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Ultrastructure of the Z organ in Xiphinema ifacolum
a)2&7519/85 $3.00+0.00 Per&wnon Press Ltd.
0
1985AusfrdianSoriefy for Pamsitolo~y
ULTRASTRUCTURE OF THE Z ORGAN IN XPHZIVEMA IFACOLUM T. BLEVE-ZACHBO, G. ZACHEO and F. LAMBERTI Istituto di Nematologia Agraria, C.N.R. Via G. Amendola 165/A-70126 Bari, Italy (Received 3 October 1984)
Abstract-Breve-ZAcHEo T., ZACHEO G. and LAMBERTI F. 1985. Ultrastructure of the 2 organ in Xipb~n~a ifuc~lum. In~ernu~i~~~l Jowrtal for Pa~as~~5l~~y IS: 453-461. The uterus in Xipbi~ern~ i~acolumcan be divided into uterus proper, a 77 m long tube and a lemon-shaped Z organ, about 28 pm long and 18 ym wide, placed between the oviduct and the uterus proper. The 2 organ consists of a thick outer muscular layer of 120 cells, arranged in 20 rings of six ceils each and a thin inner epithelium layer, lining the lumen. The epithelial cell walls, lining the lumen of the Z organ are thicker than those lining the uterus proper and are strongly folded. The crests of some of these folds carry six large apophyses, a11 about the same size and shape, these occupy the full Ierigth of the organ, becoming thicker towards the center of the lumen. There are many tubules near the surfaces of the apophyses, the contents of which can be dissolved by treatment with pepsin and pronase, indicating that they are proteins. This material probably consists of secretions which are squeezed out of the apophyses by a passing egg and may function in the formation or hardening of the egg shell. INDEX KEY WORDS: Xiphinema ifacolum; phytoparasitic nematode; Z organ; ultrastructure;
enzymatic digestion.
The reproductive system is of primary importance for the taxonomy and identification of Xiphinema as reported by Luc (1975) and Luc & Dalmasso (1975). Female genital tracts, in the genus Xiphinemu, are complex organs and comparative studies on their differentiations could represent an available tool in understanding the structure of these organs in some groups of nematodes (Coomans, 1964). The genus includes plant parasitic nematodes, which cause considerable economical losses by reducing yield directly (Webster, 1972; Southey, 1978) and by transmitting viruses (Lamberti, Taylor & Seinhorst, 1975). Among the variations present in the development and size of genital apparati in nematodes belonging to the genus, Luc (1975) described a peculiar differentiation of the uterus, referred to as “Z differentiation”. The term is derived from the “Z organ” which was described for the first time by Luc, (1958) in the species X. ebriense. This differentiated area of the gonad is always situated between the junction of the uterine pouch and the elongated proximal part of the uterus. This structure was distinguished from the other parts of the uterus and described as a restricted area with strong circular muscles and an internal sclerotized wall. In its central lumen “refringent sclerotized apophyses more or less angular in shape” are present (Luc, 1961; Coomans, 1964). Luc (1975) pointed out that similar structure cannot be related 453
to any other character and that it is typical of didelphic species of Xiphinema. In fact the “Z organ” was never observed in species having either no or a reduced anterior genital branch. Luc (1973) distinguished the “typical Z organ” in five species from the “Z pseudo organ” in 18 species of Xiphinema, in which similar structures have been described. He pointed out that the “typical Z organ” is a structure clearly distinct from the other parts of the uterus, while the structure of the “Z pseudoorgan” varies from one species to another. Studies conducted on dissected female gonads of X. ~ive~icau~af~m have shown that the “Z pseudoorgan” is a muscular tube within a globular structure (Grimaldi-De Zio, D’Addabbo-Gallo, Lamberti & Morone-De Lucia, 1979). The ultrastructure showed that the “globules” in the Z pseudo-organ are arranged in a helicoidal system, within the lumen and consist of a series of closed vesicles and tubules, containing secretions. The “globular bodies” originated from the membrane of the epithelial tissue, which lines the lumen of the Z pseudo-organ. A muscular mass of about 80 cells, arranged in rings of four cells each, encloses the secretory portion of the organ (Bleve-Zacheo, Zacheo, Melillo & Lamberti, 1984). The shape of the “typical Z organ” is known from light microscopy but its structure and function are unknown. Therefore, the structure of the “typical Z organ” in X. ~~a~~urn was studied by electron microscopy.
T.
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BLEVE-ZACHEO, G. ZACHEO and
F.
