Observations on the behaviour of second stage juveniles of Hetero

inside host roots Urs WYSS and Ulrich ZUNKE

Institut fiir Phytopathologie, Universitat Kiel, Olshausenstrasse 40, 2300 Kiel 1, Bundesrepublik Deutschland.

SUMMARY

Using special observation chambers and high resolution video-enhanced contrast microscopy, the behaviour of infective second stage juveniles of Heterodera schachtii was studied inside roots of Brassica napus seedlings. With this technique, intracellular migration of the juveniles to the site of syncytium induction near or within the vascular tissue and feeding from the initial syncytium could, for the first time, be directly observed on live animals. The function of structures inside the feeding apparatus of the nematode and in the initial syncytium, previously only recorded with the transmission electron microscope, was analysed. Feeding from the initial syncytium was invariably in a series of repeated cycles, each composed of three distinct phases : i) continuous withdrawal of nutrients from within a zone of modified cytoplasm through a feeding tube connected to the orifice of the stylet lumen. iil stylet retraction from the feeding site, gentle stylet movements within the stomatal cavity and reinsertion of the stylet-tip, accompanicd by depletion of secretory fluids from within the expanded valve membranes of the two subventral oesophageal gland ampullae. iii) release of secretory fluids from the ampulla of the dorsal œsophageal gland, forming a new feeding tube inside the zone of modified cytoplasm. Events that occurred during these three phases within the feeding apparatus of the nematode and in the initial syncytium are described and discussed.

RESUME

Observations sur le comportement des juvéniles de deuxième stade de Heterodera schachtii dans les racines de l'hôte

Le comportement des juvéniles infestants de 2""' stade de Heterodera schachtii a été étudié à l'intérieur même des racines de plantules de Brassica napus en utilisant des chambres d'observation adaptées et un dispositif de microscopie en contraste de phase, couplé à un appareil de vidéo à haute résolution. Grâce à cette technique, la migration intracellulaire des juvéniles vers le site où sera induit le syncitium, près ou à l'intérieur du tissu vasculaire, de même que la prise de nourriture àpartir de ce syncitium naissant, ont pu, pour la première fois, être observées directement, sur des animaux vivants. Le fonctionnement du systeme de prise de nourriture a pu être ainsi analysé, alors que sa structure interne et son action dans le syncitium n'étaient jusqu'ici connues que par microscopie électronique à transmission. La prise de nourriture à partir du syncitium initial consiste invariablement en une série de cycles successifs, chacun comportant trois phases distinctes : il prélèvement continu de matière nutritive à partir d'une zone de cytoplasme modifié à travers un tube nutritionnel connecté à l'orifice de la lumière du stylet; ii) recul du stylet hors du site nutritionnel, mouvements lents du stylet à l'intérieur de la cavité buccale et réinsertion de l'extrémité du stylet accompagnée par l'écoulement de sécrétions fluides à partir des valves ouvertes des ampoules des deux glandes œsophagiennes subventrales; iii) expulsion de sécrétions fluides à partir de l'ampoule de la glande œsophagienne dorsale, donnant naissance à un nouveau tube nutritionnel à l'intérieur de la zone cytoplasmique modifiée. La cinétique des processus touchant l'appareil nutritionnel et le syncitium initial durant ces trois phases est décrite et commentée.

Little is yet known about the behaviour of infective second stage juveniles (J 2) of Heterodera and Globodera spp. inside roots. Information is restricted to root invasion and intracellular migration (e.g. Mankau & Linford, 1960; Doncaster & Seymour, 1973) of which some details were also recorded in ciné films (Doncaster, Green & Shepherd, 1968; Müller, Wyss & Inst. wiss. Film, 1981; Wyss, Müller & Inst. wiss. Film, 1983).

Until recently it was virtually impossible to study the feeding behaviour of the juveniles once they had

induced their permanent feeding site (syncytium) near or within the vascular cylinder. However, with rapid advances in high resolution video-enhanced contrast recording techniques, events can now be seen deep inside the root that hitherto had remained obscure. With the method used by Wyss and Zunke (19856) a research film was produced (Wyss, Zunke & Inst. wiss. Film, 1985) showing in great detail the behaviour of infective H. sckachtii juveniles during intracellular migration and feeding from initial syncytia, as well as characteristic cell

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responses. This paper provides an analysis of events recorded in the film and of numerous observations not included in it.

Materials and methods

Freshly-hatched J 2 of Heterodera schachtii, obtained from monoxenic root cultures kept in nutrient agar medium (Müller, 1978), were transferred to roots of Brassica napus cv. Akela seedlings grown in aseptic nutrient agar in special observation chambers as described by Wyss and Zunke (1985~). For observations under the differential interference contrast microscope the chambers were placed on the microscope stage. Relevant scenes were recorded at high magnification on 2.5 cm video tapes by using video contrast enhancement (Wyss & Zunke, 1985 b) and were analysed where necessary, by single frame evaluation.

