Electron microscope studies on the development and Title of zygospores in pectosporus, a zoopagaceous capturing ( fulltext )

Author(s) SAIKAWA,Masatoshi; WAKAI,Yuka; KATSUSHIMA,Naoko

Citation 東京学芸大学紀要. 自然科学系, 63: 91-100

Issue Date 2011-09-30

URL http://hdl.handle.net/2309/112016

Publisher 東京学芸大学学術情報委員会

Rights Bulletin of tokyo Gakugei university, Division of Natural Sciences, 63: 91 - 100，2011

Electron microscope studies on the development and germination of zygospores in Zoophagus pectosporus, a zoopagaceous fungus capturing nematodes

Masatoshi Saikawa*, Yuka Wakai** and Naoko Katsushima**

Department of Environmental Sciences

(Received for Publication; May 20, 2011)

Saikawa, M., Wakai, Y. and Katsushima, N.: Electron microscope studies on the development and germination of zygospores in Zoophagus pectosporus, a zoopagaceous fungus capturing nematodes. Bull. Tokyo Gakugei Univ. Div. Nat. Sci., 63: 91-100 (2011) ISSN 1880-4330

Abstract

Zygospore development of Zoophagus pectosporus examined in ultrathin sections is reported for the first time for the family . The fusion wall made by the union between paired gametangia is known not to dissolve, but to disappear by means of widening a central pore that is made after fusion. The fusion wall becomes to be incorporated into the of a developing, immature zygosporangium. On the other hand, two gametangial septa, newly-made cross walls delimiting between the immature zygosporangium and newly-made paired suspensors, are known to have a central pore. In this study, germination of zygospore, composed of zygosporangium and zygospore-proper, is seen in electron micrographs in thin sections for the first time for the family. The protoplasm of the zygospore in germination is known to be occupied totally by numerous electron-dense large vesicles, 0.5-1.0 µm in diameter, and is continuous through that of a germ tube and a few non-septate vegetative hyphae developed from the germ tube. A nucleus is found in the protoplasm of the germinating zygospore.

Key words: , dissolution, fusion wall, gametangium, septum, zygosporangium

Department of Environmental Sciences, Tokyo Gakugei University, 4-1-1 Nukuikita-machi, Koganei-shi, Tokyo 184-8501, Japan

* advanced Support Center for Science teachers (aSCeSt), tokyo Gakugei university ** tokyo Gakugei university (4-1-1 Nukuikita-machi, koganei-shi, tokyo, 184-8501, Japan)

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Zoophagus pectosporus (Drechsler) M.W. Dick, capturing Materials and Methods nematodes and , was once named as Acaulopage pectospora Drechsler. Although all in the Zoophagus pectosporus capturing a species of nematodes Acaulopage had been known not to capture nematodes, (Rhabditis sp.) was recovered from a water-agar (WA) plate but rhizopods by means of adhesive of prostrate vegetative in a Petri dish, 90 mm in diam, incubated with about 10 g of hyphae on agar plates and, in addition, the fungus he a few pieces of leaf for a week in the room temperature observed did not produce zygospores, Drechsler (1962) (20-22 C). The leaf mold was collected in a forest in the accommodated the species in the genus in the Zygomycotina. Takaozan hill, Hachioji, Tokyo, Japan in April, 1988 by The narrow, spindle-shaped, sessile conidia of the fungus and one (NK) of the authors. The fungus was maintained until its predacious habit were thought by him to be identical with October, 1992 on WA plates by inoculation of both fungus those of Acaulopage. and nematodes into fresh WA plates at intervals of 1-2 wk. On the other hand, Zoophagus had been thought to be For initiation of multiplication of nematodes, those that have a genus in the Peronosporales, Oomycota (Sommerstorff, not yet been infected by the fungus were transferred to SAA 1911). According to Whisler and Travland (1974) (Saikawa and Kadowaki, 2002). Subculture was done at mitochondria with tubular cristae, nuclei and general intervals of 15-20 d, in which Caenorhabditis nematodes of cytoplasmic organization in Z. insidians Sommerstorff a wild type were used as hosts in place of Rhabditis sp. are comparable to similar structures in Pythium (Grove For electron microscopy, specimens were fixed in 2% et al., 1970) and other members of the Oomycota. (v/v) glutaraldehyde buffered with 0.1 M sodium phosphate However, in light and electron micrographs, Saikawa and (pH 7.2) for 1.5 h at room temperature, washed with the same

