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Electron microscopy of infection of nematodes by Dactylaria haptotyla
89 Mycoscience 35: 89-94, 1994
Electron microscopy of infection of nematodes by Dactylaria
haptotyla
Masatoshi Saikawa and Makoto Kaneko
Department of Biology, Tokyo Gakugei University, Nukuikita-machi, Koganei-shi, Tokyo 184, Japan Accepted for publication 6 January 1994 Infection of nematodes by Dactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membranebound vesicles, 0 . 2 - 0 . 5 / J m in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. W h e n the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0 . 7 / ~ m in diam, was found in serial thin sections of the knob's cytoplasm. A t the site facing the vesicle, the peripheral part of the nematode's cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.
Key Words
adhesive knob; Dactylaria haptotyla; electron microscopy; nematode; penetration vesicle.
Introduction
Nematode-trapping hyphomycetes produce trapping organs of various types, including constricting rings, nonconstricting rings, adhesive nets and adhesive knobs. Although ultrastructural aspects of various of these traps in thin sections have been reported by several authors (Heintz and Pramer, 1972; Dowsett and Reid, 1977, 1979; Dowsett et al., 1977; Nordbring-Hertz and St~lhammar-Carlemalm, 1978; Wimble and Young, 1983a, b, 1984; Saikawa, 1985), the initial stages of fungal infection just before penetration into nematodes have not yet been fully examined. The ultrastructure of the initial stages of penetration by an adhesive knob of Dactylaria haptotyla Drechsler are shown in the present study. Materials and Methods
Dactylaria haptotyla parasitizing nematodes was found on a water agar plate in October 1992. General morphology of the fungus was identical to that reported by Drechsler (1950). The plate had been incubated for about 1 month with a pinch of moss collected in the campus of Tokyo Gakugei University, Koganei, Tokyo, Japan. The fungal materials capturing nematodes were prefixed in 2.5°J0 glutaraldehyde buffered with 0.1 M sodium phosphate (pH 7.2) by microwave irradiation three times for 4sec at below 37°C (Mizuhira, 1988). After the prefixation the specimens were washed with the same buffer for 1.5 h, and postfixed in OsO4 in the buffer at 4 ° C for 12 h. After dehydration through an acetone
series, the fungat materials were embedded in Poly/Bed 812 (Polysciences, Inc., Warrington, PA, U.S.A.). Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a JEOL 100CXll electron microscope operating at 80 kV. Results and Discussion
The trapping organ of D. haptotyla, a nematode-destroying hyphomycete, is a unicellular adhesive knob, subspherical or prolate ellipsoidal in shape, mostly 7.510/~m long and 6-9/~m wide, developed on a slender stalk of 1-3 hyphal cells. Electron micrographs showed that the cytoplasm of the adhesive knob contained a number of membrane-bound vesicles, 0.2-0.5/~m in diam and of various electron densities (Figs. 1-4}, in addition to the usual cell constituents, such as nuclei, mitochondria and ribosomes. The electron-dense vesicles were rarely seen in the stalk-cell or vegetative cell cytoplasm (Figs. 1, 2). When the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive. This was seen in ultrathin sections between the knob and the cuticle as an amorphous mass with a moderate electron density (Figs. 2-7). At the same time, electrondense vesicles in the cytoplasm were reduced in number and many small vacuoles developed (Figs. 2-7). The hyphal cell wall of the fungus was composed of an outer, electron-dense layer and an inner, less dense layer. In thin sections, both layers exhibited similar thicknesses in the adhesive knob (Figs. 3, 4), while in other parts of the mycelium the former layer of celt watt was al-
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w a y s thinner than the latter. When the adhesive knob came into c o n t a c t w i t h the n e m a t o d e ' s cuticle, both layers of cell wall became obscure at the prospective site of penetration (Fig. 3), w h e r e a vesicle, 0 . 7 / z m in diam, w a s found in the c y t o p l a s m in serial ultrathin sections of the
adhesive knob (Figs. 4 - 6 ) . In addition, the periphery of the n e m a t o d e ' s cell (Figs. 4 - 6 ) , w h i c h w a s depressed by the adhesive knob, s h o w e d a high electron density at the site facing the vesicle. It is obvious that the vesicle had migrated there and discharged its enzymic c o n t e n t s
Figs. 1-3. Electron micrographs of adhesive knobs of Dactylaria haptotyta before penetration into nematodes. 1. Cytoplasm of an adhesive knob containing a number of electron-dense, membrane-bound vesicles of various electron densities (DV). Bar: 5/~m. 2. Adhesive (Ad) secreted from an adhesive knob seen between the knob and the nematode's cuticle. The knob's cytoplasm develops vacuoles (V), and electron-dense vesicles (DV) are seen in its peripheral region facing the adhesive. Bar: 5/~m. 3. Cell wall of an adhesive knob becomes obscure at the prospective site of penetration (~), where the knob's cytoplasm pushes against the nematode. Bar: 5/~m. Abbreviations used in figures: Ad, adhesive; AK, adhesive knob; CW, cell wall; DV, electron-dense vesicle; IB, infection bulb; M, mitochondrion; N, nucleus; NC, nematode's cuticle; PV, penetration vesicle; S, septum associated with Woronin bodies; V, vacuole.