Results

ROOT INVASION AND INTRACELLTJLAR MIGRATION

When added to well growing roots, the most active freshly-hatched J 2 of Heterodera schachtii soon oriented themselves towards the roots and showed the basic pattern of behaviour described by Doncaster and Seymour (1973). They tried to invade the roots mostly at their growing tip or at sites where lateral roots emerged, but also, though to a lesser degree, at any available site. Attempts to perforate the walls of epidermal cells expended much energy and required support for the body as a whole to enable sufficient pressure to be exerted towards the target of attack. Some juveniles were seen to thrust their stylets persistently at various sites on a ce11 Wall, weakening and perforating it at some spots, but failing, even after several attempts, to cut a slit through which to invade. Those that succeeded attracted other juveniles to the invasion site so that crowding, especially in thin lateral roots, was quite common.

Once inside the first cortical cell, intracellular fonvard progression to the site of syncytium induction near or within the vascular cylinder was less tedious. Ce11 walls were explored repeatedly by probing stylet thrusts which, at suitable sites, led to concentrated stylet thrusting. As described by Doncaster and Seymour (1973) for J 2 of H. cmciferae, the thrusts were highly co-orThated towards making a line of perforation holes- until a slit was formed. Fig. 1 shows some details of the characteristic " cutting cycle ". Holes (Fig. 1 A) were made by a single thrust or by several strokes, depending on the rigidity of the Wall. Then a point immediately adjacent to the hole just made was attacked and, if not pierced immediately, caused considerable bending of the stylet-tip with the lips being lifted from the point of contact (Figs 1 A, B). After any such unsuccessful

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attempt, the stylet was thrust back into the adjacent hole and the attack was renewed, eventually with success. When ce11 walls became too elastic following persistent stylet thrusting, the juveniles then selected a new, more rigid site. The stylet-tip was generally thrust 2-3 p deep through a hole (Fig. 1 C) and finally, by a line of merging holes, a slit was cut through which the juvenile entered the neighbouring ce11 (Fig. 1 D).

The time spent in cutting slits through a ce11 Wall for fonvard progression varied considerably, from less than one to 15 mn, depending on factors such as rigidity of the Wall, conditions for applying sufficient lip pressure to the Wall and the vigour of the invader. The juvenile in Fig. 1 E, for instance, had to expend less energy in cutting an entry slit than the one shown in Figs 1 A-D, as within the narrow tunnel of still rigid walls it could exert more pressure on its target. Once inside the next cortical cell, widespread exploration (Doncaster & Seymour, 1973) was resumed, i.e. the juvenile moved fonvard (Fig. 1 F) with searching movements of the head and occasional stylet probings.

Intracellular migration towards the site of syncytium induction required much energy as, apart from the short phases of fonvard movement inside the cells, the stylet was thrust incessantly with up to 150 thrusts per minute. Cut dits were often not immediately used for fonvard progression, instead the juveniles searched for other spots to be attacked by forceful stylet thrusts.

Although al1 three oesophageal glands were active, the two subventral ones in particular were packed with relatively large secretory granules (Fig. 3 A), which filled the gland extension right to the ampulla (Fig. 3 B), no signs of release of secretory fluids could be detected during intracellular migration. The metacorpus stayed largely inactive throughout and only on a very few occasions did it pump for a few seconds. Fonvard progression inside the root to the final feeding site is thus most probably by mechanical means only, i.e. by continuous accurate stylet thrusting and strong lip pressure.

Near or within the vascular cylinder locomotary activity stopped, and a group of cells within reach of head movements was intensively probed and worked on by stylet thrusts aimed at one spot for up to several minutes. The tip of the stylet was eventually carefully inserted into the hole thus made by very gentle fonvard and backward movements. Once it had penetrated 3-4 pm into the cell, stylet movements ceased and the tip remained continuously protruded. The metacorpus then began to twitch, jerking the pump lining without opening the pump chamber, followed by Co-ordinateci muscle contractions that opened the pump chamber for up to about 40 seconds. After that secretory granules were seen to flow forward and accumulate within the ampulla behind the stylet knobs, which already contained granules during intracellular migration, although smaller and not so numerous as in the

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Jzrveniles ofHeterodera schachtii inside host roofs

Fig. 1. Intracellular migration of J 2-juveniles of H. schachtii through cortical cells in roots of Brussica napus seedlings. Bar = 10 km. A : bending of stylet-tip when thrust is met by resistance of ce11 Wall. Arrows point to holes made by two previous stylet thrusts; B : same as A, at lower magnification; C : hole made in ce11 Wall by stylet thrust; D : head region, accompanied by stylet movements, enters through slit cut into cell Wall by highly co-ordinated stylet thrusts; E : a few seconds after entering a cortical cell, the Wall of the.next ce11 is attacked by stylet thrusts; F : fonvard progression inside a cortical cell.