Morikawa (1985) became aware that A. pectospora (named buffer for 1.5 h, and post fixed in OsO4 in the same buffer currently as Z. pectosporus) was identical to a water mold, at 4 C for 12 h. After dehydration through an acetone series, Z. insidians, in that it captures microscopic animals by the fungal materials were embedded in epoxy resin. Ultrathin means of adhesive apical knobs of short lateral branches sections were stained with uranyl acetate and lead citrate arising at right angles to the main non-septate at and observed with a JEOL 100CXII electron microscope regular intervals like in Z. insidians, though Z. insidians operating at 80 kV. does not capture nematodes, but loricate rotifers under water. Saikawa et al. (1988) found that A. pectospora Results captures rotifers as well as nematodes with the short trapping branches in the same way as Z. insidians when a Light microscopy culture is added, and eventually Dick (1990) moved Zoophagus pectosporus is a zoopagaceous fungus parasitic A. pectospora into a zygomycotan genus Zoophagus in mainly nematodes and rotifers. The composed of by showing zygospores of the fungus. The image of the straightly grown vegetative hyphae, 2.5-3 µm wide, captures zygospores he showed remains in doubt, because the the animals by predacious hyphal branches, referred usually fungus he studied was not a species of Zoophagus but of to as pegs (Figs. 1-4). In the latter case, a vegetative hypha Lecophagus (Morikawa et al., 1993). grows subsurface in the agar plates and produces a number it was Miura (1967) who found sexual reproduction in of pegs, 20 µm long and 5-10 µm wide, terminated with a Z. pectosporus in 52-day-old culture of the fungus taken globose knob-like excrescence, 2.5 µm in diam (Figs. 3-4). from a forest in Iriomote Island, Japan, i.e., it yielded The pegs grow ascending toward distally and only the knob zygospores often in small groups along its mycelial appears on the agar plate. When a becomes in filaments. Zygospores identical with those found by Miura contact with the knob, the peg secretes a considerable amount were produced in the mycelium of a fungus obtained from of adhesive to capture it. Because of the fungus being an Takaozan hill, Tokyo, Japan, and their developmental stages obligate parasite, hyphae of the mycelium can grow and in ultrathin sections are shown in the present study. The produce conidia asexually only when it captures nematodes. germination of zygospore of the fungus is also shown. The conidia, slender, fusiform in shape, ca. 200 µm long and 15 µm wide (Fig. 1), develop aerially at distal end of the growing hypha one after another. Conidia develop directly from

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*

Ad

*

*

* ** * * ** *

Figs. 1-5. Light micrographs of Zoophagus pectosporus. 1. after liberation from vegetative hypha. It already germinated near the apical portion and the germ tube changes directly into a peg (Pe), terminated by a knob-like excrescence (K). 2. The head region of a nematode (Ne), captured by a peg (Pe) with adhesive (Ad). 3. Aged adhesive on three knobs (asterisks). Pe, pegs; VH, vegetative hypha. 4. Immature zygosporangium (double asterisk) produced by conjugation of two gametangia arisen from a common mycelium. Pe, peg; VH, vegetative hypha; asterisks, suspensors. 5. Magnified view of the immature zygosporangium (double asterisk) in Fig. 4. Arrow, gametangial septum; asterisks, suspensors. Bars, 50 µm for Fig. 1; 10 µm for Figs. 2-5.

vegetative hyphae that grow a few micrometers below surface of gametangia can be recognized in Fig. 4 (double asterisk), of the agar plate. After liberation from vegetative hyphae, but it is not clearly seen due to the angle to the structure conidia germinate to develop vegetative hyphae, or sometimes (Fig. 5). The better images of immature zygosporangia are predacious pegs directly (Fig. 1). shown in Figs. 6 and 7, in which an eccentric, or budlike the fungus is known to be homothallic and two enlargement arising from one of the two gametangia zygophores, or reproductive hyphae, producing gametangia characteristic to the zoopagaceous fungi. After enlargement, distally arise from the same mycelium as shown in Fig. 4 the zygosporangium becomes spherical in shape, and (asterisks). An immature zygosporangium made after fusion its cell wall is covered with a secondarily-accumulated