Infection of nematodes
t o w a r d t h e c a p t u r e d n e m a t o d e t o d e g r a d e t h e cuticle, because t h e vesicle w a s n o t e l e c t r o n - d e n s e at all and its m e m b r a n e w a s w a v y in a p p e a r a n c e (Fig. 5). The vesicle could be d e r i v e d f r o m smaller e l e c t r o n - d e n s e vesicles coalesced together. W i m b l e and Y o u n g ( 1 9 8 4 ) r e p o r t e d u l t r a s t r u c t u r e s
91
of Dactylella lysipaga Drechsler, a n e m a t o d e - t r a p p i n g fungus p r o d u c i n g b o t h a d h e s i v e k n o b s and n o n c o n s t r i c t i n g rings. In thin sections of the f u n g u s a vesicle u n d o u b t e d ly identical t o t h a t f o u n d in D. haptotyla in t h e present s t u d y w a s seen at the site of p e n e t r a t i o n by an a d h e s i v e knob. In t h a t case, t h e c y t o p l a s m had a l r e a d y p e n e t r a t -
Figs. 4, 5. Electron micrographs of an adhesive knob of Dacty/aria hyptotyla just before penetration into a nematode. 4. A vesicle (PV), 0.7 #m in diam and with moderate electron density, is seen at the prospective site of penetration, where the cell wall of the knob seems almost to be digested. Cytoplasm of the nematode shows a high electron density (~) at the site facing the penetration vesicle. Bar: 1 fire. 5. Enlargement of the vesicle at the site of penetration in Fig. 4, showing the w a v y outline of the membrane. Bar: 0.5 #m.
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ed slightly into the n e m a t o d e to f o r m a developing infection bulb, w h i c h w a s surrounded by an electron-dense material (Wimble and Y o u n g , 1984, fig. 30). Saikawa (1985) found another electron-dense, barrel-shaped, plug-like structure (0.5 x 0.7/~m in size) before penetration of n e m a t o d e s by a nonconstricting-ring trap of Dactylella leptospora Drechsler. In the latter case, no sign of penetration w a s recognized in the n e m a t o d e ' s cell.
Thus, the electron micrographs in Figs. 4 - 6 in the present study s h o w an intermediate stage in the infection process b e t w e e n t h a t in D. leptospora reported by Saikaw a and t h a t in D. lysipaga by W i m b l e and Y o u n g . The vesicle for penetration, or " p e n e t r a t i o n vesicle," is an organelle that probably occurs c o m m o n l y in this stage of infection by n e m a t o d e - t r a p p i n g h y p h o m y c e t e s . A f t e r b r e a k d o w n of the n e m a t o d e ' s cuticle, the
Fig. 6. Electron micrograph of another section of the same adhesive knob of Dactylaria haptotyla as in Fig. 4 at a higher magnification. The penetration vesicle (PV) with moderate electron density at the prospective site of penetration is cut slightly off center. Peripheral cytoplasm of the nematode shows a high electron density (*) at the site facing the vesicle. Bar: 0.5 ~m.
Infection of nematodes
93
Figs. 7, 8. Electron micrographs of adhesive knobs and infection bulbs of Dactylaria haptotyla. 7. Formation of the infection bulb (IB) by an adhesive knob (AK). Cell organelles are migrating into a newly formed infection bulb in the nematode body, Bar: 1 f~m. 8, Adhesive knob (AK) and infection bulb (IB) at a later stage of infection. Most portions of fungal cells contain large vacuoles (V). Bar: 5 f~m.
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k n o b ' s c y t o p l a s m in Dactylaria haptotyla m i g r a t e d into a n e w l y f o r m e d i n f e c t i o n bulb in t h e n e m a t o d e ' s b o d y (Fig. 7), and a s e p t u m f o r m e d at t h e b o u n d a r y b e t w e e n t h e k n o b and t h e i n f e c t i o n bulb (Fig. 8). Similar f e a t u r e s s h o w i n g m i g r a t i o n of c y t o p l a s m into i n f e c t i o n bulbs h a v e a l r e a d y been r e p o r t e d b y W i m b l e and Y o u n g ( 1 9 8 4 ) .