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Fig. 2. A J 2-juvenile of H. schachtii feeding from an initial syncytial ce11 (ISC), about one day after the nematode had started to feed from this cell. The ISC is here located near xylem elements (x) in a thin lateral root of a Brussica napus seedling. Bar = 10 Pm. A : stylet-tip inserted into zone of modified cytoplasm (zmc) during feeding phase 1 (see Fig. 4). Ce11 Wall stub (cws) shows protoplast fusion of the ISC with that of a neighbouring cell; nu 1, enlarged nucleolus of hypertrophied ISC nucleus; nu 2, enlarged nucleolus of hypertrophied nucleus of neighbouring ce11 on passage beneath cws; B : 2 min after A, showing dynamics in ISC. Note changes in position and appearance of the two nuclei. Arrows indicate cytoplasmic streaming beneath cws; C : during feeding phase II (see Fig. 4), 15 min after B; stylet-tip now retracted; zmc, though not clearly recognisable, still preserved.

Fig. 3. Parts of the anterior feeding apparatus of J 2-juveniles of H. schuchtii during intracellular migration through cortical cells (A-B) and during feeding from the initial syncytium (C-H) within roots of Brmsica nuptls seedlings. Al1 bars = 10 Pm, except D = 5 Pm. A : the nuclei of the two subventral glands (nsg) and nucleus of dorsal gland (ndg). Subventral glands fïlled with secretory granules (gr), whereas dorsal gland (dg) contains fewer granules during intracellular migration; B : the extensions (sge) and ampullae (sga) of the two subventral glands packed with granules during intracellular migration; C : throughout phase 1 (see Fig. 4) secretory granules of dorsal gland (grd) and the two subventral glands (grs) accumulate behind contracted posterior constraining muscles (pcm). dg, dorsal gland; m, metacorpus; ndg, nucleus dorsal gland; D : about 10 min after beginning of phase 1. Secretory granules of dorsal gland (grd) still within gland ampulla, where cuticularized arms of gland valve (arrows) are in open position. dgv, duct dorsal gland valve opening into oesophageal lumen; stk, stylet knobs; E : expansion of membrane of subventral gland valve (mv) within metacorpus (m) during phase II (see Fig. 4), one of the two membranes shown in this plane. Secretory granules (grs) surround expanding membrane. Arrows point to still relaxed constraining muscles in front of and behind metacorpus; F : about 30 s after E; contents within expanded valve membrane have been depleted during metacorpus pumping. Anterior (acm) and posterior (pcm) constraining muscles stay contracted during pumping; G : metacorpus (m) at beginning of phase III (see Fig. 4). Granules of dorsal gland (grd) flow fonvard (arrows) through gland extension (dge) and accumulate in dorsal gland ampulla (dga). stk, stylet knobs; H : 25 min after G; secretory granules of dorsal gland still flow fonvard (arrows) through gland extension (dge). A large '' vacuole " (v) has been formed about 5 pm in front of metacorpus (m). " Vacuole in procorporal Wall tissue lies beneath oesophageal lumen lining (out of focus in region of '' vacuole ").

ampullae of the two subventral glands. After about 45 mn the metacorpus started to pump in a regular rhythm, characteristic for food ingestion.

We were able to observe these critical moments of feeding behaviour, at the very beginning of syncytium induction, only on two occasions and then not very clearly, as the anterior feeding apparatus (from the oral opening to the oesophago-intestinal valve) extended into different optical planes, and the pressure exerted by the 40 x and 100 x oil immersion objectives disturbed the nematodes such that they soon stopped feeding. I t was much easier to search for juveniles that had already initiated their permanent feeding site. With much luck a juvenile was found in a thin lateral root where the initial syncytium (about one day after induction) and the anterior feeding apparatus were in the same optical plane (Figs 2 A-C). The behaviour of this juvenile could be observed in great detail over a period of three days. The descriptions that follow are partially based on this juvenile and are complemented by observations on fifteen other juveniles whose syncytium and parts of their feeding apparatus could also be studied at high magnification.

FEEDING BEHAVIOUR AND HOST RESPONSES IN INITIAL SYNCYTIAL CELLS

Pattern of feeding behaviour The pattern of feeding behaviour of J 2 feeding from

initial syncytial cells was found to be composed by a series of repeated cycles, each with three distinct phases (1-1111, as illustrated in Fig. 4. In phase 1 the tip of the stylet stayed protruded 3-4 pm deep in the initial syncytial ce11 while the metacorpus pulsated continuously at a rate of 5-7 contractions per second that dilated the pump chamber. This phase lasted, on average, for about one hour.

At the beginning of phase II the metacorpus stopped pumping and the tip of the stylet was retracted into the stomatal cavity. This was soon followed by intermittent periods of slow and gentle stylet movements. Four to 11 mn (n = 14*) after retraction, the stylet-tip was reinserted into the initial syncytial cell. Reinsertion was accompanied by metacorpus pulsations. Two other juveniles, including the one shown in Fig. 2, showed slightly different behaviour. Periods of stylet movements within the stomatal cavity were followed by two to five periods of metacorpus pulsations. Twelve to 36 mn after retraction, the stylet was reinserted into the initial syncy- tial cell, also accompanied by metacorpus pulsations. Phase III was characterized by the stylet-tip staying pot-uded 3-4 p a into the initial syncytial ce11 and bjy total inactivity of the metacorpus muscles. This lasted, on average, for 20 mn and was followed by a repeat of phase 1 with the stylet-tip remaining protruded in the initial syncytial cell.

