane of focus, because it was seen both on glass surfaces (lateral rotation when the cells were viewed from above) and on nematodes (anterior-posterior rotation when the cells were viewed in profile). Flagellar retraction was completed in less than 2 min and it involved approximately two ‘ complete ’ rotations of the cell contents, gauged by movement of the MLC. However, the abnormal, globose zoospores (e.g. Fig. 10) sometimes showed up to eight rotations of the cell contents, continuing after the visible part of the flagellum had been retracted. In a few of these globose cells the retraction was incomplete because very fine strands (possibly microtubular arrays) remained outside the cell. Often the globose zoospores showed less conspicuous amoeboid crawling and produced larger, balloon-like pseudopodia than did the normal elongated zoospores (compare Fig. 3 with Figs 2 and 4). In many cases the cysts resulting from globose zoospores did not germinate. Germination and further development

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Fig. 9. Sequence of photographs of flagellar retraction by a normal elongated zoospore encysting on a glass surface. (a) The flagellum (out of the plane of focus) has kinked (arrow). (b) After 19 s, the flagellum has arced. (c, d, e) After 52, 85 and 104 s, the flagellum is being retracted (flagellar tip arrowed) while the major organelles are rotating in the cell. ( f ) After 129 s, the flagellum is fully retracted. (g) After 40 min ; the cyst has germinated ; conspicuous vacuoles are seen in the posterior of the cell, while a germ-tube has emerged (tip arrowed, out of focus) next to the glass surface. Bar ¯ 10 µm.

Cysts were spherical or slightly discoid, 3±8–4±1 µm diam., when viewed from above on glass surfaces (Figs 9, 10) but were flattened against the surface when viewed in profile on nematodes (Figs 6, 8). They were laterally compressed rather than rounded where zoospores had massed on a nematode as a result of chemotaxis. About 20 min after encystment, phasebright vacuoles began to appear near the posterior of the cyst, and these vacuoles enlarged progressively and fused with one another. During this time the cyst sometimes swelled slightly (e.g. Fig. 13). Germination occurred by outgrowth of a germtube from the region of cyst attachment to a glass or nematode surface (Figs 9, 10, 11). Sometimes the outgrowth was seen as early as 20 min after flagellar retraction, especially on nematodes or in diffusates around dead nematodes where zoospores encysted rapidly. In the absence of nematodes, when zoospore encystment was delayed, the outgrowth often was seen at 70–90 min after flagellar retraction. All these observations were made in sealed water films, where development might have been oxygen-limited. Indeed, the last zoospores to encyst in the absence of nematodes were those near air bubbles on the slides, indicating that encystment in the absence of nematodes occurred in response to oxygen limitation.

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(a)

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Figs 11–12. Cysts of C. anguillulae that have germinated in sealed water films. Bar ¯ 10 µm. Fig. 11. In absence of nutrients, the cyst (C) produced a germ-tube which stopped growing and formed an intercalary vesicle (V) containing a lipid globule. The cyst contents are partly evacuated into the vesicle. Fig. 12. In the presence of nematode diffusates, the cyst (C) contents have been translocated into the vesicle (V) which has swollen and contains many lipid bodies. A branching rhizoidal system has developed from the vesicle. Bacterial cells (B) are contaminants from the nematode. Fig. 10. Sequence of photographs of flagellar retraction by a globose zoospore encysting on a glass surface. (a) The flagellum has kinked (arrow). (b) After 25 s, the flagellum has arced and is being retracted while the internal organelles are rotating. (c, d ) After 39 and 54 s, further stages of flagellar retraction (flagellar tip arrowed) accompanied by further rotation of cell contents. ( f ) After 35 min ; the cyst (out of median plane of focus) has germinated by a germtube (GT) next to the glass surface. Bar ¯ 10 µm.