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* *

*

* *

Figs. 6-10. Light micrographs of Zoophagus pectosporus. 6. Immature zygosporangium under its eccentric enlargement. It arose from one of the two gametangia. Arrows show gametangial septa delimiting the zygosporangium from suspensors (asterisk). Arrowhead, the portion of the fusion wall. 7. Another example of the immature zygosporangium. Double arrow shows the septum between suspensor (asterisk) and vegetative hypha. 8. Zygosporangium after its enlargement, showing somewhat verrucose in outline. Asterisks, suspensors. 9. A portion of aged vegetative hyphae (VH) of mycelium with which a fully developed zygosporangium (Zy) is connected through a suspensor (asterisk). Arrow, septum. 10. Matured zygosporangium, spherical in shape. The cell wall shows bumpy in appearance (arrows). Bars, 10 µm for Figs. 6-8, 10; 50 µm for Fig. 9. substance showing it somewhat rough in outline (Fig. 8). The (0.5-1.0 µm) before secretion of adhesive. After secretion, zygospore becomes to have a thick cell wall composed of a adhesive derived from the large vesicles shows an amorphous thin outer- and a thick inner layer after hyphae associating mass of electron dense homogenous material that contains a the structure are totally evacuated. The outer layer of number of bubbles (Figs. 11, 12). The cell wall of the knob the cell wall of matured zygospore is covered with the becomes doubled after secretion of adhesive and an aliquot secondarily-accumulated amorphous substance showing of the adhesive remains in the intervening space between the bumpy in outline (Figs. 9, 10), in which the term zygospore outer and inner layers of cell wall (Fig. 12). is used for the combination of zygospore proper and Except for the image of a zygospore of Z. pectosporus zygosporangium (Benjamin, 1959). appeared recently in Mycoscience (Saikawa, 2011, fig. 12), sexual reproductive structures in the family Zoopagaceae Electron microscopy in ultrathin sections have not yet been reported so far. Electron microscopic images of the fungus in capturing The protoplasm in zygophores, or branches to become nematodes are almost identical with those of the strain gametangia, contains a number of electron dense small, obtained from Saikawa River, Kanazawa, Japan in 1982 and large vesicles as that in the vegetative hyphae (Fig. 13). (Saikawa and Morikawa, 1985) and of the rotifer-capturing Unlike general appearance for the large vesicles they Zoophagus insidians studied by Whisler and Travland are not homogeneous due mainly to the difficulty of (1974) and that by Saikawa and Goho (2010), i.e., the trap’s fixation (Fig. 13). At right before the conjugation of two cytoplasm is occupied by large electron-dense vesicles gametangia in different mating types, a common fusion

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HM Ad

SV

HM

Figs. 11, 12. Electron micrographs of Zoophagus pectosporus capturing nematodes with predacious pegs. 11. Adhesive (Ad) seen between the peg (Pe) and the nematode (Ne) as an electron-dense, amorphous mass. It contains a number of foamy mass. Another electron dense substance (arrow) is also seen beneath the cuticle of nematode (Ne). 12. Amorphous mass of adhesive (Ad) exuded from the knob of a peg (Pe). The mass is composed of an electron-dense, homogeneous material (HM) and a number of groups of small vesicles (SV) of less electron density. Homogenous material is also included in the knob. Although the cell wall of the portion of the peg is composed of an outer electron- dense- and an inner less dense layer (arrow), that of the knob is doubled (double arrows). Adhesive is recognized also in the intervening space. Bars, 10 µm for Fig. 11; 1 µm for Fig. 12.

wall is established between the two (Fig. 14, arrowheads) thick cell wall (Fig. 20, asterisks) is distinguishable from the and, at almost the same time, the gametangial septum zygosporangial cell wall. delimiting from each of the suspensors is made between the germination of zygospores in the family Zoopagaceae the gametangium and immature zygosporangium (Figs. 14, is shown for the first time in the present study (Fig. 21). 15, 17, arrows), in which the cross wall has been made by So far we thought the image to be of the zygosporangium centripetal-, or inward growth of cell wall of the zygophores. formation by three gametangia, though the structures are If the section is cut at the central part of the septum, a tiny extremely narrower than the real gametangia as shown perforation of the simple-pore type is seen (Figs. 15, 16), here. However, the outwardly-curved appearance of the where the pore is not associated with parenthesomes but zygosporangial cell wall at its broken portion would exhibit a bridge-like structure of electron-dense, homogenous that it is the image of germination (Fig. 21, arrow). During substance (Fig. 16, asterisk). Before maturation of the development and germination of zygosporangium of Z. zygosporangium, a rudiment of the fusion wall is seen in pectosporus, more than one nucleus is not found in thin thin sections close to the zygosporangial cell wall (Fig. 17). sections (Fig. 22). The immature zygosporangium becomes spherical in shape by its enlargement and deposits of secondary wall material Discussion are seen on the zygosporangial cell wall (Figs. 18, 19). After evacuation of protoplasm from suspensors and vegetative ultrastructural features of asexual of fungi in hyphae, the zygosporangium associating with the structures the family Zoopagaceae have been reported in becomes in a resting stage (Fig. 20) until its germination. rhabdospora Drechsler (Saikawa, 1986) and in Acaulopage In a matured zygosporangium zygospore proper with its dichotoma Drechsler, A. tetraceros Drechsler, S. cephalote