LAMBERTI
I.I.P. VOL. 15. 1985
MATERIALS AND METHODS
Gonads from living females of a population of X. ifacolum from Nigeria and one from Sri Lanka were dissected with an eye-scalpel in 5% glutaraldehyde at 4°C in 0.1 M cacodylate buffer at pH 7.4. They were then transferred into fresh buffered glutaraldehyde and left for 6 h at 4°C. Fixed material was washed with buffer overnight and post-fixed in 2% osmium tetroxide in the same buffer for 6 h at 4°C. After post-fixation, the gonads were rinsed in buffer and stained overnight in 0.5% uranyl acetate in distilled water. The specimens were dehydrated in an ascending series to absolute ethanol and embedded in Spurr’s medium. Some gonads were embedded in glycol methacrylate @MA), as described bv Leduc & Bernhard (1967). which dermits-differential staining. Sections 2 rm thick were stained with periodic acid Schiff (PAS), mounted in xylene and observed under the light microscope.‘Enzymatic digestions were performed on gonads dissected from living females in glutaraldehyde, as previously indicated, immediately rinsed in buffer and transferred into enzymatic solutions. The following two enzymes were tested: (i) 0.5% pepsin (Sigma) in 0.1~ HCl at pH 1.6; (ii) 1% protease (Sigma type VIII) in Hz0 at pH 8. After incubation for 12 h at 37”C, the material was fixed and embedded in Spurr’s resin as previously described. Sections were cut with a LKB ultratome III, stained with uranyl acetate and lead citrate and examined under a Philips 400 T transmission electron microscope at 80 kV. RESULTS
Observations on a population of X. ifacolum from Nigeria and one from Sri Lanka showed that the
gonads in both populations have the same structure. Therefore the observations are presented without reference to the separate populations. In X. ifacolum the two branches of the genital system are equal size in development; in each branch the “typical Z organ” is present (Fig. 1). Longitudinal sections showed that the uterus in X. ifacolum consists of two parts, the uterus proper and the Z organ connected by a narrow tube (Figs. 2, 3). The uterus proper is larger than the Z organ; the uterus is about 77 pm long; the Z organ is lemon shape about 28 pm long and 18 ,um wide. A constriction separates the oviduct from the Z organ and another one the Z organ from the uterus proper; the latter constriction being the narrow tube mentioned before. The wall of the Z organ consists of 120 cells of parallel muscles, encircling the lumen. They are arranged in 20 parallel rings, each ring consisting of six cells, whose fibres are mainly transverse (Figs. 4, 8). The muscle rings are tightly packed; nuclei and
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1 FIG. 1. Diagram of the anterior gonad of Xiphinema ifacolum; ova = ovary; ovi = oviduct; Z = Z organ; U = uterus; V = vulva. Scale bar = 20 pm. sarcoplasm are only outside the organ in contrast to the ovijector and the sarcoplasm has few cellular organelles (Figs. 4, 5). A single layer of actively syn-
thetizing epithelial cells lies against the muscular cells and lines the lumen of the Z organ. The cell wall
FIG. 2. Electron micrograph of a longitudinal section through the uterus and the Z organ of X. ifacolum. Note the cuticle (c), somatic muscles @mu), the intestine (I) and a portion of the gonad, showing the Z organ and its connection with the uterus proper (LJ). Muscles (mu) in the Z organ are stronger than in the uterus. Membranes (me) in the uterus are more plicated than those of the Z organ. Scale bar = 6 pm. FIG. 3. Electron micrograph of longitudinal section of X. ifacolum treated with pepsin. The digestion of the muscles exhibits the structure of the membranes (me) of the uterus, its connection with the Z organ, the lumen (1) and apophyses (ap) in the Z organ. The plasma membrane of epithelial cells is evident (arrows); also chromatin of the nuclei (nu) of the muscle cells is still visible. I = intestine. Scale bar =4.5 pm.
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lining the lumen of the empty uterus is highly convoluted; the cell wall in the Z organ shows more regular folds into the lumen (Figs. 4, 9). The structure of the wall, lining the lumen, is clearly evident in Fig. 3, where enzymatic digestion emphasizes how the membranes of the epithelial tissue are arranged into the lumen of the uterine cavity; in the Z organ the cell wall lining the lumen is distinctly thickerthan that lining the lumen of the uterus proper. In longitudinal section the folds of the epithelial membrane wall form a network into the lumen (Fig. 4); large excrescences are formed on the crests of some of the folds, extending into the lumen (Figs. 5, 6). The extension of the excrescences can be followed in Figs. 6 and 7. Several epithelial cells, forming a single fold, contribute to the formation of an excrescence (Fig. 3). These excrescences are the “sclerotized refringent apophyses” previously described by Luc (1958). Serial cross-sections of the Z organ showed that the number of apophyses in each section becomes larger towards the centre of the organ, with the maximum of six (Fig. 8). The figures indicate that they are slightly staggered, two by two symmetric and opposite (Figs. 8, 9). Each apophysis is connected to more than one fold (Fig. 9) and is largest in the wider portion of the lumen. Each apophysis appears as an electron dense mass equal to the wall of the epithelial tissue lining the lumen. They present a system of tubules, more abundant near their surface and distributed parallel to the axis of the organ. The tubule system opens on the surfaces of the apophyses. The tubules, circular in shape, contain less electron dense material than the cell wall (Figs. 9, 10). The lumen of the Z organ contains fluid material, finely granular with a few scattered paracrystals (Fig. lo-inset) and numerous bacteria and rickettsia (Figs. 8, 9) as already described by other authors in the organs of other nematodes (Shepherd, Clark & Kempton, 1973; Endo, 1979; Bird & Akhurst, 1983). They are present in the gonads of both populations examined. The apophyses did not take up PAS stain, demonstrating that they do not contain carbohydrates.