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519 The germination outgrowth was a narrow tube, about 1 µm diam., with a rounded, blunt tip. It grew to a length of four or five cyst diameters, then stopped and a swollen vesicle developed in an intercalary position (Fig. 11) or sometimes terminally. The vesicle expanded while the cyst progressively vacuolated and its protoplasm moved into the vesicle. Eventually the cyst was left as an empty ghost (Fig. 12). Further development of the vesicle depended on exogenous nutrients ; if these were available in nematode diffusates, or added peptone, then the vesicle swelled and developed conspicuous lipid bodies (Fig. 12). Branching rhizoids were then formed by regrowth of the original germ-tube tip (e.g. Fig. 13) or from the swollen vesicle (Fig. 12). The germ-tubes sometimes penetrated a nematode and produced vesicles in the host (Fig. 14). However, in most instances the emergence and growth of a germ-tube caused the cyst to be detached from the nematode surface (Fig. 13). Interpretation of this was complicated by the fact that freezeinactivated nematodes almost invariably ruptured near the pharyngial bulb or occasionally near the anus after about 20–40 min, owing to their high internal hydrostatic pressure. At this stage the zoospores had already settled and encysted near the body openings ; but during rupture of the nematodes, part of the intestine burst rapidly through the body wall, and this caused cysts to be dislodged instantaneously from the nematode surface. For example, all the cysts in Fig. 8 were dislodged by explosive rupture of the nematode before they had germinated ; the cysts in Fig. 13 were similarly ‘ sprung ’ from the surface when the nematode ruptured near the bulb. In contrast, cysts were not dislodged from nematode surfaces when the cover-slip was moved sideways with the tip of a scalpel. Also, cysts that had begun to germinate on glass slides were not dislodged by repeatedly pressing on the cover-slip or, in one test, by moving a cover slip sideways so that the meniscus of an air bubble moved forwards and backwards twice across the cysts at 500 µm s−" (measured from successive video frames). Thus, the cysts adhered to nematode and glass surfaces in normal conditions. When cysts that had germinated on glass slides were stained with DAPI and examined under a fluorescence microscope, a single nucleus was always seen near the anterior of the cysts. But the nucleus seemed to have no fixed relationship to the site of germ-tube outgrowth ; instead, its presence in the anterior region seemed to be caused by the development of a large vacuole in the posterior region.

14 Figs 13–14. Germinating cysts of C. anguillulae on a nematode surface. Bar ¯ 10 µm. Fig. 13. Cysts from zoospores that were attracted to the mouth region of a nematode, but subsequently dislodged by rupture of the nematode bulb (to right of photograph, not shown). Each cyst contains a conspicuous vacuole and has formed an outgrowth from the region of contact with the nematode surface. Fig. 14. Cysts (C) from zoospores that accumulated near the anus of a nematode. Some cysts are empty (E), others contain large vacuoles. Some of the cyst outgrowths penetrated the nematode and have produced large, lipid-containing vesicles (V) in the host.

DISCUSSION The use of sealed water films, coupled with videomicroscopy, enabled us to make repeated observations of single zoospores and zoospore cysts, and to observe behavioural changes in these small cells that would be difficult to observe or quantify by routine light microscopy. Several new and interesting features were revealed in this way. Zoospores of C. anguillulae orientated precisely during encystment on surfaces and then germinated from a region of contact with the surface. This was a non-specific response, observed on both glass and nematode surfaces. As a prelude

