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Multiplication of Tipula and Chilo iridescent viruses in cells of Antheraea eucalypti
NOTES
278
per spore, monospore cultures of them should have a minimum of variability. Monospore cultures were prepared as follows: Small circles (diam i 2 mm) were engraved on the bottom lid of petri dishes with the aid of a Leitz marker objective
(Type MAKKI, photograph 3). Sterile, filtered, water agar was poured into warm, sterile petri dishes to give a very thin film of agar. The center of the circle was marked with an ink dot on the underside of the petri dish. Small droplets containing, on the average, one Metarrhizium-spore were delivered on the agar above the ink dots by means of a sterile Pasteur pipette. The dots were then removed and the number of spores in each circle mounted under phase contrast with a magnification of 16x (objective), 8X (ocular). The field corresponded with the field of the engraved circles. Circles with only one spore were marked again with an ink dot to aid in their transfer to Sabouraud maltose agar with a sterile loop. K. H. VEEN Agricultural University Department of Entomology Wageningen, The Netherlands
FIG. n. Device dish with engraved
for monospore circles. Photo
Multiplication
cultures; petri by G. Eimers.
Received March 9, 1967
of
Tipula
and
Cells
of
Antheraea
The continuous line of Antheraea eucdypti ovarian cells which was established by T. D. C. Grace (Nature, 195, 788-789, 1962) is susceptible to infection by Sericesthb iridescent virus (A. J. D. Bellett and E. H. Mercer, Virology, 24, 645-653, 1964). This note reports the successful infection of this cell line with Tip&a iridescent virus (TIV), and with Chile iridescent virus (CIV) described by M. Fukaya and S. Nasu (Appl. Entomol. Zool., 1, 69-72, 1966). The suspensionsof the two viruses were prepared as follows: Larvae of the greater Gallerk mellonella, which had wax moth,
Chile
Iridescent
Viruses
in
eucalypti
been infected with either TIV or CIV, were surface-sterilized with 1% hyamine. Their abdominal legs were clipped and the oozing hemolymph, containing fragments of fat-body tissue, was collected in capillary tubes. The hemolymph was added to a modification of the Grace’s culture medium described previously (T. Hukuhara, J. Sericult. Sci. Japan, 35, 349-354, 1966). The cell cultures were incubated overnight with the virus suspension (1.5-2.0 ml) at 20-21°C. After this period the cultures were supplied with fresh culture medium. For light- and electron-microscope examinations the TIV-infected cells were fixed
NOTES
279
FIG. 1. Antheraea eucalypti cells fixed 21 days after inoculation with TIV. A. Light micrograph of infected cells showing intracytoplasmic inclusions. The arrow points to one of them. 1100 X. B. Electron rnicrograph of an ultrathin section of an infected cell showing groups of virus particles in the cytoplasm. Uranyl acetate stain. 10,000 X. C. Infected cell similar to B at a higher magnification. 40,000 >;. S, nucleus: P51, plasma membrane; V, virus particle.
280
NOTES
FIG. 2. Electron micrographs of ultrathin sections of Anthernea eucalypti cells fixed 6 days after inoculation with CIV. Uranyl acetate and lead nitrate stain. A. Infected cell showing the virus particles in the cytoplasm and protrusions of the plasma membrane. 10,000 X. B. Spherical body filled with large numbers of virus particles which is presumed to be an infected cell or its debris. 10,000 x. C. Infected cell showing protrusions in various stages of their development. 20,000 X. E, extracellular body; N, nucleus; PM, plasma membrane; V, virus particle.
