Electron microscope studies on the development and Title germination of zygospores in Zoophagus pectosporus, a zoopagaceous fungus capturing nematodes( 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 Zoopagaceae. 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 cell wall 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: Acaulopage, 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) － 91 － Bulletin of Tokyo Gakugei University, Division of Natural Sciences, Vol. 63 (2011) SAIKAWA et al.: Zoophagus pectosporus Zoophagus pectosporus (Drechsler) M.W. Dick, capturing Materials and Methods nematodes and rotifers, was once named as Acaulopage pectospora Drechsler. Although all species in the genus 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 mold 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 hypha 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 rotifer 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 mycelium 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 nematode 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 － 92 － Bulletin of Tokyo Gakugei University, Division of Natural Sciences, Vol. 63 (2011) SAIKAWA et al.: Zoophagus pectosporus * Ad * * * ** * * ** * Figs. 1-5. Light micrographs of Zoophagus pectosporus. 1. Conidium 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 － 93 － Bulletin of Tokyo Gakugei University, Division of Natural Sciences, Vol. 63 (2011) SAIKAWA et al.: Zoophagus pectosporus * * * * * 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.