After digestion of portions of the nematode gonad in vivo, with pepsin, the muscles and cell content are completely removed; only the cell walls and apophyses of the Z organ remain visible in electron micrographs; these membranes remain intact even after long digestion (Figs. 2, 3). In Fig. 3, also, the chromatin of muscle cells is still visible. The apophyses of the Z organ are not hydrolized, while the material present in the tubules and the microorganisms in the lumen are partially digested. A similar pattern was obtained through digestion with pronase. DISCUSSION
The Z organ of X. ifacolum consists of 120 muscle cells, arranged in 20 rings at right angles to the axis of the uterus, larger in the central part of the organ and progressively becomes reduced at the two ends to form two constrictions. According to our investigations the structure of the Z organ of X. ifacolum agrees with Luc’s description of the organ as “bulbe a musculature circulaire puissante” (Luc, 1961). The muscle rings are arranged in such a way that they form a very compact muscular unity. The muscle organization in the Z organ greatly differs from that in the “Z pseudoorgan” in X. diversicaudatum; in the latter no difference is observed between the muscles in the Z pseudo-organ and those in the uterus proper (BleveZacheo et al., 1984). There are twenty rings in both species, each ring consists of four cells in X. diversicaudatum and six in X. ifacolum. The 20 rings in X. diversicaudatum together form a tube 70 pm in length and those in X. ifacolum form a lemon shape body 28 pm in length. As a result, the structure is much more compact in X. ifacolum than in X. diversicaudatum. The apophyses, formed on the crests of some folds of epithelial cell walls, occupy a large part of the lumen of the organ, leaving a tube shape, space free in the centre. In the longitudinal sections the apophyses show concave and curved margins which seem to fit into each other. The apophyses seem to consist of spongy material which is pressed against the wall of the lumen when an egg passes. The
FIGS. 4, 5, 6, 7. Micrographs of longitudinal serial sections from the dorsal to the middle region of the Z organ. (Fig. 4). A strong muscular mass (mu) delimits the cavity of the Z organ; two constrictions (co) separate the organ from the oviduct and from the uterus respectively. The wall of the lumen (1) shows that membranes (me) of the epithelial cells extend on both sides of the organ forming a network. Note the cytoplasm of epithelial cells (ec) inside the membranes. smu=somatic muscles; I=intestine. Scale bar =3 q. (Fig. 5). The top of an apophysis (ap) on the crest of folded membranes (me) is visible. Note the nuclei (nu) of the muscle cells (mu) as in Fig. 4. Scale bar = 15 inn. (Fig. 6). The apophysis (ap) as in Fig. 5 shows its widest size. Other two apophyses are present in the section and their origin is evident from the membranes (me) of the epithelial cells (ec). The figure shows a concavity in the structure of an apophysis fitting together with a convexity of another one (arrows). The lumen (1) of the organ contains granular material and microorganisms (mo). Scale bar = 1.8 inn. (Fig. 7). Section in the middle region of the Z organ. The apophysis, as in Figs. 5, 6 (double arrow) whose connections with the membranes (me) are not visible, is recognizable. Note the nuclei (nu) of the muscle cells (mu) as in Figs. 4, 5, 6; co = constriction; I = intestine; smu = somatic muscles. Scale bar = 2.9 inn. ’
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FIG. 8. Electron micrograph of cross-section in the wider portion of the Z organ. A ring of six muscle cells (l-6) encircles the lumen of the organ, lined by the epithelial folded membrane; six apophyses, orginating from some folds of the membrane (me), occupy the lumen (1). Their disposition suggest how their margins can form a channel, when the organ enlarges, during the egg passage: mo = microorganisms. Scale bar = I4 nrn. Fm. 9. Electron micrograph of a cross-section in the middle portion of the Z organ. The figure shows apophyses (ap) connected to two and three folds of the epithelial membrane (ec) respectively. Numerous microorganisms (mo) are evident into the lumen (1) of the organ; t = tubules; mu’= muscle cells. Scale bar = O-7 pm.