Literature cited Dowsett, J. A. and Reid, J. 1977. Transmission and scanning electron microscope observations on the trapping of nematodes by Dactylaria candida. Can. J. Bot. 56: 2 9 6 3 - 2 9 7 0 . Dowsett, J. A. and Reid, J. 1979. Observations on the trapping of nematodes by Dactylaria scaphoides using optical, transmission and scanning-electron-microscopic techniques. Mycologia71: 379-391. Dowsett, J. A., Reid, J. and Caeseele, L. 1977. Transmission and scanning electron microscope observations on the trapping of nematodes by Dactylaria brochopaga. Can. J. Bot. 55: 2 9 4 5 - 2 9 5 5 .
Drechsler, C. 1950. Several species of Dactylella and Dactylaria that capture free-living nematodes. Mycologia 42: 177. Heintz, C.E. and Pramer, D. 1972. Ultrastructure of nematode-trapping fungi. J. Bacteriol. 1 1 0 : 1 1 6 3 - 1 1 7 0 . Mizuhira, V. 1988. Application of microwave irradiation to the biological tissue fixation method. Pure Chemicals "Daiichi" 19: 103-116. (in Japanese). Nordbring-Hertz, B. and St~lhammar-Carlemalm, M. 1978. Capture of nematodes by Arthrobotrys oligospora, an electron microscope study. Can. J. Bot. 56: 1297-1307. Saikawa, M. 1985. Ultrastructural features of the nonconstricting-ring trap in Dactytetta leptospora, Trans. Mycol. Soc. Japan 26: 209-213. Wimble, D. B. and Young, T. W. K. 1983a. Septum structure in Dactylella lysipaga. Mycologia 75: 174-175. Wimble, D. B. and Young, T. W. K. 1983b. Structure of adhesive knobs in Dactylella lysipaga. Trans. Br. Mycol. Soc. 80: 515-519. Wimble, D. B. and Young, T, W. K. 1984. UItrastructure of the infection of nematodes by Dactylella lysipaga. Nova Hedw. 40: 9-29.
Electron microscopy of infection of nematodes by Dactylaria
haptotyla
Masatoshi Saikawa and Makoto Kaneko
Department of Biology, Tokyo Gakugei University, Nukuikita-machi, Koganei-shi, Tokyo 184, Japan Accepted for publication 6 January 1994 Infection of nematodes by Dactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membranebound vesicles, 0 . 2 - 0 . 5 / J m in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. W h e n the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0 . 7 / ~ m in diam, was found in serial thin sections of the knob's cytoplasm. A t the site facing the vesicle, the peripheral part of the nematode's cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.
Key Words
adhesive knob; Dactylaria haptotyla; electron microscopy; nematode; penetration vesicle.
Introduction
Nematode-trapping hyphomycetes produce trapping organs of various types, including constricting rings, nonconstricting rings, adhesive nets and adhesive knobs. Although ultrastructural aspects of various of these traps in thin sections have been reported by several authors (Heintz and Pramer, 1972; Dowsett and Reid, 1977, 1979; Dowsett et al., 1977; Nordbring-Hertz and St~lhammar-Carlemalm, 1978; Wimble and Young, 1983a, b, 1984; Saikawa, 1985), the initial stages of fungal infection just before penetration into nematodes have not yet been fully examined. The ultrastructure of the initial stages of penetration by an adhesive knob of Dactylaria haptotyla Drechsler are shown in the present study. Materials and Methods
Dactylaria haptotyla parasitizing nematodes was found on a water agar plate in October 1992. General morphology of the fungus was identical to that reported by Drechsler (1950). The plate had been incubated for about 1 month with a pinch of moss collected in the campus of Tokyo Gakugei University, Koganei, Tokyo, Japan. The fungal materials capturing nematodes were prefixed in 2.5°J0 glutaraldehyde buffered with 0.1 M sodium phosphate (pH 7.2) by microwave irradiation three times for 4sec at below 37°C (Mizuhira, 1988). After the prefixation the specimens were washed with the same buffer for 1.5 h, and postfixed in OsO4 in the buffer at 4 ° C for 12 h. After dehydration through an acetone
series, the fungat materials were embedded in Poly/Bed 812 (Polysciences, Inc., Warrington, PA, U.S.A.). Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a JEOL 100CXll electron microscope operating at 80 kV. Results and Discussion