Zoospore attachment and cyst germination in Catenaria anguillulae to encystment, the zoospores showed chemotaxis to the body orifices of nematodes and then underwent a period of amoeboid crawling, presumably searching for a suitable site for docking on the host. C. anguillulae is thus a further example of a zoosporic parasite with a clearly defined homing sequence (Deacon & Donaldson, 1993) involving attraction to a host, selection of an encystment site, orientated adhesion, encystment and orientated cyst germination. This pattern has now been described for fungi or fungus-like organisms of five taxa : Plasmodiophora brassicae (Aist & Williams, 1971), Rozella allomycis (Chytridiales ; Held, 1973), Pythium and Phytophthora spp. (Peronosporales ; Deacon & Donaldson, 1993), Saprolegnia spp. (Saprolegniales ; Durso, Lehnen & Powell, 1993) and C. anguillulae (Blastocladiales). So it is a common feature of zoosporic parasites. Flagellar retraction in C. anguillulae occurred by rotation of the cell contents while the cell periphery seemed to remain fixed by adhesion to a surface. Posteriorly uniflagellate zoospores of the chytridiomycota are reported to have various methods of flagellar retraction, including ‘ body-twist ’, ‘ lash-around ’ and ‘ straight-in ’ (Koch, 1968 ; Held, 1973). The method in C. anguillulae resembled the body-twist method described for Blastocladiella emersonii (Koch, 1968) but only the cell contents rotated. Moreover, this seemed to be tightly controlled because it consistently involved approximately two rotations of the cell contents of the normal, elongated zoospores. The abnormal globose zoospores, however, exhibited consistently more rotations. The shape of these cells indicated that some of their cytoskeletal elements had been disrupted, presumably during their prolonged motility (they were the last zoospores to encyst) and repeated transitions between swimming and amoeboid phases. This suggestion is supported by the observation that pseudopodia sometimes did not detach easily from the glass surface when amoeboid cells resumed swimming, and in some cases the pseudopodium was stretched into a filament that trailed permanently behind the zoospore. Also, in several cases the globose zoospores retracted the main part of the flagellum during encystment but fine strands remained outside of the cell at the site of flagellar retraction. In the conditions of this study the mode of development from cysts differed from the original description of C. anguillulae (Sorokin, 1876, reproduced in Sparrow, 1960) where cysts were reported to germinate by producing a delicate rhizoid and then to form a tubular outgrowth from the opposite pole. In our work the cysts always produced a narrow germ-tube from one pole, and this produced an intercalary (or occasionally terminal) vesicle which expanded as the cyst contents emptied into it. Butler & Buckley (1927) also observed this when zoospores of C. anguillulae encysted on fluke eggs. Sometimes the germ-tube penetrated a nematode and produced a swollen vesicle inside the host, leaving the empty cyst case on the host surface. Then the vesicle enlarged progressively and accumulated lipids as nutrients were absorbed from the host. The vesicle resembled an infection bulb typical of the early stage of development of many nematophagous fungi (Dijksterhuis et al., 1994). Assimilative hyphae or rhizoids then developed from the vesicle, but there was little further development even during

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3 days of observation, presumably because of oxygen depletion under the sealed cover-slips. The amoeboid cells of C. anguillulae showed non-specific adhesion to glass and nematode surfaces. Tunlid et al. (1991) reported that zoospores of C. anguillulae adhered to a germanium crystal, and by infrared spectroscopy the adhesins were shown to have proteinaceous components. This is consistent with reports of glycoprotein components on the zoospore surface of several chytridiomycota, although these components are poorly characterised (review by Powell, 1994). Cell-surface glycoproteins often serve as receptors that mediate responses to environmental stimuli, so they might be responsible both for non-specific adhesion of amoeboid cells and for specific recognition of encystment sites by C. anguillulae. We found that zoospores encysted non-specifically on glass or nematode surfaces after 60 min in sealed water films, but they encysted rapidly and specifically after chemotaxis to nematode body orifices. The non-specific encystment seemed to be caused by oxygen depletion because zoospores remained motile for longest near air bubbles. Specific encystment might have been mediated by recognition of nematode surface components near the body orifices. There is evidence of site-specific adhesion of the spores of some nematophagous fungi, indicating the existence of site-specific components on nematode surfaces (Jansson & Nordbring-Hertz, 1983). In any case, the orientation of zoospores of C. anguillulae during amoeboid crawling and encystment showed clearly that surface-recognition was mediated by the soma and not the flagellum, which was always held away from surfaces. Held (1973) reported similar behaviour in Rozella allomycis, and the report by Aist & Williams (1971) suggests that Plasmodiophora brassicae also recognizes encystment sites by receptors on the soma. This contrasts with the behaviour of oomycota (Pythium, Phytophthora and possibly Saprolegnia spp.) where the zoospores settle with the flagella adjacent to a surface, and flagellum-specific monoclonal antibodies can induce encystment in vitro (Hardham & Suzaki, 1986). Similarly, the flagella of the alga Chlamydomonas bear the sexual agglutinins involved in conjugation of mating pairs (Musgrave & van den Ende, 1987). In C. anguillulae the cysts adhered non-specifically to glass and nematode surfaces, similar to the non-specific cyst adhesion of other chytridiomycota (Dalley & Sonneborn, 1982 ; Powell, 1994), Pythium and Phytophthora (Sing & Bartnicki-Garcia, 1975) and Saprolegnia (Durso et al., 1993). The cyst adhesins of oomycota are proteins or glycoproteins released from peripheral vesicles of the zoospores in the initial stages of encystment (Lehnen & Powell, 1989 ; Hardham, 1992). Zoospores of some chytridiomycota have vesicles that disappear after encystment (Powell, 1994). C. anguillulae does not seem to have them (Chong & Barr, 1974) but an extracellular cyst coat polymer has been detected by electron microscopy (Tunlid et al., 1991) ; presumably it functions as an adhesin but its origin and properties are unknown. The cysts of C. anguillulae always germinated from a region of attachment to a surface, but we do not know if the germination site is pre-determined. In Pythium the existence of a pre-determined site of germ-tube outgrowth was shown