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FIG. 10. Electron micrograph of a cross-section of the Z organ. Muscles (mu) show the intersections of their fibres (mf). The cytoplasm of epithelial cells (ec) rich in organelles is evident in the folded membrane (me) lining the lumen (1). Three apophyses (ap) show how they enlarge from the origin on the crest of the membranes to the centre of the organ. Many tubules (t), containing secretory material and opening into the lumen are present on their surface; nu = nucleus. Scale bar = 1 pm. Very few paracrystals (PC) are observed in the lumen (Inset-Scale bar = 1.5 pm),
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secretions present in the spongy material is squeezed out and spread on the surface of the egg. The absence of sphincters on both oviduct-Z organ and Z organ-uterus connections and the shortness of the structure of the Z organ suggest that the egg passes through that quite rapidly; that could explain why an egg was never observed in the Z organ of X. ifacolum but frequently in the Z pseudo-organ of X. diversicaudatum. Our observations as well as those by Luc (1975) show that the structure of the Z organ is very constant; no variation is observed among 30 specimens from two different populations. The dorsoventral cross-sections through the axis of the uterus of two specimens are almost completely equal (Fig. 3). Treatment of specimens with enzymes shows that what Luc (1961,1975) and Coomans (1964) described as “sclerotized apophyses” are to be considered proliferations of the cell wall of single layered epithehum, lining the lumen of the Z organ, consisting of glycoproteins, like the cell wall. So the origin of the apophyses seems to be the same as that of the globular bodies described by Bleve-Zacheo et al. (1984) in the pseudo Z organ of X diversicaudatum. Long enzymatic treatment did not affect, as in X. diversicaudatum, apophyses and lumen wall, indicating they contain no proteins. Digestion was observed in the content of the tubules, present on the surface of the apophyses and in the material stored around the apophysis margins and the lumen cavity. The effect of enzymatic treatment on the content of the tubules suggests the presence of proteins (Fasold & Gundlach, 1963; Weintraub & Ragetli, 1968), secreted by the epithelial cell and transferred, through the tubules to the surface of the apophyses and then onto the egg, during its passage. Comparing the organization of this Z organ with that of the pseudo Z organ in X. diversicaudatum it seems that in the latter the egg is kept in the pseudo Z organ by the two sphincters and is enveloped by the secretions, present in the lumen and released by the globules, pressed by the egg. Since the egg does not stop in the Z organ of X. ifa~o~um, the process in transferring secretions appears to occur more rapidly during the compression and the expulsion of the egg through the constriction of the organ. Apparently the Z organ and the pseudo Z organ are specialized parts of the uterus, secreting materials involved in the formation of the egg shell (Coomans, 1964; McClure & Bird, 1976). Acknowledgements-The Lerario
for photographic
authors assistance.
are
grateful
461
organ in X. ifacohitn
to
Mr.
REFERENCES BIRD A.
F. & AKHURST R. .I. 1983. The nature of the intestinal vesicle in nematodes of the family Steiner-
nematidae. rnternational JournaI for Parasifology 13: 599-606.
BLEVE-ZACHEO T., ZACHEO G., MELILLO M. T. & LAMBERTI F. 1984. On the structure of the 2 pseudo organ in
Xiph~nema di~e~icaadatum. rnfernationa~Journal for Parasitology 14: 404-4 13.
COOMANS A.
1964. Structure of the female gonads in members of the Dorylaimina. Nemafoiogica 10: 601-622. ENDO B. Y. f979. The ultrastructure and distribution of an intracellular Bacterium-like microorganism in tissue of larvae of the soybean cyst nematode, Heterodera
giycines. Journal of UltrastructureResearch 67: l-14.
FA~~LD H. & GUNDLACH C. 1963. Characterization of peptides and proteins with enzymes. In Methods of Enzymatic Analysis (Edited by BERGMEYER H. V.). Academic Press, New York. GRIMALDI-DE ZIO S., D’ADDABBO-GALLO M., LAMBERT~F. & MORONE-DE LUCIA M. R. 1979. The “Z” differentiation in Xiphinema: a hypothesis of its function in relation to amphigony. Nematologica 25: 36-41. LAMBERT~ F., TAYLOR C. E. & SEINHORST J. W. 1975. Nematode Vectors of Plant Viruses. Plenum Press, London and New York. LEDUC E. & BERNHARD W. 1967. Recent modifications of the glycol methacrylate embedding procedure. Journal of
UltrastructureResearch 19: 196-203.
Luc M. 1958. Xiphinema de I’Ouest africain: description de cinq nouvelle especes (Nematoda: Dorylaimidae).
Nematologica 3: 57-72. Luc M. 1961. Structure de la gonade femelle chez quelques especes du genre Xiphinema Cobb, 1913 (Netnatoda: Dorylaimoidea). Nematoiogica 6: 144-154. LLJC M. 1973. Redescription de Xiphinema hatley Luc, 1958 et description de six nouveiles especes de Xiphinema Cobb, 1893 (Nematoda: Dorylaimoidea). Cahiers de
I’Uffice de la Recherche ~ientif~que et T~hnique Ontre-Mer, Biologic 21: 45-65. Lot M. 1975. Taxonomy of Xiphinema Cobb, 1913. In Nematode Vectors of Ptunt Viruses (Edited by LAMBERTI F., TAYLOR C. E. & SEINHORST J. W.). on. 39-52. Plenum Press, London and New York. ‘. _. Luc M. SL DALMASSOA. 1975. Considerations on the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) and a “lattice” for the identification of species. Cakiers de
/‘Office de la Recherche Scientifique et Technique Uutre-Mer, Biologie, 10: 303-327.
MCCLURE M. A. & BIRD A. F. (Nematoda) egg shell: formation
1976. The tylenchid of the egg shell in
Meloidogyne javanica. Parasitology72: 29-39.
SHEPHERD A. M., CLARK S. A. & KEMPTON A. 1973. An intracellular micro-organism associated with tissues of
Heterodera spp. Nemato~agica19: 31-34.
SOUTHEY J. F. 1978. Plan? Nematology. Her Majesty’s Stationery Office, ADAS, London. WEBSTER J. M. 1972. Economic Nematology. Academic Press, London and New York. WEINTRAUB M. & RAGETL~ H. W. 1968. Intracellular characterization of bean yellow mosaic virus-induced inclusions by differential enzyme digestion. Journal of
Cell Biology 38: 316-328.