* Designates number of J 2 examined.

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Analysis of the three feeding phases

Phase I : During this phase food was ingested by a continuous pumping action of the metacorpus. Occasionally the pumping rhythm slowed, but it soon returned to its steady state. Constraining muscles in front of and behind the metacorpus remained contracted (Fig. 4 A). Secretory granules, continuously synthesized in front of the nuclei of the dorsal and two subventral glands, accumulated behind the constraining muscles posterior to the pulsating metacorpus (Fig. 3 C). Some of the granules of the dorsal gland apparently fused to form larger globules (Fig. 3 C). The ampulla and supporting extension of the dorsal gland still contained many secretory granules (Figs 2 A-B, 3 D) showing agitated movement. The cuticularized arms of the valve within the ampulla remained in most cases in an open position (Fig. 3 D).

Food was withdrawn through a feeding tube, which remained connected to the ventrally orientated stylet orifice close to the tip (Fig. 5 C). The tube was always surrounded by a zone of modified cytoplasm (Fig. 5 C).

Phase II : As soon as the pumping action of the metacorpus stopped, both sets of constraining muscles relaxed simultaneously. The secretory granules that had accumulated behind the posterior muscles now surged fonvard through the gland extensions towards the ampullae (Fig. 4 B). A few seconds later the stylet was retracted into the stomatal cavity and thus lost contact with the feeding tube (Fig. 4 B). Retraction was immediately followed by a few irregular metacorpus pulsations. About two minutes later the stylet was gently moved within the stomatal cavity without emerging through the oral orifice. Concurrent with the accumulation of secretory granules in the ampullae of the two subventral glands, the membrane of the valves started to expand and within about two minutes became greatly inflated. This expansion was difficult to detect and was only clearly evident when the metacorpus was as ideally orientated as in Fig. 3 E. Reinsertion of the stylet-tip into the initial syncytial ce11 by gentle jabs was accompanied by rather irregular metacorpus pulsations, which lasted for around 50 seconds and during which the pump chamber was dilated about twice per second. The pulsations were always preceded by a simultaneous contraction of the constraining muscles in front of and behind the metacorpus (Fig. 4 C; 3 F). Within a few seconds of the onset of pumping, the expanded valve membrane collapsed (Fig. 4 D, Fig. 3 F). The plug material beneath the lips (Fig. 4, Fig. 5 E) was seen to be deformed slightly while the tip of the stylet was being pushed through it with ease. As soon as the tip was inserted 3-4 pm into the initial syncytial cell, metacorpus pulsations stopped.

As previously mentioned, phase II was not identical in al1 juveniles. In particular ln the case of the juvenile which was examined most intensively and recorded in a

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Fig. 4. Schematic representation of events occurring in parts of the anterior feeding apparatus of J 2-juveniles of H. schachtii during feeding phases 1-111 (see text). Same juvenile as that shown in Fig. 2, feeding from an initial syncytial ce11 (ISC), about one day after nematode had started to feed. A : PHASE 1, stylet continuously inserted beyond plug material (p) into zone of modified cytoplasm of ISC. Feeding tube (ft) in contact with orifice of stylet lumen. Number of granules in dorsal gland ampulla (dga) reduced. Metacorpus continuously pumping (indicated by arrow pointing to dilated pump chamber). Anterior (acm) and posterior (pcm) constraining muscles contracted. Secretory granules from the three oesophageal glands accumulate behind pcm; B : PHASE

1 II, about 2 min after termination of phase 1. Stylet retracted, performs gentle movements (arrows) in stomatal cavity. Feeding tube (ft) lost contact with stylet-tip. Metacorpus at rest (indicated by arrow pointing to closed pump chamber). Constraining muscles relaxed. Secretory granules flow forward through dorsal gland extension (dge) and accumulate in dorsal gland ampulla (dga). Granules also accumulate in subventral gland ampulla (sga, only one of two ampullae shown). Inside ampulla, membrane of gland valve starts to expand; C : PHASE II, a few min after B. Stylet at beginning of or during gentle fonvard protraction (arrow), tip still behind plug material. Membrane of subventral gland valve -mv) expanded. Metacorpus just started to pump (arrow), both constraining muscles contracted; D : Phase II, a few after C. Metacorpus still pumping (arrow). Contents of mv now depleted. Stylet-tip penetrates plug material; E : PHASE III, a few min before phase 1 recommences. Stylet-tip inserted in ISC, new feeding tube (ft) formed. Metacorpus at rest (arrow), constraining muscles relaxed. Dorsal gland extension -dge) and ampulla (dga) filled with granules, also ampulla of subventral gland. '' Vacuole (v) in front of metacorpus much expanded.

