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Electron microscope study of an intranuclear protozoan parasite in archaeocytes of the fresh water sponge Ephydatia fluviatilis L. (Porifera)
Arch. Protistenkd. 137 (1989): 247-253 VEB Gustav Fischer Verlag lena
Zoologisches Institut der Universitat Bonn, BRD
Electron Microscope Study of an Intranuclear Protozoan Parasite in Archaeocytes of the Fresh Water Sponge Ephydatia fluviatilis L. (Porifera) By NORBERT WEISSENFELS,
ERICH SCHOLTYSECK
& ROLF ENTZEROTH
With 9 Figures
Summary An intranuclear protozoan parasite in archaeocytes of the fresh water sponge Ephydatia fluviatilis is described. The parasites only attack the archaeocytes of this spongillid, invade their cytoplasm and later enter the nucleus. In the further development the parasite grows and undergoes nuclear divisions leading to a plasmodium with 30-40 nuclei. The parasite gradually displaces the host cell nucleus, which eventually becomes restricted to a small peripheral band surrounding the growing parasite. The following developmental phase may be considered as "formation of spores" or "sporulation". The body of the parasite ("sporont") undergoes fragmentation into lobes containing one to several nuclei, leading to the formation of spore-like structures ("spores"). Differentiated "spores" are limited by two membranes and contain a small nucleus, endoplasmic reticulum, and mitochondria-like structures. The completely differentiated spores invade new archaeocytes. The parasite kills the sponge about three months after invasion.
Introduction During studies of sponge contraction of Ephydatia fluviatilis (WEISSENFELS 1984) in the fall of 1984 unexpected disturbances in the growth of the sponge occulTed. In order to find out what happened, the material was examined microscopically. During this study, surprisingly, we found an intranuclear eucaryotic parasite in sponge archaeocytes. The light and electron microscope studies allowed the description of a nearly continuous sequence of the developmental stages of the parasite, which will be presented in this publication.
Material and Methods Gemmules of sponges collected from the river Sieg (tributary of the Rhine) on Nov. 9th 1982 were stored in the refrigerator. Two years later groups of five gemmules hatched in polypropylene dishes followed by cultivation in a well-aerated aquarium at 16°C (WEISSENFELS 1984). Two months old sponges were fixed in J % OS04 + 1 % K2CrZ07 in 0.02 M cacodylate buffer. Subsequent preparations including embedding in styrol-methacrylate have been previously described (WEISSENFELS 1982). Semi- and ultrathin sections were made with an LKB Ultratome III and observed with a Zeiss EM9 S-2. Phasecontrast microscopy was carried out with a Leitz-Dialux microscope fitled with a Wild photoautomatic apparatus (PMPS 5l/45).
Results In the light microscope the cultured two months old fresh water sponges showed an unusual appearance. There was an abnormal increase of cells with granules (granulocytes). Higher magnifications of archaeocytes showed (Figs. I, 2, 3), that the nuclei in many cases are swollen and contain inclusions not normally present. These inclusions are located in a membrane-bound area which for the most part occupies the nucleus of the host cell. Compared with uninfected sponges, these inclusions are considered to be intranuclear parasites with a more dense envelope
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et al.
Figs. [-3. Light micrographs of developed stages of an intranuclear parasite in archaeocytes of EphydatiaJluviatilis. Phase-contrast, semithin methacrylate sections. Fig. I. Section through a parasite (P) which has partly displaced the host cell (H) nucleus (NH) to a peripheral band. The parasite cytoplasm contains nuclei (-» with nucleoli. Fig. 2. Section through a parasitic stage (P) with a lobe shaped cytoplasm (.-+) which contains nuclei. H == host cell. Fig. 3. Section through parasitized (P) archaeocytes (H) containing numerous "spores" (--+-) inside a capsule (SC).