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1985AusfrdianSoriefy for Pamsitolo~y
ULTRASTRUCTURE OF THE Z ORGAN IN XPHZIVEMA IFACOLUM T. BLEVE-ZACHBO, G. ZACHEO and F. LAMBERTI Istituto di Nematologia Agraria, C.N.R. Via G. Amendola 165/A-70126 Bari, Italy (Received 3 October 1984)
Abstract-Breve-ZAcHEo T., ZACHEO G. and LAMBERTI F. 1985. Ultrastructure of the 2 organ in Xipb~n~a ifuc~lum. In~ernu~i~~~l Jowrtal for Pa~as~~5l~~y IS: 453-461. The uterus in Xipbi~ern~ i~acolumcan be divided into uterus proper, a 77 m long tube and a lemon-shaped Z organ, about 28 pm long and 18 ym wide, placed between the oviduct and the uterus proper. The 2 organ consists of a thick outer muscular layer of 120 cells, arranged in 20 rings of six ceils each and a thin inner epithelium layer, lining the lumen. The epithelial cell walls, lining the lumen of the Z organ are thicker than those lining the uterus proper and are strongly folded. The crests of some of these folds carry six large apophyses, a11 about the same size and shape, these occupy the full Ierigth of the organ, becoming thicker towards the center of the lumen. There are many tubules near the surfaces of the apophyses, the contents of which can be dissolved by treatment with pepsin and pronase, indicating that they are proteins. This material probably consists of secretions which are squeezed out of the apophyses by a passing egg and may function in the formation or hardening of the egg shell. INDEX KEY WORDS: Xiphinema ifacolum; phytoparasitic nematode; Z organ; ultrastructure;
enzymatic digestion.
The reproductive system is of primary importance for the taxonomy and identification of Xiphinema as reported by Luc (1975) and Luc & Dalmasso (1975). Female genital tracts, in the genus Xiphinemu, are complex organs and comparative studies on their differentiations could represent an available tool in understanding the structure of these organs in some groups of nematodes (Coomans, 1964). The genus includes plant parasitic nematodes, which cause considerable economical losses by reducing yield directly (Webster, 1972; Southey, 1978) and by transmitting viruses (Lamberti, Taylor & Seinhorst, 1975). Among the variations present in the development and size of genital apparati in nematodes belonging to the genus, Luc (1975) described a peculiar differentiation of the uterus, referred to as “Z differentiation”. The term is derived from the “Z organ” which was described for the first time by Luc, (1958) in the species X. ebriense. This differentiated area of the gonad is always situated between the junction of the uterine pouch and the elongated proximal part of the uterus. This structure was distinguished from the other parts of the uterus and described as a restricted area with strong circular muscles and an internal sclerotized wall. In its central lumen “refringent sclerotized apophyses more or less angular in shape” are present (Luc, 1961; Coomans, 1964). Luc (1975) pointed out that similar structure cannot be related 453
to any other character and that it is typical of didelphic species of Xiphinema. In fact the “Z organ” was never observed in species having either no or a reduced anterior genital branch. Luc (1973) distinguished the “typical Z organ” in five species from the “Z pseudo organ” in 18 species of Xiphinema, in which similar structures have been described. He pointed out that the “typical Z organ” is a structure clearly distinct from the other parts of the uterus, while the structure of the “Z pseudoorgan” varies from one species to another. Studies conducted on dissected female gonads of X. ~ive~icau~af~m have shown that the “Z pseudoorgan” is a muscular tube within a globular structure (Grimaldi-De Zio, D’Addabbo-Gallo, Lamberti & Morone-De Lucia, 1979). The ultrastructure showed that the “globules” in the Z pseudo-organ are arranged in a helicoidal system, within the lumen and consist of a series of closed vesicles and tubules, containing secretions. The “globular bodies” originated from the membrane of the epithelial tissue, which lines the lumen of the Z pseudo-organ. A muscular mass of about 80 cells, arranged in rings of four cells each, encloses the secretory portion of the organ (Bleve-Zacheo, Zacheo, Melillo & Lamberti, 1984). The shape of the “typical Z organ” is known from light microscopy but its structure and function are unknown. Therefore, the structure of the “typical Z organ” in X. ~~a~~urn was studied by electron microscopy.
T.
454
BLEVE-ZACHEO, G. ZACHEO and
F.