The trapping organ of D. haptotyla, a nematode-destroying hyphomycete, is a unicellular adhesive knob, subspherical or prolate ellipsoidal in shape, mostly 7.510/~m long and 6-9/~m wide, developed on a slender stalk of 1-3 hyphal cells. Electron micrographs showed that the cytoplasm of the adhesive knob contained a number of membrane-bound vesicles, 0.2-0.5/~m in diam and of various electron densities (Figs. 1-4}, in addition to the usual cell constituents, such as nuclei, mitochondria and ribosomes. The electron-dense vesicles were rarely seen in the stalk-cell or vegetative cell cytoplasm (Figs. 1, 2). When the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive. This was seen in ultrathin sections between the knob and the cuticle as an amorphous mass with a moderate electron density (Figs. 2-7). At the same time, electrondense vesicles in the cytoplasm were reduced in number and many small vacuoles developed (Figs. 2-7). The hyphal cell wall of the fungus was composed of an outer, electron-dense layer and an inner, less dense layer. In thin sections, both layers exhibited similar thicknesses in the adhesive knob (Figs. 3, 4), while in other parts of the mycelium the former layer of celt watt was al-
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w a y s thinner than the latter. When the adhesive knob came into c o n t a c t w i t h the n e m a t o d e ' s cuticle, both layers of cell wall became obscure at the prospective site of penetration (Fig. 3), w h e r e a vesicle, 0 . 7 / z m in diam, w a s found in the c y t o p l a s m in serial ultrathin sections of the
adhesive knob (Figs. 4 - 6 ) . In addition, the periphery of the n e m a t o d e ' s cell (Figs. 4 - 6 ) , w h i c h w a s depressed by the adhesive knob, s h o w e d a high electron density at the site facing the vesicle. It is obvious that the vesicle had migrated there and discharged its enzymic c o n t e n t s
Figs. 1-3. Electron micrographs of adhesive knobs of Dactylaria haptotyta before penetration into nematodes. 1. Cytoplasm of an adhesive knob containing a number of electron-dense, membrane-bound vesicles of various electron densities (DV). Bar: 5/~m. 2. Adhesive (Ad) secreted from an adhesive knob seen between the knob and the nematode's cuticle. The knob's cytoplasm develops vacuoles (V), and electron-dense vesicles (DV) are seen in its peripheral region facing the adhesive. Bar: 5/~m. 3. Cell wall of an adhesive knob becomes obscure at the prospective site of penetration (~), where the knob's cytoplasm pushes against the nematode. Bar: 5/~m. Abbreviations used in figures: Ad, adhesive; AK, adhesive knob; CW, cell wall; DV, electron-dense vesicle; IB, infection bulb; M, mitochondrion; N, nucleus; NC, nematode's cuticle; PV, penetration vesicle; S, septum associated with Woronin bodies; V, vacuole.
Infection of nematodes
t o w a r d t h e c a p t u r e d n e m a t o d e t o d e g r a d e t h e cuticle, because t h e vesicle w a s n o t e l e c t r o n - d e n s e at all and its m e m b r a n e w a s w a v y in a p p e a r a n c e (Fig. 5). The vesicle could be d e r i v e d f r o m smaller e l e c t r o n - d e n s e vesicles coalesced together. W i m b l e and Y o u n g ( 1 9 8 4 ) r e p o r t e d u l t r a s t r u c t u r e s
91
of Dactylella lysipaga Drechsler, a n e m a t o d e - t r a p p i n g fungus p r o d u c i n g b o t h a d h e s i v e k n o b s and n o n c o n s t r i c t i n g rings. In thin sections of the f u n g u s a vesicle u n d o u b t e d ly identical t o t h a t f o u n d in D. haptotyla in t h e present s t u d y w a s seen at the site of p e n e t r a t i o n by an a d h e s i v e knob. In t h a t case, t h e c y t o p l a s m had a l r e a d y p e n e t r a t -
Figs. 4, 5. Electron micrographs of an adhesive knob of Dacty/aria hyptotyla just before penetration into a nematode. 4. A vesicle (PV), 0.7 #m in diam and with moderate electron density, is seen at the prospective site of penetration, where the cell wall of the knob seems almost to be digested. Cytoplasm of the nematode shows a high electron density (~) at the site facing the penetration vesicle. Bar: 1 fire. 5. Enlargement of the vesicle at the site of penetration in Fig. 4, showing the w a v y outline of the membrane. Bar: 0.5 #m.