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Fig. 4. Electron micrograph of an archaeocyte (H) harbouring in the nucleus (NH) a parasite (P). The parasite contains a nucleus (NP) and endoplasmic reticulum (ER) beside other cytoplasmic structures, and is in close contact with the host cell nucleolus (Nu). D = dictyosome. Fig. 5. Section through a binucleated (NP) intranuclear parasite (P) within a host cell (H) with small granules (G) and dietyo somes (D). Note the chromatin plaques (CP) at the periphery of the parasite's nucleus. NH = host cell nucleus.
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et al.
than the nuclear envelope of the host cell. After thorough analysis of the different stages a progressive developmental sequence could be established beginning with parasites containing several nuclei (Fig. 1, arrow) in their cytoplasm. The parasite is surrounded by a small band of the host cell nucleus (NH). The further development is characterized by cytoplasm a condensation followed by fragmentation into subunits (Fig. 2, arrow). The irregular shaped subunits in Fig. 2 are considered as an intermediate stage that finally leads to numerous ovoid stages containing dark bodies ("spores") as shown in Fig. 3 (arrows). Electron micrographs show that only archaeocytes act as host cells (H) which harbour the eucaryotic parasite in the nucleus (NH) (Fig. 4). The nucleus of the parasite (NP) has a typical two-membranous envelope. The endoplasmic reticulum (ER) is well developed but typical mitochondria could not be found. Sometimes the intranuclear parasite (P) can be found in direct contact to the nucleolus (Nu) of the host cell (Fig. 4). The parasitized archaeocyte has well developed dictyosomes (D). A section through a parasite containing two nuclei (NP) with chromatin plaques (CP) at the periphery is shown in Fig. 5. The parasite (P) is located within the nucleus of the host cell (NH). The host cell cytoplasm is characterized by an increased golgi activity shown on electron micrographs by the presence of numerous dictyosomes (D) (Fig. 5). Secretory granules (G) of different sizes are found in the cytoplasm of the host cell (H). Without demonstrating all following developmental stages (it is presumed that the parasite nucleus undergoes caryokinesis), Fig. 6 shows a multinucleated parasite (P) with 18 nuclei (NP) present in the section plane. The cytoplasm of the parasite is highly differentiated. The parasite (P) is separated from the host cell nucleus (NH) by a prominent thick layer of electron dense material. The cytoplasm of the host cell in the vicinity of the nucleus contains dictyosomes (D). Further development of the parasite is characterized by a change in the structural appearance of the parasite caused by the formation of lobes producing portions of cytoplasm which contain one to several nuclei (NP) interconnected by small cytoplasmic bridges (arrows in Fig. 7). The host cell nucleus (NH) is present in the form of a small band. A number of active dictyosomes (D) situated close to the parasitized nucleus and various granules (G) are present in the host cell (H). The development finally leads to the formation of numerous mononucleated ovoid shaped bodies which resemble "spores" (S) and measure about 2.0- 2.5 Ilm in the larger extension (Fig. 8). The ribosome-rich cytoplasm of these "spores" is characterized by electron dense structures (arrows in Fig. 8). Stages with "spores" can be found in sponges beginning two months after infection. Three months after starting the sponge cultures (Fig. 9) single "spores" (S) could be observed extracellularly and also in the cytoplasm of newly infected host cells (H) between the cell membrane (CM) and the host cell nucleus (NH). It was only in this stage that in spores mitochondria-like structures (M) could be demonstrated. Three months after primary infection the sponges stopped growing and began to degenerate.
Discussion This is the first report of a protozoan parasite in fresh water sponges. The parasites are pathogenic which was shown by the fact that all of the sponge cultures infected with the organism died. WEISSENFELS (1984) has repeatedly studied development of Ephydatia Jluviatilis without
Fig. 6. Section through the central part of a multinucleated (NP) parasite (P) with well structurated cytoplasm containing numerous vesicles (V). NH = host cell nucleus; D = Dictyosome. Fig. 7. Section through a parasite (P) within a host cell (H) nucleus (NH). The cytoplasm of the parasite underwent plasmotomy and shrunk to small portions connected by plasmabridges (-....-). The areas contain one to several nuclei (NP). D = dictyosome; G = granules.