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GW

SV

GW

SV

ZW LV

LV ZW

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Figs. 13-16. Electron micrographs of Zoophagus pectosporus. 13. A zygophore (ZP) developed from a vegetative hypha (VH). 14. Conjugation of two gametangia (Ga). The protoplasm in both gametangia contains small vesicles (SV). GW, gametangial wall; arrow, gametangial septum; arrowheads, fusion wall. 15. Immature zygosporangium (Zy). The fusion wall (arrowheads) made by cell walls of two gametangia is in incorporation into the zygosporangial cell wall (ZW). LV, large vesicle; SV, small vesicles; arrows, gametangial septa; asterisk, a tiny central pore of septum. 16. Photographic enlargement of the pore in Fig. 15, associated with a bridge-like structure of an electron-dense, homogenous substance (asterisk). Bars, 2 µm for Figs. 13-15; 1 µm for Fig. 16.

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SV SV

ZW LV LV

ZW *

LV

LV *

LV

ZW SV

V LV * * V

ZW ZW

W ZPW

Figs. 17-20. Electron micrographs of zygosporangium in Zoophagus pectosporus in successive stages in maturation. 17. U-shaped immature zygosporangium (Zy). The protoplasm contains both large (LV) and small vesicles (SV). The remnant of the fusion wall (asterisk) is recognized. ZW, zygosporangial wall; Arrow, gametangial septum. 18. Immature, but fully grown zygosporangium (Zy). The cell wall (ZW) shows verrucose in appearance by warts (arrows). LV, large vesicles; Su, suspensor; SV, small vesicles. 19. Matured zygosporangium (Zy). Electron-dense, homogenous substance accumulates irregularly on the zygosporangial cell wall (ZW) as warts (W). LV, large vesicles; SV, small vesicles; V, vacuoles. 20. Zygosporangium in a resting state. Zygospore proper (ZP), distinguishable from the zygosporangial cell wall (ZW), has a thick cell wall (ZPW, asterisks). The layer of secondarily-accumulated materials on the zygosporangial wall shows even in thickness. LV, large vesicles. Bars, 5 µm for Figs. 17-19; 10 µm for Fig. 20.

Drechsler and Zoophagus tentaclum Karling (Saikawa, 2011) types make a common fusion wall between the two (Fig. 14, up to now. On the other hand, only one figure of the structure arrowheads) and at almost the same time two gametangial representing the sexual reproduction has been reported septa, each delimiting from the suspensor are made between by Saikawa (2011). It was a matured zygosporangium of the immature zygosporangium and the gametangium Z. pectosporus showing the existence of a single nucleus in it. (Figs. 14, 15, 17, arrows). The fusion wall is found in thin In the present paper we add information on the development sections to disappear not by dissolution, but by making a pore and germination of zygospores in the same fungus. In thin at the central portion of the wall, and the pore then widens sections, it is found that two gametangia of different mating gradually during maturation of the zygosporangium by

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ZPW

ZW LV

*

LV N

W

LV

Figs. 21, 22. Electron micrographs of zygosporangium in Zoophagus pectosporus under germination. 21. Protoplasmic flow from the portion of the zygospore-proper in the zygospore through the germination bud (GB). Although the bud produced three vegetative hyphae, only one of which (VH) is seen in this thin section. The outwardly-curved appearance of the zygosporangial cell wall at the broken portion (arrows) shows that the zygosporangium is in germination. The protoplasm of the zygospore-proper is covered with a thin cell wall (ZPW). LV, large vesicles; ZW, zygosporangial cell wall. 22. A nucleus (N) seen in a germinating zygospore. LV, Large vesicles. Bars, 10 µm for Fig. 21; 1 µm for Fig. 22. incorporating the wall of septum into the zygosporangial cell Saikawa (2010) found the germination of zygospores in wall. Such ultrastructural features have also been reported A. lophospora Drechsler cultured on a WA plate that had been in (Ehrenb.: Fr.) Vuill. by Ho and Chen incubated for ca. 1 mo after formation of the zygospores, (1998). In addition, a tiny pore is found at the central portion and found that the protoplasmic movement of granules of a newly-made gametangial septum, in which the pore became active and was accompanied by the disappearance is not accompanied with parenthesomes but with a bridge- of the thick-wall layer beneath the zygosporangial cell like structure of electron-dense, homogeneous substance. wall. In electron micrographs of both resting (Fig. 20) and Although Ho and Chen (1998) showed the “vesiculate body” germinating (Fig. 21) zygospores of Z. pectosporus in in the cell wall of the gametangial septum in R. stolonifer, the ultrathin sections, we confirm that the morphological changes body would be a portion close to the septal pore. Both of the in Z. pectosporus zygospore exhibit quite similar to those disappearance of fusion wall and existence of the septal pore in A. lophospora. More than one nucleus had never been in the gametangial septum will be the general appearance observed in zygospores of Z. pectosporus after maturation as in electron microscopy for developing zygosporangium of mentioned by Saikawa (2011) in A. lophospora. zygomycotan fungi. Using an optical microscope, Hirotani-Akabane and

