Ultrastructures ofNosema typographiWeiser 1955 (Microspora: Nosematidae) of the Bark BeetleIps typographusL. (Coleoptera; Scolytidae)

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

70, 156–160 (1997)

IN974677

NOTE Ultrastructures of Nosema typographi Weiser 1955 (Microspora: Nosematidae) of the Bark Beetle Ips typographus L. (Coleoptera; Scolytidae) The spruce bark beetle Ips typographus L. is the most important pest of spruce in Northern and Central Europe. Its outbreaks are connected with local snow and wind damage in old forest stands. During an outbreak in 1953–1954, a survey was made for pathogens and parasites of I. typographus in Czechoslovakia (Weiser, 1954, 1955). Two microsporidia infecting adults were described: Chytridiopsis typographi and Nosema typographi. At that time, electron microscopy was not available. The ultrastructure of C. typographi was described later (Purrini and Weiser, 1984, 1985). In our recent study of natural enemies affecting populations of I. typographus in Central Europe (Wegensteiner and Weiser, 1994; Weiser and Wegensteiner, 1995), we rediscovered N. typographi and here we present details of its ultrastructure. Adult I. typographus were collected during their spring attacks from trap trees in several localities in Austria, Czech Republic, and Germany. Beetles were removed from their nuptial chambers in the bark and dissected in the laboratory. The gut and parts of the fat body, the Malpighian tubules, and gonads were inspected and, when infected, were fixed in 3% glutaraldehyde in cacodylate buffer (pH 7.2). The material was postfixed in 2% OsO4 and processed for embedding in Vestopal W. Semithin sections stained in toluidine blue were used for histological evaluation. Ultrathin sections were stained with uranyl acetate and lead citrate and inspected with a Philips CX12 electron microscope. Dry smears were fixed in methanol and stained with Giemsa. Nuclei of mature spores were differentiated with 10 min hydrolysis in 1 N HCl at 70°C and restained with Giemsa. Fresh material was compared with type material which consisted of smears stained with Giemsa and histological material of N. typographi deposited in the author’s collection. It originated in collections from Horsˇovsky´ Ty´n from 1955. The new material was from the locality Ra´jec-Jestrˇebı´, Czech Republic, collected on July 6, 1995, and reared in the insectary of the Institute

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of Forest Entomology in Vienna. Beetles hatched on August 24, 1995. Another material was collected on the locality Tamsweg (Achner Kogel), Austria, in May 1995 and Flatz (near Neukirchen, South of Vienna), Austria, during May and June 1995. Additional material with the same infection was collected in the Schwarzwald mountains near Freiburg, SW Germany. This material was reared in the insectary and adults were hatched in August 1995. The type materials of N. typographi were smears of infected fat body of a male beetle. Broad oval spores 2.5 6 0.5 3 4.0 6 0.5 µm (n 5 100) were present. Some spores with extruded polar filaments were observed. The maximum length of the filament was 120 µm. Spores were larger after extrusion of the polar filament, 2.5–3 3 4.5–5 µm (n 5 12). The HCl-hydrolysis of the type material brought evidence of two round nuclei in a diplokaryotic arrangement. The material from the four new localities was identical in spore shape and size and in the site of host infections. The heaviest infections were localized in the fat body. However, the circular and longitudinal muscles of the gut and flight muscles were infected, too. Minor groups of spores were present in some parts of the midgut epithel. The infection was also present in the egg follicles and in maturing eggs in female beetles. Merogonial stages were round, 3–5 µm in diameter, with centrally located diplokarya. Sporonts formed short ribbons with two diplokarya and divided in elongated sporoblasts 3 3 5 µm. Dimensions of mature spores in all new materials were 4.5 6 0.5 3 2.2 6 0.3 µm (average of 50 measurements of spores from each locality). Meronts (Figs. 1 and 2) had diplokaryotic nuclei and have large cisternae of endoplasmic reticulum within their cytoplasm. The plasmalemma is thin, with a fine surface deposit which in some areas has tubular extensions (Figs. 1 and 2, t). Early sporonts have a surface coat characterized by interspersed lenticular thickenings of the membrane (Fig. 3, s). The surface membrane

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FIG. 1. Meronts of Nosema typographi with diplokaryon (N), large cytoplasmic vacuoles, and a thin surface coat. Tubules (t) are secreted on its surface. Bar, 1 µm. FIG. 2. Two meronts with tubular extensions of the plasmalemma (t). Nuclei (N). Bar, 1 µm. FIG. 3 Sporont (S) with lenticular thickenings of the covering membrane (s) and distinct lamellae of endoplasmic reticulum close to the nucleus (N). Bar, 1 µm. FIG. 4. Sporoblast with thickened cell wall (e) and polar sac (P) with polar filament forming. Tubules of the Golgi system (G). Bar, 1 µm. 157

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FIG. 5. Sporoblast during formation of the polar filament inside the coiled polar sac (P). In some sections two filament cross sections are in the sac (arrowhead). The cell wall (e) is more condensed. Nuclei not in adhesion (N). Bar, 0.5 µm. FIG. 6. Anterior end of a mature spore of N. typographi. The nucleus (N) is closed in the ribosomal basket (r). The polaroplast is composed of parallel fine lamellae (T) in both parts. The ending of the polar filament (P) is thickened pestle-like and inserted in the anchoring disc (a)