J. W. Deacon and Geeta Saxena experimentally (Mitchell & Deacon, 1986), and by relating the site of outgrowth to the former site of the water-expulsion vacuole (WEV) of the zoospore (Jones et al., 1991). In Phytophthora palmivora the germination site is near the point of flagellar insertion (and WEV) of the zoospores (Paktitis et al., 1986). In P. cinnamomi the germination site corresponds to a localised region where vesicles near the base of the flagella release a cytochemically distinct adhesin during encystment (Hardham & Gubler, 1990). This also seems to be true for Saprolegnia ferax, although other factors might modify the precise site of emergence (Durso et al., 1993). For these oomycota, therefore, the precise orientation of the zoospore during encystment serves to locate the germination site close to a host or other surface. The precise orientation of encystment by Catenaria zoospores suggests that the site of germination also might be fixed. But we could not relate the germination site to any spatially fixed cellular markers because the cell contents were rearranged during flagellar retraction. Finally, this study revealed a remarkable uncoupling of flagellar beating during the amoeboid phase of Catenaria zoospores. The flagellum stopped beating as soon as amoeboid movement began, and started beating rapidly while the last pseudopodium or pseudopodial projection was being withdrawn. The force of flagellar beating caused the spore to detach from a surface and swim off before the pseudopodium was fully retracted. Zoospores made these transitions several times, suggesting the existence of a rapidly reversible motor switch. This seemed to be linked to the degree of contact with a surface. Several workers have emphasized the close association of the mitochondrion with the flagellar basal apparatus in zoospores of chytridiomycota (e.g. Chong & Barr, 1973, 1974), the implication being that this is significant in providing energy for flagellar function. However, the major visible organelles, including the mitochondrion, did not change their normal spatial relationship to one another or to the flagellum during amoeboid crawling. So the switch in motor activity would not seem to involve a change in energy provision. Protrusions from the surface of animal cells are thought to be driven by actin polymerization (Condeelis, 1993) and this is assumed also for non-turgor driven tip growth of Saprolegnia (Money, 1994). The switch between swimming and amoeboid phases of zoospores could therefore involve changes in the cytoskeleton or activities of motor proteins, mediated by cell contact with a surface. G. Saxena is grateful to the University of Delhi for granting study leave, and to the University of Edinburgh for providing facilities for this work. We thank Frank Johnston for excellent photographic support. REFERENCES Aist, J. R. & Williams, P. H. (1971). The cytology and kinetics of cabbage root hair penetration by Plasmodiophora brassicae. Canadian Journal of Botany 49, 2023–2034. Allan, R. H., Thorpe, C. J. & Deacon, J. W. (1992). Differential tropism to living and dead root hairs by the biocontrol fungus Idriella bolleyi. Physiological and Molecular Plant Pathology 41, 217–226. Bartnicki-Garcia, S. & Lippman, E. (1969). Fungal morphogenesis : cell wall construction in Mucor rouxii. Science 165, 302–304.

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