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research film (Wyss, Zunke & Inst. wiss. Film, 1985) it was much prolonged (up to 36 mn). Four to five periods of metacorpus pulsations, each lasting about one minute, occurred at intervals while the stylet remained retracted. However, these pulsations and the one accompanying stylet reinsertion led to a rapid collapse of the extended valve membrane within the ampullae of the subventral glands observed in other specimens.

Phase III : During this phase the stylet-tip remained protruded 3-4 pm into the initial syncytial ce11 and, while the muscles of the metacorpus stayed completely inac- tive, secretory granules of the three fland cells again flowed forward through the extensions into the ampullae (Fig. 4 E, Fig. 3 G). However, no expansion of the valve membranes within the ampullae of the two subventral glands was recorded at this stage. Nor did an expansion become clearly visible at the valve within the dorsal gland ampulla, as it was probably obscured by the massive aggregation of secretory granules around it. The supply of granules from the site of synthesis in the dorsal gland was continuous, and the gland extension and ampulla, bath packed with granules, increased greatly in size. This overcrowding caused numerous granules to flow backwards again.

Soon after phase III started, a “ vacuole ” or vesicle ” became visible within the cells of the procorpus Wall tissue about 5 pm in front of the metacorpus. It increased steadily in size and, towards the end of phase III, had reached the volume shown in Fig. 3 H. Throughout phase III secretory fluids were seen to emanate from the stylet orifice and gradually harden into a feeding tube (Fig. 5 A-B).

Phase III was again followed by phase 1 (continuous food ingestion). When pumping started, no twitching of the metacorpus, i.e. spasmodic contractions of individual groups of radial metacorpus muscle fibrils, was observed. However, pumping lasted up to one minute until the Co-ordinated muscle contractions became strong enough to dilate the pump chamber. From then onwards the vacuole ’’ in the procorpus shrank rapidly, and after about two minutes it was no longer discernible. A few minutes later a sudden change in body size was noted when the juvenile defecated.

Host responses in initial syncytials cells

As mentioned before, events during syncytium induction could not be observed clearly. However, about one day after feeding had commenced an initial syncytium had already been formed by the partial dissolution of walls in the enlarged syncytial ce11 and by protoplast fusion with neighbouring cells (Figs 2, 4). Metabolic activity was greatly enhanced as revealed by a remarkable increase in cytoplasmic density, pronounced acceleration in cytoplasmic streaming and increases in

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nuclear and nucleolar volumes (Fig. 5 F-G). Cytoplasmic streaming, especially during phase II and III was so strong that the hypertrophied nuclei constantly changed their position and shape (Fig. 2). The dome of modified cytoplasm, which usually harboured several feeding tubes (Fig. 5 D) remained preserved during al1 feeding phases and acted like a barrier to ce11 organelles that flowed past it. At the site of stylet penetration a plug of unidentified material between the nematode’s lips and the dome of modified cytoplasm (Figs 4, 5 E) persisted throughout feeding.

About three days after induction, several cells had become incorporated into a steadily growing syncytium, which now appeared like one huge elongated ce11 packed with uniformly streaming cytoplasm and containing several greatly enlarged nuclei as well as Wall fragments. A few days after the juvenile shown in Fig. 2 had died (about five days after syncytium induction), the contents of the syncytium disintegrated into a mass of coagulated cytoplasm (Fig. 5 H).

Discussion

INTUCELLULAR MIGRATION AND ESTABLISHMENT OF A PERMANENT FEEDING SITE

As also reported for Heterodera trifolii (Mankau & Linford, 1960), H. glycines (Endo, 1964) and H. cnrci3perae (Doncaster & Seymour, 1973), forward progression by H. schachtii J 2 through the root to the site of syncytium induction in the vascular region is strictly intracellular and is not accompanied by metacorpus pulsations. Although the three oesophageal glands are then active, the two subventral glands especially being packed with relatively large granules, glandular secretions obviously do not assist ce11 Wall rupture. Bird (1967) suggested that the granules in the subventral glands play a rôle in root penetration by infective J 2 of the root-knot nematode Meloidogyne javanica. According to Endo (1975) the J 2 of Meloidogyne spp. migrate through the root tissue chiefly intercellularly. During this kind of migration the metacorpus of M . incognita has been seen to pulsate on many occasions (Zunke, unpubl. observ.). However, as will be discussed later, it seems improbable that subventral gland secretions could pass forward and escape through the orifice of the stylet lumen. Interestingly, in males of H. schachtii, which in countless observations were never seen to attack roots and whose metacorpus muscles were degenerate, we have observed that the extensions of al1 three oesophageal glands were packed with granules, as has also been reported in males of H. glycines (Baldwin, Hirschmann & Triantaphyllou, 1977).