LAMBERTI
I.I.P. VOL. 15. 1985
MATERIALS AND METHODS
Gonads from living females of a population of X. ifacolum from Nigeria and one from Sri Lanka were dissected with an eye-scalpel in 5% glutaraldehyde at 4°C in 0.1 M cacodylate buffer at pH 7.4. They were then transferred into fresh buffered glutaraldehyde and left for 6 h at 4°C. Fixed material was washed with buffer overnight and post-fixed in 2% osmium tetroxide in the same buffer for 6 h at 4°C. After post-fixation, the gonads were rinsed in buffer and stained overnight in 0.5% uranyl acetate in distilled water. The specimens were dehydrated in an ascending series to absolute ethanol and embedded in Spurr’s medium. Some gonads were embedded in glycol methacrylate @MA), as described bv Leduc & Bernhard (1967). which dermits-differential staining. Sections 2 rm thick were stained with periodic acid Schiff (PAS), mounted in xylene and observed under the light microscope.‘Enzymatic digestions were performed on gonads dissected from living females in glutaraldehyde, as previously indicated, immediately rinsed in buffer and transferred into enzymatic solutions. The following two enzymes were tested: (i) 0.5% pepsin (Sigma) in 0.1~ HCl at pH 1.6; (ii) 1% protease (Sigma type VIII) in Hz0 at pH 8. After incubation for 12 h at 37”C, the material was fixed and embedded in Spurr’s resin as previously described. Sections were cut with a LKB ultratome III, stained with uranyl acetate and lead citrate and examined under a Philips 400 T transmission electron microscope at 80 kV. RESULTS
Observations on a population of X. ifacolum from Nigeria and one from Sri Lanka showed that the
gonads in both populations have the same structure. Therefore the observations are presented without reference to the separate populations. In X. ifacolum the two branches of the genital system are equal size in development; in each branch the “typical Z organ” is present (Fig. 1). Longitudinal sections showed that the uterus in X. ifacolum consists of two parts, the uterus proper and the Z organ connected by a narrow tube (Figs. 2, 3). The uterus proper is larger than the Z organ; the uterus is about 77 pm long; the Z organ is lemon shape about 28 pm long and 18 ,um wide. A constriction separates the oviduct from the Z organ and another one the Z organ from the uterus proper; the latter constriction being the narrow tube mentioned before. The wall of the Z organ consists of 120 cells of parallel muscles, encircling the lumen. They are arranged in 20 parallel rings, each ring consisting of six cells, whose fibres are mainly transverse (Figs. 4, 8). The muscle rings are tightly packed; nuclei and
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1 FIG. 1. Diagram of the anterior gonad of Xiphinema ifacolum; ova = ovary; ovi = oviduct; Z = Z organ; U = uterus; V = vulva. Scale bar = 20 pm. sarcoplasm are only outside the organ in contrast to the ovijector and the sarcoplasm has few cellular organelles (Figs. 4, 5). A single layer of actively syn-
thetizing epithelial cells lies against the muscular cells and lines the lumen of the Z organ. The cell wall
FIG. 2. Electron micrograph of a longitudinal section through the uterus and the Z organ of X. ifacolum. Note the cuticle (c), somatic muscles @mu), the intestine (I) and a portion of the gonad, showing the Z organ and its connection with the uterus proper (LJ). Muscles (mu) in the Z organ are stronger than in the uterus. Membranes (me) in the uterus are more plicated than those of the Z organ. Scale bar = 6 pm. FIG. 3. Electron micrograph of longitudinal section of X. ifacolum treated with pepsin. The digestion of the muscles exhibits the structure of the membranes (me) of the uterus, its connection with the Z organ, the lumen (1) and apophyses (ap) in the Z organ. The plasma membrane of epithelial cells is evident (arrows); also chromatin of the nuclei (nu) of the muscle cells is still visible. I = intestine. Scale bar =4.5 pm.
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.P. VOL.15. 1985
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455
456
T.
BLEVE-ZACHEO,
G.
ZACHEO
and F.LAMBERTI
I.J.P.VOL.
1.5.1985
I.J.P.
VOL.
15. 1985
451
2 organ in X. ifacolum
lining the lumen of the empty uterus is highly convoluted; the cell wall in the Z organ shows more regular folds into the lumen (Figs. 4, 9). The structure of the wall, lining the lumen, is clearly evident in Fig. 3, where enzymatic digestion emphasizes how the membranes of the epithelial tissue are arranged into the lumen of the uterine cavity; in the Z organ the cell wall lining the lumen is distinctly thickerthan that lining the lumen of the uterus proper. In longitudinal section the folds of the epithelial membrane wall form a network into the lumen (Fig. 4); large excrescences are formed on the crests of some of the folds, extending into the lumen (Figs. 5, 6). The extension of the excrescences can be followed in Figs. 6 and 7. Several epithelial cells, forming a single fold, contribute to the formation of an excrescence (Fig. 3). These excrescences are the “sclerotized refringent apophyses” previously described by Luc (1958). Serial cross-sections of the Z organ showed that the number of apophyses in each section becomes larger towards the centre of the organ, with the maximum of six (Fig. 8). The figures indicate that they are slightly staggered, two by two symmetric and opposite (Figs. 8, 9). Each apophysis is connected to more than one fold (Fig. 9) and is largest in the wider portion of the lumen. Each apophysis appears as an electron dense mass equal to the wall of the epithelial tissue lining the lumen. They present a system of tubules, more abundant near their surface and distributed parallel to the axis of the organ. The tubule system opens on the surfaces of the apophyses. The tubules, circular in shape, contain less electron dense material than the cell wall (Figs. 9, 10). The lumen of the Z organ contains fluid material, finely granular with a few scattered paracrystals (Fig. lo-inset) and numerous bacteria and rickettsia (Figs. 8, 9) as already described by other authors in the organs of other nematodes (Shepherd, Clark & Kempton, 1973; Endo, 1979; Bird & Akhurst, 1983). They are present in the gonads of both populations examined. The apophyses did not take up PAS stain, demonstrating that they do not contain carbohydrates.