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Fig. 8. Section through a central part of a host cell (H) nucleus (NH) containing numerous "spores" (S) within a spore capsule (SC). The "spores" contain a nucleus (NP), numerous ribosomes, endoplasmic reticulum and several threadlike electron dense structures (-). Note the host cell cytoplasm with membrane-bound electron dense granules (G). Fig. 9. The peripheral part of an archaeocyte (H) infected by a "spore" (S) which is not located in a parasitophorous vacuole. NH = host cell nucleus; M = mitochondrium-like structure of the parasite; CM = cell membrane of the host cell.
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detecting symbiotic or parasitic organisms within the sponge cells. Diseases of commercial and noncommercial marine sponges have been frequently recorded, but the causative agent of some of these diseases were fungi, bacteria and viruses (V ACE LET & GALLISSIAN 1978). The described parasite here is located within the nucleus of archaeocytes and is characterized as an eucaryote by a well defined nucleus limited by a nuclear envelope. The parasite can be clearly differentiated from bacteria which are common in demosponges and calcareous sponges and from symbiotic Cyanophyceae (VACELET & DONADEY 1977). The parasite apparently enters the host cell via the cytoplasm. It could not be determined whether cell entry occurs actively or passively by phagocytosis, the latter is more likely because archaeocytes are known to phagocytize. It is not known how the parasites become located within the host cell nucleus. No evidence for structures like endospore, exospore, polar filament, polaroplast were found, separating the here described organism from typical microspora (LEVINE et al. 1980). VAVRA, to whom we have send original electron micrographs, cannot precisely identify the organism, but his feeling is, that it might be a microsporidian of the primitive type close to the genus Metchnikovella (HILDEBRAND & VIVIER 1971; VIVIER & SCHREVEL 1973), which typically occurs in gregarines.
Zusammenfassung Die Parasiten wurden in den Archiiocyten von Ephydatia fluviatilis vorgefunden. Sie dringen in ihr Cytoplasma, spiiter in ihren Kern ein, wachsen heran und machen Kernteilungen durch, die zu einem Plasmodium mit 30 bis 40 Zellkernen fiihren. Hierbei verdriingen die Parasiten den Innenraum des Wirtszellkerns bis auf einen schmalen, peripheren Bereich rings urn den Parasiten. Die anschlieBende Entwicklungsphase kann als Sporenbildung (Sporulation) bezeichnet werden. Hierbei fiihrt die Fragmentierung des Parasiten-Zellkiirpers zu "Sporen". Ausdifferenzierte "Sporen" weisen 2 Membranen auf und enthalten einen kleinen Zellkern, ein endoplasmatisches Retikulum und mitochondrien-iihnliche Strukturen. Fertige Sporen dringen erneut in Archiiocyten ein. Die Parasiten tiiten den Schwamm etwa 3 Monate nach Befall.
Acknowledgements Dedicated in thankfulness to Prof. Dr. Dr. h. c. ERICH SCHOLTYSECK who passed away on the 18th of April 1985 before completion of the work. We thank Dr. N. D. LEVINE (University of Illinois at Urbana-Champaing, U.S.A.), Dr. 1. VAVRA (Charles University, Prague, Czechoslovakia) and Prof. Dr. H. M. SEITZ (lnstitut flir Medizinische Parasitologie der Universitiit Bonn, F.R.G.) for their helpful suggestions concerning the systematic position of the described parasite; Dr. P. AUGUSTINE and Dr. B. CHOBOTAR for reviewing and M. NGUYEN for typing the manuscript. The work was supported by funds of the German Research Council (DFG). We thank Mrs. M. GElS, Mrs. U. MOLLER and Mrs. 1. NOsSLE for technical assistance.