At root invasion and during intracellular root migration the J 2 of H. schachtii (and probably those of

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Fig. 5. J 2-juvenile of H. schuchtii (same as in Fig. 2) feeding from the initial syncytium in root of a Brussica nupus seedling. Ail bars = 10 Pm. A-B : feeding tube formation during phase III (see Fig. 4). Dorsal gland ampulla (dga) filled with secretory granules. A : secretions (s) emanate through orifice of stylet lumen and touch a feeding tube (ft) formed in the previous feeding cycle; B : 15 min after A; secretions have hardened into a new feeding tube that pushed the previous tube aside, few min before ingestion will start; C : now at beginning of ingestion (phase 1), with focus on " border " (arrows) between zone of modified cytoplasm (zmc) and surrounding fast-streaming cytoplasm; D : several hours later, focus on three feeding tubes (ft) within zone of modified cytoplasm; E : focus on plug material (p) surrounding base of inserted stylet-tip; F : one of the highly active nuclei (n) in the initial syncytium, about two days after induction. nu, nucleolus; G : in comparison the normal size of a nucleus in a neighbouring cell, not yet affected by the nematode; H : disintegrated syncytium (ds), a few days after juvenile had died at beginning of second moult.

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al1 other Heterodera and Globodera spp.) adhere to a programmed behaviour, i.e. the highly Co-ordinated stylet thrusting for cutting a slit in the eggshell at hatching (Doncaster & Shepherd, 1967) is used again on the surface of and within the root, and in later stages by males emerging from their ensheating larval cuticles (Doncaster & Seymour, 1973). This programmed pattern is, however, abandoned once the juveniles have located the site of syncytium induction in the region of the vascular tissue. Ce11 walls are then attacked by stylet thrusts aimed at one spot, and once a hole has been made in the Wall of the ce11 successfully perforated, the stylet-tip is very carefully inserted into it, perhaps to avoid damage to the plasmalemma or, what would be worse, to the tonoplast. In a recent electronmicroscopic study the tip of the inserted stylet was seen to be surrounded by the plasmalemma (Wyss, Stender & Lehmann, 1984).

It is still unknown which stimuli guide the invading juveniles so quickly to the region of the vascular cylinder, the only site where a successful host-parasite relationship for egg producing females can become established. As is well known (e.g. Jones, 198la) Wall ingrowths develop where the expanding syncytium abuts xylem elements, as a response to the vital solute exchange between the apoplast and symplast from which developing and egg producing females derive their nutrients. Males, which stop feeding at the end of the third developmental stage (Müller, Rehbock & Wyss, 198 1), do not require this apoplast-symplast association (Wyss, Stender & Lehmann, 1984). Endo (1964) has previously noted that where syncytia did not extend to the stele only males of H. glycines developed.

FEEDING FROM THE INITIAL SYNCYTIUM

Feeding from the initial syncytium occurs in a series of repeated cycles, each composed of three distinct phases. During the first and by far the longest, nutrients are withdrawn from within a zone of modified cytoplasm through a feeding tube connected to the orifice of the stylet lumen. As early as 191 1 Né mec found in syncytia of H. schachtii threadlike mitochondria which he later described as threadlike protein crystals (Némec, 1932). Tubular filaments within syncytia, sometimes seen to be attached to the stylet-tip of H. trifolii, were thought to be related in some way to nematode feeding (Mankau & Linford, 1960). Only recently were the feeding tubes produced by H. schachtii (Wyss, Stender & Lehmann, 1984) and other cyst nematode species (Rumpenhorst, 1984) examined in more detaiï. Rumpenhorst (1984) found in the initial syncytial cells bundles of tubes which were an indication of repeated production. This was confirmed in the present study. As Rumpenhorst (1984) could not detect a permanent connection between stylet and tube, he assumed that the nematodes feed from an extensible “ feeding ampulla ” just beneath a conical

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ring that envelops the stylet-tip. Feedings tubes were thought to transport root ce11 secretions to this ampulla. We could, however, never detect such an “ ampulla ” and propose that the observations on living nematodes may be more reliable, i.e. nutrients are withdrawn directly from the feeding tubes, being constantly replenished with solutes derived from the zone of modified cytoplasm that surrounds them.

This zone stays free of ceil organelles such as mitochondria and plastids during al1 feeding phases. In syncytia of Raphanus sativus var. oleiformis the zone appears less dense. than that of the surrounding cytoplasm (Wyss, Stender & Lehmann, 1984). In syncytia of potato, fed on by G. pallida, the organelle-free zone contains a uniform endoplasmic reticulum which, apparently, passes through the feeding tubes (Rumpenhorst, 1984). Swirls of endoplasmic reticulum were observed around feeding tubes of the sedentary endoparasite Rotylenchulus reniformis (Rebois, 1980), and a membraneous network, interpreted as a proliferation of the plasmalemma, around the feeding tubes of the migratory ecto-endoparasite Scutellonema brachyururn (Schuerger & McClure, 1983). The food cells of Helicotylenchus spp. within the root cortex contained a “ salivary droplet ” which enveloped the inserted stylet-tip, and whorls of rough endoplasmic reticulum as well as a “ salivary tube ” were seen around it (Jones, 1978). These and the in vivo observations on the feeding of ectoparasitic epidermal feeders [(e.g. Paratylenchzls projectus, P. dianthus (Rhoades & Linford, 1961)] and Tylenchorhynchus dubius (Wyss, 1973) indicate that for food withdrawal al1 tylenchid nematodes (whose stylet orifice barely exceeds 100 nm), whether primitive or highly evolved in mode of parasitism, must, in some way, modify the cytoplasm around the inserted stylet-tip.