After digestion of portions of the nematode gonad in vivo, with pepsin, the muscles and cell content are completely removed; only the cell walls and apophyses of the Z organ remain visible in electron micrographs; these membranes remain intact even after long digestion (Figs. 2, 3). In Fig. 3, also, the chromatin of muscle cells is still visible. The apophyses of the Z organ are not hydrolized, while the material present in the tubules and the microorganisms in the lumen are partially digested. A similar pattern was obtained through digestion with pronase. DISCUSSION
The Z organ of X. ifacolum consists of 120 muscle cells, arranged in 20 rings at right angles to the axis of the uterus, larger in the central part of the organ and progressively becomes reduced at the two ends to form two constrictions. According to our investigations the structure of the Z organ of X. ifacolum agrees with Luc’s description of the organ as “bulbe a musculature circulaire puissante” (Luc, 1961). The muscle rings are arranged in such a way that they form a very compact muscular unity. The muscle organization in the Z organ greatly differs from that in the “Z pseudoorgan” in X. diversicaudatum; in the latter no difference is observed between the muscles in the Z pseudo-organ and those in the uterus proper (BleveZacheo et al., 1984). There are twenty rings in both species, each ring consists of four cells in X. diversicaudatum and six in X. ifacolum. The 20 rings in X. diversicaudatum together form a tube 70 pm in length and those in X. ifacolum form a lemon shape body 28 pm in length. As a result, the structure is much more compact in X. ifacolum than in X. diversicaudatum. The apophyses, formed on the crests of some folds of epithelial cell walls, occupy a large part of the lumen of the organ, leaving a tube shape, space free in the centre. In the longitudinal sections the apophyses show concave and curved margins which seem to fit into each other. The apophyses seem to consist of spongy material which is pressed against the wall of the lumen when an egg passes. The
FIGS. 4, 5, 6, 7. Micrographs of longitudinal serial sections from the dorsal to the middle region of the Z organ. (Fig. 4). A strong muscular mass (mu) delimits the cavity of the Z organ; two constrictions (co) separate the organ from the oviduct and from the uterus respectively. The wall of the lumen (1) shows that membranes (me) of the epithelial cells extend on both sides of the organ forming a network. Note the cytoplasm of epithelial cells (ec) inside the membranes. smu=somatic muscles; I=intestine. Scale bar =3 q. (Fig. 5). The top of an apophysis (ap) on the crest of folded membranes (me) is visible. Note the nuclei (nu) of the muscle cells (mu) as in Fig. 4. Scale bar = 15 inn. (Fig. 6). The apophysis (ap) as in Fig. 5 shows its widest size. Other two apophyses are present in the section and their origin is evident from the membranes (me) of the epithelial cells (ec). The figure shows a concavity in the structure of an apophysis fitting together with a convexity of another one (arrows). The lumen (1) of the organ contains granular material and microorganisms (mo). Scale bar = 1.8 inn. (Fig. 7). Section in the middle region of the Z organ. The apophysis, as in Figs. 5, 6 (double arrow) whose connections with the membranes (me) are not visible, is recognizable. Note the nuclei (nu) of the muscle cells (mu) as in Figs. 4, 5, 6; co = constriction; I = intestine; smu = somatic muscles. Scale bar = 2.9 inn. ’
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FIG. 8. Electron micrograph of cross-section in the wider portion of the Z organ. A ring of six muscle cells (l-6) encircles the lumen of the organ, lined by the epithelial folded membrane; six apophyses, orginating from some folds of the membrane (me), occupy the lumen (1). Their disposition suggest how their margins can form a channel, when the organ enlarges, during the egg passage: mo = microorganisms. Scale bar = I4 nrn. Fm. 9. Electron micrograph of a cross-section in the middle portion of the Z organ. The figure shows apophyses (ap) connected to two and three folds of the epithelial membrane (ec) respectively. Numerous microorganisms (mo) are evident into the lumen (1) of the organ; t = tubules; mu’= muscle cells. Scale bar = O-7 pm.