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ed slightly into the n e m a t o d e to f o r m a developing infection bulb, w h i c h w a s surrounded by an electron-dense material (Wimble and Y o u n g , 1984, fig. 30). Saikawa (1985) found another electron-dense, barrel-shaped, plug-like structure (0.5 x 0.7/~m in size) before penetration of n e m a t o d e s by a nonconstricting-ring trap of Dactylella leptospora Drechsler. In the latter case, no sign of penetration w a s recognized in the n e m a t o d e ' s cell.
Thus, the electron micrographs in Figs. 4 - 6 in the present study s h o w an intermediate stage in the infection process b e t w e e n t h a t in D. leptospora reported by Saikaw a and t h a t in D. lysipaga by W i m b l e and Y o u n g . The vesicle for penetration, or " p e n e t r a t i o n vesicle," is an organelle that probably occurs c o m m o n l y in this stage of infection by n e m a t o d e - t r a p p i n g h y p h o m y c e t e s . A f t e r b r e a k d o w n of the n e m a t o d e ' s cuticle, the
Fig. 6. Electron micrograph of another section of the same adhesive knob of Dactylaria haptotyla as in Fig. 4 at a higher magnification. The penetration vesicle (PV) with moderate electron density at the prospective site of penetration is cut slightly off center. Peripheral cytoplasm of the nematode shows a high electron density (*) at the site facing the vesicle. Bar: 0.5 ~m.
Infection of nematodes
93
Figs. 7, 8. Electron micrographs of adhesive knobs and infection bulbs of Dactylaria haptotyla. 7. Formation of the infection bulb (IB) by an adhesive knob (AK). Cell organelles are migrating into a newly formed infection bulb in the nematode body, Bar: 1 f~m. 8, Adhesive knob (AK) and infection bulb (IB) at a later stage of infection. Most portions of fungal cells contain large vacuoles (V). Bar: 5 f~m.
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k n o b ' s c y t o p l a s m in Dactylaria haptotyla m i g r a t e d into a n e w l y f o r m e d i n f e c t i o n bulb in t h e n e m a t o d e ' s b o d y (Fig. 7), and a s e p t u m f o r m e d at t h e b o u n d a r y b e t w e e n t h e k n o b and t h e i n f e c t i o n bulb (Fig. 8). Similar f e a t u r e s s h o w i n g m i g r a t i o n of c y t o p l a s m into i n f e c t i o n bulbs h a v e a l r e a d y been r e p o r t e d b y W i m b l e and Y o u n g ( 1 9 8 4 ) .
Literature cited Dowsett, J. A. and Reid, J. 1977. Transmission and scanning electron microscope observations on the trapping of nematodes by Dactylaria candida. Can. J. Bot. 56: 2 9 6 3 - 2 9 7 0 . Dowsett, J. A. and Reid, J. 1979. Observations on the trapping of nematodes by Dactylaria scaphoides using optical, transmission and scanning-electron-microscopic techniques. Mycologia71: 379-391. Dowsett, J. A., Reid, J. and Caeseele, L. 1977. Transmission and scanning electron microscope observations on the trapping of nematodes by Dactylaria brochopaga. Can. J. Bot. 55: 2 9 4 5 - 2 9 5 5 .
Drechsler, C. 1950. Several species of Dactylella and Dactylaria that capture free-living nematodes. Mycologia 42: 177. Heintz, C.E. and Pramer, D. 1972. Ultrastructure of nematode-trapping fungi. J. Bacteriol. 1 1 0 : 1 1 6 3 - 1 1 7 0 . Mizuhira, V. 1988. Application of microwave irradiation to the biological tissue fixation method. Pure Chemicals "Daiichi" 19: 103-116. (in Japanese). Nordbring-Hertz, B. and St~lhammar-Carlemalm, M. 1978. Capture of nematodes by Arthrobotrys oligospora, an electron microscope study. Can. J. Bot. 56: 1297-1307. Saikawa, M. 1985. Ultrastructural features of the nonconstricting-ring trap in Dactytetta leptospora, Trans. Mycol. Soc. Japan 26: 209-213. Wimble, D. B. and Young, T. W. K. 1983a. Septum structure in Dactylella lysipaga. Mycologia 75: 174-175. Wimble, D. B. and Young, T. W. K. 1983b. Structure of adhesive knobs in Dactylella lysipaga. Trans. Br. Mycol. Soc. 80: 515-519. Wimble, D. B. and Young, T, W. K. 1984. UItrastructure of the infection of nematodes by Dactylella lysipaga. Nova Hedw. 40: 9-29.