We cannot yet explain how ce11 organelles, which in this study continuously streamed past the dome of modified cytoplasm, are kept away from this zone. This was also observed in later stages of the nematode’s development (Wyss, Zunke & Inst. wiss. Film, 1985). Food is withdrawn by a very rapid, regular and continuous pumping action of the metacorpus. For the J 2 this action lasts, on average, for about one hour; for later developmental stages it is much longer (Müller, Rehbock & Wyss, 1981).

Feeding is associated with a release of waste products, which start to form a subcrystalline layer around the cuticle within a few hours after the permanent feeding site has become estabiished (Zunke, 1986). Continuous pumping is also associated with a steady increase in body volume and is then followed by a rest period (Phases II and III of the present study). At the end of this the body volume suddenly decreases dramatically (Müller, Rehbock & Wyss, 19Sl), shown to result from defecation (Wyss, unke & Inst. wiss. Film, 1985),

Revue Nématol., 9 (2) : 153-165 (1986)

Juveniles of Heterodera schachtii inside host roots

which actually occurs according to this analysis a few minutes after metacorpus pulsations have started.

Seymour (1983) provides an extensive account of the action of the metacorpus of the tylenchid nematode Ditylenchus dipsaci. According to his interpretation, the set of radial muscles at the junction of the procorpus with the metacorpus (Shepherd & Clark, 1983) restrain forward displacement of the relatively non-muscular anterior tissues when the metacorpus starts pumping. Multidirectional muscle elements constraining muscles ”) were also found in front of as well as behind the metacorpus of H. glycines J 2 (Endo, 1984). As shown in this study, these muscles contract simultaneously whenever the metacorpus starts pumping, and we assume, like Shepherd, Clark and Hooper (1980) and Seymour (1983) that they provide stability to the pumping metacorpus. When the muscles contract, the secretory granules within the extensions of the three oesophageal glands are squeezed both forward and backward in the dorsal, and mainly backward in the subventral gland extensions. As the muscles remain contracted throughout pumping, the secretory granules, constantly synthesized in the glands, accumulate behind the posterior constraining muscles. When continuous food ingestion stops, probably after a critical limit in body volume has been reached, the granules flow rapidly fonvard through the extensions towards the ampullae of the glands, perhaps assisted by microtubules as suggested by Endo (1984).

We can find no explanation as to why, after food ingestion, the stylet is consistently retracted into the stomatal cavity, as we also observed for later

, developmental stages. The subsequent gentle stylet movements may probably prevent a clogging of the oral

; orifice by secretory fluids released from the dorsal gland ,! ampulla. As the stylet-tip was not seen to be protruded

;?: beyond the oral orifice, the plug material between ,$! nematode lips and zone of modified cytoplasm (e.g. ; Fig. 5 E) was most likely not affected. According to

Endo (1978) this material, thought to seal the ce11 Wall after stylet withdrawal at moulting, is secreted from the amphidial canals, the openings of the inner labial

: receptors and the stylet vestibule. When the stylet-tip is reinserted into the initial syncytial cell, usually a few minutes after withdrawal, it meets no resistance from the

’ plug material, which is deformable and thus probably seals the penetration hole whenever the stylet is retracted. Apart from stylet retraction and movements within the stomatal cavity, phase II of the feeding cycle is characterized by the expansion of the valve membranes within the ampullae of the two subventral glands and quick depletion of valve contents as soon as metacorpus pulsations start to dilate the pump chamber.

The first detailed report. on the possible mechanisms involved in the emission of oesophageal gland secretions by a tylenchid nematode through an expansible quadriradiate valvular end-apparatus was provided by

Revue Nématol., 9 (2) : I53-I65 (I986)

Anderson and Byers (1975), when they studied the ultrastructure of the procorporal oesophagus region in Tylenchorhynchus dubius. From further studies (Baldwin, Hirschmann & Triantaphyllou, 1977; Shepherd & Clark, 1983; and especially Endo, 1984) the structure of the valves within the gland ampullae is now well known. In J 2 of H. glycines (Endo, 1984), the cuticularized tetraradiate support arms of the subventral gland valves are lined by the membranes of the gland ampullae. In an open position the membranes are shown in section to balloon outwards and the valves are filled with homogeneous electron-dense material, most likely derived from the adjacent secretory granules. It has been suggested that the ampullar contents (granules) are liquefïed by secretory nerves found in close association with them (Anderson & Byers, 1975; Endo, 1984). We may therefore assume, with good reason, that the valve membranes expand as they become filled with secretory fluids. These fluids then pass through the short cuticularized valve ducts into a triangular vestibule behind the metacorpus pump chamber (Endo, 1984). When the metacorpus starts pumping, the fluids are forced backward towards the intestine through the expansible triradiate oesophageal lumen of the isthmus. As might be expected, the contents of the expanded valve membranes are quickly emptied by a few metacorpus pulsation that dilate the pump chamber.