Literature HILDEBRAND, H., & VIVIER, E. (1971): Observations ultrastructurales sur Ie sporoblaste de Metchnikovella wohlfarth! n. sp. Protistologica 7: 13 [-\39. LEVINE, N. D., CORLISS, 1.0., Cox, F. E. G., DEROUX, G., GRAIN, 1., HONIGBERG. B. M., LEEDALE, F. c., POLJANSKY, G., SPRAGUE. V., VAVRA, 1., & WALLACE, F. G. (1980): A new revised classification of the protozoa. J. Protozool. 27: 37-58. VACELET, 1., & DONADEY, C. (1977): Electron microscope study of the association between some sponges and bacteria. 1. Exp. Mar. BioI. Eeal. 30: 30\-314. - & GALLISSIAN, M.-l. (1978): Virus-like particles in cells of the sponge Verongia cavernicola (Demospongiae, Dictyoceratida) and accomanying tissue changes. 1. Invert. Pathol. 31: 246- 254. VIVIER, E., & SCHREVEL, 1. (1973): Etudes en microscopie photonique et I'electronique de differentes stades du cycle de Metchnikovella hovasse! et observations sur la position systematique des Metchnikovellidae. Protistologica9: 95-118.
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WEISSENFELS, N. (1982): Bau und Funktion des SiiBwasserschwamms Ephydatia fluviatilis L. (Porifera) IX. Rasterelektronenmikroskopische Histologie und Cytologie. Zoomorphology 100: 75-87. (1984): Bau und Funktion des SiiBwasserschwamms Ephydatia fluviatiUs L. (Porifera) XI. Nachweis eines endogenen Kontraktionsrhythmus durch Infrarot-Retlexion. Zoomorphology 104: 292-297. (1985): An intranuclear protozoan Parasite in archaeocytes of the fresh water sponge Ephydatia fluviatilis L. (Porifera). Third International Conference on the Biology of Sponges. Woods Hole. Authors's address: Prof. Dr. NORBERT WEISSENFELS, Zoologisches Institut der Universitat Bonn, Entwicklungsgeschichtliche Abteilung, Poppelsdorfer SchloB, D-5300 Bonn I.
Zoologisches Institut der Universitat Bonn, BRD
Electron Microscope Study of an Intranuclear Protozoan Parasite in Archaeocytes of the Fresh Water Sponge Ephydatia fluviatilis L. (Porifera) By NORBERT WEISSENFELS,
ERICH SCHOLTYSECK
& ROLF ENTZEROTH
With 9 Figures
Summary An intranuclear protozoan parasite in archaeocytes of the fresh water sponge Ephydatia fluviatilis is described. The parasites only attack the archaeocytes of this spongillid, invade their cytoplasm and later enter the nucleus. In the further development the parasite grows and undergoes nuclear divisions leading to a plasmodium with 30-40 nuclei. The parasite gradually displaces the host cell nucleus, which eventually becomes restricted to a small peripheral band surrounding the growing parasite. The following developmental phase may be considered as "formation of spores" or "sporulation". The body of the parasite ("sporont") undergoes fragmentation into lobes containing one to several nuclei, leading to the formation of spore-like structures ("spores"). Differentiated "spores" are limited by two membranes and contain a small nucleus, endoplasmic reticulum, and mitochondria-like structures. The completely differentiated spores invade new archaeocytes. The parasite kills the sponge about three months after invasion.
Introduction During studies of sponge contraction of Ephydatia fluviatilis (WEISSENFELS 1984) in the fall of 1984 unexpected disturbances in the growth of the sponge occulTed. In order to find out what happened, the material was examined microscopically. During this study, surprisingly, we found an intranuclear eucaryotic parasite in sponge archaeocytes. The light and electron microscope studies allowed the description of a nearly continuous sequence of the developmental stages of the parasite, which will be presented in this publication.