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Acknowledgment Miura, K. 1967. On Acaulopage pectospora. J. Jpn. Bot. 42: 202-206. Morikawa, C., Saikawa, M. and Barron, G. L. 1993. Fungal predators i (MS) thank Prof. Dr. Masaki Matsukawa, Department of rotifers-a comparative study of Zoophagus, Lecophagus and of Environmental Sciences, Tokyo Gakugei University for Cephaliophora. Mycol. Res. 97: 421-428. recommendation this paper to be published in the Bulletin. Saikawa, M. 2011. Ultrastructural studies on zygomycotan fungi in the Zoopagaceae and Cochlonemataceae. Mycoscience 52: 83-90. References Saikawa, M. and Goho, M. 2010. An electron microscope study on the peg-like predacious organ in Zoophagus insidians capturing Benjamin, R. K. 1959. The merosporangiferous Mucorales. Aliso 4: rotifers. Bull. Tokyo Gakugei Univ. Div. Nat. Sci., 62: 63-69. 321-433. Saikawa, M. and Kadowaki, T. 2002. Studies on Acaulopage dichotoma Dick, M. W. 1990. The systematic position of Zoophagus insidians. and A. tetraceros (, ) capturing amoebae. Mycol. Res. 94: 347-354. Nova Hedwig. 74: 365-371. Drechsler, C. 1962. A nematode-capturing phycomycete with distally Saikawa, M. and Morikawa, C. 1985. Electron microscopy on a adhesive branches and proximally imbedded fusiform conidia. nematode-trapping fungus, Acaulopage pectospora. Can. J. Bot. Amer. J. Bot. 49: 1089-1095. 63: 1386-1390. Grove, S. N., Bracker, B. C. E. and Morre, J. D. 1970. An ultrastructural Saikawa, M., Yamaguchi, K. and Morikawa, C. 1988. Capture of rotifers basis for hyphal tip growth in Pythium ultimum. Amer. J. Bot. 57: by Acaulopage pectospora, and further evidence of its similarity 245-266. to Zoophagus insidians. Mycologia 80: 880-884. Hirotani-Akabane, E. and Saikawa, M. 2010. Germination and Sommerstorff, H. 1911. Ein Tiere fangender Pilz (Zoophagus insidians, morphology of zygospores in two Cochlonema and one nov. gen., nov. sp.). Österreichische Botanische Zeitschrift 61: Acaulopage species. Mycologia 102: 39-43. 361-373. Ho, H. M. and Chen, Z. C. 1998. Ultrastructural study of wall ontogeny Whisler, H. C. and Travland, L. B. 1974. The rotifer trap of Zoophagus. during zygosporogenesis in Rhizopus stolonifer (Mucoraceae), an Arch. Microbiol. 101: 95-107. amended model. Bot. Bull. Acad. Sin. 39: 269-277.

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センチュウ捕食性接合菌Zoophagus pectosporusにおける 接合胞子の発生と発芽に関する電顕的研究

犀川 政稔・若井 由佳・勝島 尚子

環境科学分野

要 旨

Zoophagus pectosporus における接合胞子発生の超薄切片像を，ゾウパーゲ科菌類として初めて報告する。一対の配 偶子嚢が接着してできる接着壁は溶解ではなく，接着後壁の中央に生じた小孔が広がることによって消滅することが わかった。そのさい接着壁は発生中の接合胞子嚢の細胞壁に取り込まれた。一方，接合胞子嚢と新しくできた支持体 との間の配偶子嚢隔壁は中央に 1 つの小孔をもつことがわかった。また，本研究では接合胞子の発芽の電顕像が，こ の科として初めて見ることができた。発芽中の接合胞子の原形質は直径 0.5 ～ 1.0 µm の高電子密度ベシクルで満たさ れており，それは発芽管へと，そして発芽管からは数本の栄養菌糸へと連続していた。発芽中の原形質内には核が 1 つだけ見つかった。

キーワード: アカウロパーゲ，溶解，配偶子嚢接着壁，配偶子嚢，隔壁，接合胞子嚢

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