It could be argued that secretory fluids from the subventral glands may also be forced forward towards the feeding site. However, because the oesophageal lumen in tylenchid nematodes is thick-walled and still circular in cross section just in front of the metacorpus pump chamber, but relatively thin-walled and triradiate (and thus expansible) behind it (e.g. Shepherd & Clark, 1983; Endo, 1984), fluids from within a dilated pump chamber are expelled backwards on pump closure, as explained by Doncaster (1971). As an expansion of the valve membranes of the two subventral glands was only recorded in phase II of a complete feeding cycle, it appears that the emission of secretory fluids from these glands is restricted to relatively short periods. A discharge is definitely not possible during food ingestion, as the supply of granules to the ampullae is at that time blocked by the barrier imposed by the constantly contracted posterior constraining muscles.

Phase III of the feeding cycle is characterized by total inactivity of the metacorpus muscles and the discharge of secretory fluids through the orifice of the reinserted stylet-tip. The ampulla of the dorsal gland, packed with granules, is then considerably distended. As already mentioned, an expansion of the valve membrane was not observed, probably because the massive aggregation of constantly moving granules obscured it. Anderson and Byers (1975) discussed the probable pressure relationships within the ampulla of the dorsal gland and procorporal oesophageal lumen during salivation and

163

U. Wyss & U. Zunke

ingestion. As in Our observations the density of the granules becomes reduced during ingestion and the cuticularized arms of the valve then become visible in a still open position, it may well be that in J 2 of H. schachtii some glandular secretions are ingested together with nutrients derived from the syncytium.

The dorsal gland secretions discharged through the stylet orifice become gradually transformed into a feeding tube, perhaps by a reaction with cytoplasm, as suggested by Rumpenhorst (1984). As feeding tubes obviously have to be formed anew during phase III of the feeding cycle, the rather long and coiled tubes found in syncytia of later developmental stages may represent a fusion of tubes. Fig. 5 A & B show how a freshly formed tube appears to fuse with a tube from a previous cycle. The chemical nature of the tube material is unknown, although there are some indications that a diffuse protein fraction, repeatedly found by micro-electrophoresis only in syncytia of older developmental stages (Krauthausen & Wyss, 1984) may be associated with tube material. Whatever the nature of the dorsal gland secretions, they have at least three functions : stimulation of syncytium formation, modification of the cytoplasm at the permanent feeding site and provision of the means for efficient food withdrawal. Once induced, syncytium maintenance and transfer ce11 formation may be simply under the control of a source-sink continuum (Jones, 1981b).

We cannot yet explain how the “ vacuole ” or “ vesicle ” in the oesophageal Wall tissue about 5 Pm in front of the metacorpus is formed during phase III of the feeding cycle. It is definitely not an artefact as, during this phase, it was observed in al1 juveniles (later developmental stages not examined). Its quick disappearance soon after ingestion had started is even more puzzling. While studying the ultrastructure of the oesophagus of H. glycines J 2, Endo (1984) did not examine the procorpus in the region where the “ vacuole ” is formed but noticed that, in the anterior region of the metacorpus, a membrane complex was attached around 2/3 of the oesophageal lumen Wall (see Fig. 11 in Endo, 1984). The dorsal gland extension adjacent to part of this membrane complex was bordered dorsally by a pair of nerve processes and an enlarged neurosecretory cell. In this plane of the metacorpus of D. dipsaci, Shepherd and Clark (1983) found an extensive array of parallel lamellae, interpreted them as being extensions of the ce11 membrane and suggested that they were probably involved in ion uptake and secretion; again, however, the “ vacuole jJ region of the procorpus was not illustrated. In Pratylenchus penetrans, IGsiel, Himmelhoch and Zuckerman (1976) recorded a membrane complex surrounding the procorpbral oesophageal lumen within the region where the “ vacuole ” is formed in J 2 of H. schachtii. To speculate in the absence of clear evidence, perhaps “ vacuole ” formation in the procorpus Wall tissue nlay somehow be

164

linked with an adjacent membraneous network and with, as yet unsubstantiated, neurosecretion. A detailed electronmicroscopic examination will hopefully clarifiy “ vacuole ” formation and, more important, its function during salivation.

ACKNOWLEDGEMENTS

The authors thank Dr. Audrey M. Shepherd, previously Nematology Department, Rothamsted Experimental Station, England and Dr. David G. McNamara, East Malling Research Station, England for critically reviewing the manuscript.

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Accepté pour publication le 7 janvier 1986.

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