Material and Methods Gemmules of sponges collected from the river Sieg (tributary of the Rhine) on Nov. 9th 1982 were stored in the refrigerator. Two years later groups of five gemmules hatched in polypropylene dishes followed by cultivation in a well-aerated aquarium at 16°C (WEISSENFELS 1984). Two months old sponges were fixed in J % OS04 + 1 % K2CrZ07 in 0.02 M cacodylate buffer. Subsequent preparations including embedding in styrol-methacrylate have been previously described (WEISSENFELS 1982). Semi- and ultrathin sections were made with an LKB Ultratome III and observed with a Zeiss EM9 S-2. Phasecontrast microscopy was carried out with a Leitz-Dialux microscope fitled with a Wild photoautomatic apparatus (PMPS 5l/45).
Results In the light microscope the cultured two months old fresh water sponges showed an unusual appearance. There was an abnormal increase of cells with granules (granulocytes). Higher magnifications of archaeocytes showed (Figs. I, 2, 3), that the nuclei in many cases are swollen and contain inclusions not normally present. These inclusions are located in a membrane-bound area which for the most part occupies the nucleus of the host cell. Compared with uninfected sponges, these inclusions are considered to be intranuclear parasites with a more dense envelope
248
N.
WEISSENFELS
et al.
Figs. [-3. Light micrographs of developed stages of an intranuclear parasite in archaeocytes of EphydatiaJluviatilis. Phase-contrast, semithin methacrylate sections. Fig. I. Section through a parasite (P) which has partly displaced the host cell (H) nucleus (NH) to a peripheral band. The parasite cytoplasm contains nuclei (-» with nucleoli. Fig. 2. Section through a parasitic stage (P) with a lobe shaped cytoplasm (.-+) which contains nuclei. H == host cell. Fig. 3. Section through parasitized (P) archaeocytes (H) containing numerous "spores" (--+-) inside a capsule (SC).
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Fig. 4. Electron micrograph of an archaeocyte (H) harbouring in the nucleus (NH) a parasite (P). The parasite contains a nucleus (NP) and endoplasmic reticulum (ER) beside other cytoplasmic structures, and is in close contact with the host cell nucleolus (Nu). D = dictyosome. Fig. 5. Section through a binucleated (NP) intranuclear parasite (P) within a host cell (H) with small granules (G) and dietyo somes (D). Note the chromatin plaques (CP) at the periphery of the parasite's nucleus. NH = host cell nucleus.
250
N.
WEISSENFELS
et al.
than the nuclear envelope of the host cell. After thorough analysis of the different stages a progressive developmental sequence could be established beginning with parasites containing several nuclei (Fig. 1, arrow) in their cytoplasm. The parasite is surrounded by a small band of the host cell nucleus (NH). The further development is characterized by cytoplasm a condensation followed by fragmentation into subunits (Fig. 2, arrow). The irregular shaped subunits in Fig. 2 are considered as an intermediate stage that finally leads to numerous ovoid stages containing dark bodies ("spores") as shown in Fig. 3 (arrows). Electron micrographs show that only archaeocytes act as host cells (H) which harbour the eucaryotic parasite in the nucleus (NH) (Fig. 4). The nucleus of the parasite (NP) has a typical two-membranous envelope. The endoplasmic reticulum (ER) is well developed but typical mitochondria could not be found. Sometimes the intranuclear parasite (P) can be found in direct contact to the nucleolus (Nu) of the host cell (Fig. 4). The parasitized archaeocyte has well developed dictyosomes (D). A section through a parasite containing two nuclei (NP) with chromatin plaques (CP) at the periphery is shown in Fig. 5. The parasite (P) is located within the nucleus of the host cell (NH). The host cell cytoplasm is characterized by an increased golgi activity shown on electron micrographs by the presence of numerous dictyosomes (D) (Fig. 5). Secretory granules (G) of different sizes are found in the cytoplasm of the host cell (H). Without demonstrating all following developmental stages (it is presumed that the parasite nucleus undergoes caryokinesis), Fig. 6 shows a multinucleated parasite (P) with 18 nuclei (NP) present in the section plane. The cytoplasm of the parasite is highly differentiated. The