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of maturing sporoblasts (Fig. 4) has a thin episporal membrane with a finely granulated internal layer. The polar filament (Fig. 4, P) is formed in a voluminous polar sac which is deposed in one broad turn close to the wall. Maturing sporoblasts (Fig. 5) are typical ‘‘shrunken’’ stages. Their exospore (e) has a distinct smooth episporal membrane, the granular layer is reduced, and the polar filament is formed in the polarsac coiled in this stage in 11–12 turns, some presenting two cross sections of the formed polar filament (Fig. 5). The filament is formed in the whole sac at the same time in all its length. Mature spores have a distinct rigid electron-lucent endospore wall (Figs. 6 and 7, E). This is attenuated at the anterior pole where the anchoring disc is attached to the endospore. The exospore of the mature spore is of equal thickness, formed of finely granulated electrondense material, and is bound by a thin episporal membrane (Fig. 7). The nuclei are diplokaryotic and are surrounded by an array of polyribosomes. The polaroplast (Fig. 6, T) is lamellar in both parts. The polar filament is fixed in a bulbous anchoring disc (Fig. 7, a), with a thin peripheral umbrellar part which covers the proximal area of the polaroplast. Another layer extends from the anchoring disc, covers the polar filament, and proceeds along the filament as its covering layer (Fig. 6). The bulbous end of the polar filament is thickened in a short cylindric structure with a distinct clear zone communicating with the translucent layer of the polar filament. The mature polar filament is coiled (Fig. 7) in 16/17 turns in one single row and is isofilar, with two last turns with postponed maturation. The Golgi system is present as a coil of tubules in the posterior part of the spore. Some spores in section preparations are empty (Fig. 8), with granular remains inside, without visible polar opening and without deformation of exospore and endospore walls. The type material to old descriptions of N. typographi is nearly identical in size and shape to fresh spores obtained from new localities; the measurements indicated in the 1955 publication included young spores and spores after extrusion of the polar filament which are larger. Mature spores are single and not enclosed in any parasite- or host-produced membrane. Measurements of the length of the polar filament in the type

material up to 120 µm correspond with 14–20 turns, if the inner space for the coil is calculated. The tubular secretions on the outer membrane of the meront and the lenticular thickenings of the wall in sporonts are analogous to such structures in other typical Nosema spp. (N. algerae; Va´vra and Undeen, 1970). This analogy includes the lamellar structure of the polaroplast. The polar sac is most typical during the early period of formation of the polar filament. During spore maturation it is reduced to a system of thin membranes including the outer (umbrella-like) cover of the polaroplast and its lining of the polar filament, as indicated by Va´vra (1976). The anchoring part of the polar filament and the organization of the lamellar parts of the polaroplast also conform with those of other Nosema spp. Empty spores in many microsporidia are the primary spores, serving for the internal spread of infection inside the primary host. In N. typographi they differ slightly in size, if this is not just the result of release of the spore content. With the extrusion of the filament, the spore wall remains regular and rather rigid, compared with deformations of spores during the process of maturation. The information on ultrastructures of N. typographi broadens the original description and does not cause any conflicts among later described microsporidia from bark beetles of conifers. The only other Nosema, N. calcarati from Pityogenes calcaratus (Purrini and Halperin, 1982), has broad oval spores and does not infect the fat body of its host. KEY WORDS: Nosema typographi; bark beetles; Ips typographus. This research was supported by Grant GZ 45.244/2-46a/92 of the Austrian Ministry of Science and Research. REFERENCES Purrini, K., and Halperin, J. 1982. Z. Angew. Entomol. 94, 87–92. Purrini, K., and Weiser, J. 1984. Zool. Anzeiger 212, 369–376. Purrini, K., and Weiser, J. 1985. J. Invertebr. Pathol. 45, 66–74. Va´vra, J. 1976. In ‘‘Comparative Pathobiology’’ (L. A. Bulla and T. C. Cheng, Eds.), Vol. I, pp. 1–86. Va´vra, J., and Undeen, A. H. 1970. J. Protozool. 17, 240–249. Wegensteiner, R., and Weiser, J. 1994. J. Invertebr. Pathol. 65, 203–205. Weiser, J. 1954. Acta Czech. Zool. Soc. 18, 217–224.

with a bulbous center and thin lateral umbrella (u). The endospore (E) is thick, attenuated on the anterior pole, and the exospore (e) is formed of granular electron-dense material, without the epispore membrane. The anterior part of the internal membrane covering the polar filament is visible (arrowhead). Bar, 0.5 µm. FIG. 7. Longitudinal section of a mature spore with a rigid endospore (E) attenuated at the anterior pole. Exospore (e) of uniform thickness, closed in the epispore membrane (m); nuclei (N) in adhesion in the center; polar filament (P) with pestle-formed ending in the anchoring disc (a), coiled in 16/17 coils; Golgi tubules coiled in the posterior end (G). Bar, 1 µm. FIG. 8. Empty spore after release of the germ, with rigid exospore (e) and an endospore (E). Remains of the propulsing system of the polaroplast (T) are in its interior. Bar, 0.5 µm.

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Weiser, J. 1955. Acta Czech. Zool. Soc. 19, 374–380. Weiser, J., and Wegensteiner, R. 1995. Z. Angew. Zool. 80, 425–434.

JAROSLAV WEISER* RUDOLF WEGENSTEINER† ZDENEˇ K ZˇIZˇ KA‡ *Institute of Entomology Czech Academy of Sciences ˇ eske´ Budeˇjovice, Czech Republic Branisˇovska´ 31 37005 C

†Institute of Forest Entomology Forest Pathology and Forest Protection, BOKU Hasenauerstrasse 38 A-1190 Vienna, Austria ‡Institute of Microbiology Czech Academy of Sciences Vı´denˇska´ 1083 140 00 Praha 4, Czech Republic Received June 5, 1996; accepted March 26, 1997