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FIG. 10. Electron micrograph of a cross-section of the Z organ. Muscles (mu) show the intersections of their fibres (mf). The cytoplasm of epithelial cells (ec) rich in organelles is evident in the folded membrane (me) lining the lumen (1). Three apophyses (ap) show how they enlarge from the origin on the crest of the membranes to the centre of the organ. Many tubules (t), containing secretory material and opening into the lumen are present on their surface; nu = nucleus. Scale bar = 1 pm. Very few paracrystals (PC) are observed in the lumen (Inset-Scale bar = 1.5 pm),
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secretions present in the spongy material is squeezed out and spread on the surface of the egg. The absence of sphincters on both oviduct-Z organ and Z organ-uterus connections and the shortness of the structure of the Z organ suggest that the egg passes through that quite rapidly; that could explain why an egg was never observed in the Z organ of X. ifacolum but frequently in the Z pseudo-organ of X. diversicaudatum. Our observations as well as those by Luc (1975) show that the structure of the Z organ is very constant; no variation is observed among 30 specimens from two different populations. The dorsoventral cross-sections through the axis of the uterus of two specimens are almost completely equal (Fig. 3). Treatment of specimens with enzymes shows that what Luc (1961,1975) and Coomans (1964) described as “sclerotized apophyses” are to be considered proliferations of the cell wall of single layered epithehum, lining the lumen of the Z organ, consisting of glycoproteins, like the cell wall. So the origin of the apophyses seems to be the same as that of the globular bodies described by Bleve-Zacheo et al. (1984) in the pseudo Z organ of X diversicaudatum. Long enzymatic treatment did not affect, as in X. diversicaudatum, apophyses and lumen wall, indicating they contain no proteins. Digestion was observed in the content of the tubules, present on the surface of the apophyses and in the material stored around the apophysis margins and the lumen cavity. The effect of enzymatic treatment on the content of the tubules suggests the presence of proteins (Fasold & Gundlach, 1963; Weintraub & Ragetli, 1968), secreted by the epithelial cell and transferred, through the tubules to the surface of the apophyses and then onto the egg, during its passage. Comparing the organization of this Z organ with that of the pseudo Z organ in X. diversicaudatum it seems that in the latter the egg is kept in the pseudo Z organ by the two sphincters and is enveloped by the secretions, present in the lumen and released by the globules, pressed by the egg. Since the egg does not stop in the Z organ of X. ifa~o~um, the process in transferring secretions appears to occur more rapidly during the compression and the expulsion of the egg through the constriction of the organ. Apparently the Z organ and the pseudo Z organ are specialized parts of the uterus, secreting materials involved in the formation of the egg shell (Coomans, 1964; McClure & Bird, 1976). Acknowledgements-The Lerario
for photographic
authors assistance.
are
grateful
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organ in X. ifacohitn
to
Mr.
REFERENCES BIRD A.
F. & AKHURST R. .I. 1983. The nature of the intestinal vesicle in nematodes of the family Steiner-
nematidae. rnternational JournaI for Parasifology 13: 599-606.
BLEVE-ZACHEO T., ZACHEO G., MELILLO M. T. & LAMBERTI F. 1984. On the structure of the 2 pseudo organ in
Xiph~nema di~e~icaadatum. rnfernationa~Journal for Parasitology 14: 404-4 13.
COOMANS A.
1964. Structure of the female gonads in members of the Dorylaimina. Nemafoiogica 10: 601-622. ENDO B. Y. f979. The ultrastructure and distribution of an intracellular Bacterium-like microorganism in tissue of larvae of the soybean cyst nematode, Heterodera
giycines. Journal of UltrastructureResearch 67: l-14.
FA~~LD H. & GUNDLACH C. 1963. Characterization of peptides and proteins with enzymes. In Methods of Enzymatic Analysis (Edited by BERGMEYER H. V.). Academic Press, New York. GRIMALDI-DE ZIO S., D’ADDABBO-GALLO M., LAMBERT~F. & MORONE-DE LUCIA M. R. 1979. The “Z” differentiation in Xiphinema: a hypothesis of its function in relation to amphigony. Nematologica 25: 36-41. LAMBERT~ F., TAYLOR C. E. & SEINHORST J. W. 1975. Nematode Vectors of Plant Viruses. Plenum Press, London and New York. LEDUC E. & BERNHARD W. 1967. Recent modifications of the glycol methacrylate embedding procedure. Journal of
UltrastructureResearch 19: 196-203.
Luc M. 1958. Xiphinema de I’Ouest africain: description de cinq nouvelle especes (Nematoda: Dorylaimidae).
Nematologica 3: 57-72. Luc M. 1961. Structure de la gonade femelle chez quelques especes du genre Xiphinema Cobb, 1913 (Netnatoda: Dorylaimoidea). Nematoiogica 6: 144-154. LLJC M. 1973. Redescription de Xiphinema hatley Luc, 1958 et description de six nouveiles especes de Xiphinema Cobb, 1893 (Nematoda: Dorylaimoidea). Cahiers de
I’Uffice de la Recherche ~ientif~que et T~hnique Ontre-Mer, Biologic 21: 45-65. Lot M. 1975. Taxonomy of Xiphinema Cobb, 1913. In Nematode Vectors of Ptunt Viruses (Edited by LAMBERTI F., TAYLOR C. E. & SEINHORST J. W.). on. 39-52. Plenum Press, London and New York. ‘. _. Luc M. SL DALMASSOA. 1975. Considerations on the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) and a “lattice” for the identification of species. Cakiers de
/‘Office de la Recherche Scientifique et Technique Uutre-Mer, Biologie, 10: 303-327.
MCCLURE M. A. & BIRD A. F. (Nematoda) egg shell: formation
1976. The tylenchid of the egg shell in
Meloidogyne javanica. Parasitology72: 29-39.
SHEPHERD A. M., CLARK S. A. & KEMPTON A. 1973. An intracellular micro-organism associated with tissues of
Heterodera spp. Nemato~agica19: 31-34.
SOUTHEY J. F. 1978. Plan? Nematology. Her Majesty’s Stationery Office, ADAS, London. WEBSTER J. M. 1972. Economic Nematology. Academic Press, London and New York. WEINTRAUB M. & RAGETL~ H. W. 1968. Intracellular characterization of bean yellow mosaic virus-induced inclusions by differential enzyme digestion. Journal of
Cell Biology 38: 316-328.