parasite (P) is separated from the host cell nucleus (NH) by a prominent thick layer of electron dense material. The cytoplasm of the host cell in the vicinity of the nucleus contains dictyosomes (D). Further development of the parasite is characterized by a change in the structural appearance of the parasite caused by the formation of lobes producing portions of cytoplasm which contain one to several nuclei (NP) interconnected by small cytoplasmic bridges (arrows in Fig. 7). The host cell nucleus (NH) is present in the form of a small band. A number of active dictyosomes (D) situated close to the parasitized nucleus and various granules (G) are present in the host cell (H). The development finally leads to the formation of numerous mononucleated ovoid shaped bodies which resemble "spores" (S) and measure about 2.0- 2.5 Ilm in the larger extension (Fig. 8). The ribosome-rich cytoplasm of these "spores" is characterized by electron dense structures (arrows in Fig. 8). Stages with "spores" can be found in sponges beginning two months after infection. Three months after starting the sponge cultures (Fig. 9) single "spores" (S) could be observed extracellularly and also in the cytoplasm of newly infected host cells (H) between the cell membrane (CM) and the host cell nucleus (NH). It was only in this stage that in spores mitochondria-like structures (M) could be demonstrated. Three months after primary infection the sponges stopped growing and began to degenerate.
Discussion This is the first report of a protozoan parasite in fresh water sponges. The parasites are pathogenic which was shown by the fact that all of the sponge cultures infected with the organism died. WEISSENFELS (1984) has repeatedly studied development of Ephydatia Jluviatilis without
Fig. 6. Section through the central part of a multinucleated (NP) parasite (P) with well structurated cytoplasm containing numerous vesicles (V). NH = host cell nucleus; D = Dictyosome. Fig. 7. Section through a parasite (P) within a host cell (H) nucleus (NH). The cytoplasm of the parasite underwent plasmotomy and shrunk to small portions connected by plasmabridges (-....-). The areas contain one to several nuclei (NP). D = dictyosome; G = granules.
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Fig. 8. Section through a central part of a host cell (H) nucleus (NH) containing numerous "spores" (S) within a spore capsule (SC). The "spores" contain a nucleus (NP), numerous ribosomes, endoplasmic reticulum and several threadlike electron dense structures (-). Note the host cell cytoplasm with membrane-bound electron dense granules (G). Fig. 9. The peripheral part of an archaeocyte (H) infected by a "spore" (S) which is not located in a parasitophorous vacuole. NH = host cell nucleus; M = mitochondrium-like structure of the parasite; CM = cell membrane of the host cell.
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detecting symbiotic or parasitic organisms within the sponge cells. Diseases of commercial and noncommercial marine sponges have been frequently recorded, but the causative agent of some of these diseases were fungi, bacteria and viruses (V ACE LET & GALLISSIAN 1978). The described parasite here is located within the nucleus of archaeocytes and is characterized as an eucaryote by a well defined nucleus limited by a nuclear envelope. The parasite can be clearly differentiated from bacteria which are common in demosponges and calcareous sponges and from symbiotic Cyanophyceae (VACELET & DONADEY 1977). The parasite apparently enters the host cell via the cytoplasm. It could not be determined whether cell entry occurs actively or passively by phagocytosis, the latter is more likely because archaeocytes are known to phagocytize. It is not known how the parasites become located within the host cell nucleus. No evidence for structures like endospore, exospore, polar filament, polaroplast were found, separating the here described organism from typical microspora (LEVINE et al. 1980). VAVRA, to whom we have send original electron micrographs, cannot precisely identify the organism, but his feeling is, that it might be a microsporidian of the primitive type close to the genus Metchnikovella (HILDEBRAND & VIVIER 1971; VIVIER & SCHREVEL 1973), which typically occurs in gregarines.
Zusammenfassung Die Parasiten wurden in den Archiiocyten von Ephydatia fluviatilis vorgefunden. Sie dringen in ihr Cytoplasma, spiiter in ihren Kern ein, wachsen heran und machen Kernteilungen durch, die zu einem Plasmodium mit 30 bis 40 Zellkernen fiihren. Hierbei verdriingen die Parasiten den Innenraum des Wirtszellkerns bis auf einen schmalen, peripheren Bereich rings urn den Parasiten. Die anschlieBende Entwicklungsphase kann als Sporenbildung (Sporulation) bezeichnet werden. Hierbei fiihrt die Fragmentierung des Parasiten-Zellkiirpers zu "Sporen". Ausdifferenzierte "Sporen" weisen 2 Membranen auf und enthalten einen kleinen Zellkern, ein endoplasmatisches Retikulum und mitochondrien-iihnliche Strukturen. Fertige Sporen dringen erneut in Archiiocyten ein. Die Parasiten tiiten den Schwamm etwa 3 Monate nach Befall.
Acknowledgements Dedicated in thankfulness to Prof. Dr. Dr. h. c. ERICH SCHOLTYSECK who passed away on the 18th of April 1985 before completion of the work. We thank Dr. N. D. LEVINE (University of Illinois at Urbana-Champaing, U.S.A.), Dr. 1. VAVRA (Charles University, Prague, Czechoslovakia) and Prof. Dr. H. M. SEITZ (lnstitut flir Medizinische Parasitologie der Universitiit Bonn, F.R.G.) for their helpful suggestions concerning the systematic position of the described parasite; Dr. P. AUGUSTINE and Dr. B. CHOBOTAR for reviewing and M. NGUYEN for typing the manuscript. The work was supported by funds of the German Research Council (DFG). We thank Mrs. M. GElS, Mrs. U. MOLLER and Mrs. 1. NOsSLE for technical assistance.
Literature HILDEBRAND, H., & VIVIER, E. (1971): Observations ultrastructurales sur Ie sporoblaste de Metchnikovella wohlfarth! n. sp. Protistologica 7: 13 [-\39. LEVINE, N. D., CORLISS, 1.0., Cox, F. E. G., DEROUX, G., GRAIN, 1., HONIGBERG. B. M., LEEDALE, F. c., POLJANSKY, G., SPRAGUE. V., VAVRA, 1., & WALLACE, F. G. (1980): A new revised classification of the protozoa. J. Protozool. 27: 37-58. VACELET, 1., & DONADEY, C. (1977): Electron microscope study of the association between some sponges and bacteria. 1. Exp. Mar. BioI. Eeal. 30: 30\-314. - & GALLISSIAN, M.-l. (1978): Virus-like particles in cells of the sponge Verongia cavernicola (Demospongiae, Dictyoceratida) and accomanying tissue changes. 1. Invert. Pathol. 31: 246- 254. VIVIER, E., & SCHREVEL, 1. (1973): Etudes en microscopie photonique et I'electronique de differentes stades du cycle de Metchnikovella hovasse! et observations sur la position systematique des Metchnikovellidae. Protistologica9: 95-118.
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WEISSENFELS, N. (1982): Bau und Funktion des SiiBwasserschwamms Ephydatia fluviatilis L. (Porifera) IX. Rasterelektronenmikroskopische Histologie und Cytologie. Zoomorphology 100: 75-87. (1984): Bau und Funktion des SiiBwasserschwamms Ephydatia fluviatiUs L. (Porifera) XI. Nachweis eines endogenen Kontraktionsrhythmus durch Infrarot-Retlexion. Zoomorphology 104: 292-297. (1985): An intranuclear protozoan Parasite in archaeocytes of the fresh water sponge Ephydatia fluviatilis L. (Porifera). Third International Conference on the Biology of Sponges. Woods Hole. Authors's address: Prof. Dr. NORBERT WEISSENFELS, Zoologisches Institut der Universitat Bonn, Entwicklungsgeschichtliche Abteilung, Poppelsdorfer SchloB, D-5300 Bonn I.