281
NOTES
WI the 21st day after inoculation, and the CIV-infected cells on the 6th day in the following manner: The cells adhering to the coverslips in the culture vessels were fixed with Bouin’s fluid and stained with Harris hematoxylin; the cells, free-floating in the medium, were centrifuged for 15 minutes at 1500 rev/min, fixed with glutaraldehyde-OsOq, embedded in Epon. and sectioned. Light-microscope studies of the cells from TIV- and CIV-infected cultures showed that most of them contained, in their cytoplasm, one to several spherical inclusions, which stained intensely with hematoxylin, and were surrounded by a clear halo (Fig. 1A). Such bodies were not found in the cells from uninfected cultures. Ultrathin sections of TIV- and (XV-infected cells revealed the presence of virus particles in the cytoplasm but never in the nucleus ( Fig. 1B and Fig. 2A). These virus particles are identical in appearance with those noted in infected cells in susceptible insects. Sections of CIV-infected cells showed a number of protrusions of the plasma membrane, varying in size (Fig. 2A, C). Some appeared as a light bulging of the mcmhrane where a single virus particle
Mycotoxin
Production
by
\I>-cotosins from entomogenous fungi Ilnve been demonstrated and investigated hv marry workrrs ( C. Toumanoff, Coml,t. filWll. icacl. sci.. 187, :3913$X3, 192s; c. E. Burnside, c7.S. Dept. Agr. Tech. Bull. 149, 13 pp., I930; Y. Kodaira, Res. Rep. Fat. Textile Sericult. Shinshti IyniL-., 4, 4749, 1954; and D. W. Roberts, J. Zmertebrate Pa&d., 8, 212-221, 222-227, 1966). In the cease of entomophthoraceous fungi, the present note is the first to report the demonstration of the presence of a mvcotoxin in the culture filtrate.
was seen. Others had protruded farther, and were assuming a fingerlike shape, in which several virus particles were arranged in a row. Still others seemed to be breaking away from the membrane. Small extracellular bodies consisting of one to three virus particles enveloped by a membrane were often seen near the surface of CIVinfected cells. These bodies were apparently formed by the budding process in the cell membrane. Also, in CIV-infected cultures, spherical bodies (25 p) filled with large numbers of virus particles were sometimes found ( Fig. 2B). Presumably, these represent infected cells or their debris. TOSIHIKO HUKUHARA~ YOKO HASHIMOTO
Laboratory Faculty of Unioersit y Bunkyo-ku,
of Sericulture Agriculture of Tokyo Tokyo, Japan
Received March 14, 1967 ’ The authors are grateful to Dr. hl. Fukayn of the Faculty of Agriculture, Tokyo University of Education, for providing the larvae of the greater wax moth infected with Chile iridescent viros.
Species
of
Entomoph.thora
The entomophthoraceous fungi protluced mycotoxins in both stationary and shake cultures. The standard culture filtratc~ of Elm?zop/fthora was obtained &rr iuoculating species of Entomophthom in 50 ml of a modified peptone medium (Bactopeptone 10 g; dipotassium phosphate 4.3 g; potassium biphosphate 3.4 g; and distilled water added to make 1000 ml) held in a 2%~ml Erlenmeyer flask. The inoculated flask was shaken ( 160 oscillations/min ) at room temperature ( 22”27°C ) for 96 hr. The fungus mvcelia were
278
per spore, monospore cultures of them should have a minimum of variability. Monospore cultures were prepared as follows: Small circles (diam i 2 mm) were engraved on the bottom lid of petri dishes with the aid of a Leitz marker objective
(Type MAKKI, photograph 3). Sterile, filtered, water agar was poured into warm, sterile petri dishes to give a very thin film of agar. The center of the circle was marked with an ink dot on the underside of the petri dish. Small droplets containing, on the average, one Metarrhizium-spore were delivered on the agar above the ink dots by means of a sterile Pasteur pipette. The dots were then removed and the number of spores in each circle mounted under phase contrast with a magnification of 16x (objective), 8X (ocular). The field corresponded with the field of the engraved circles. Circles with only one spore were marked again with an ink dot to aid in their transfer to Sabouraud maltose agar with a sterile loop. K. H. VEEN Agricultural University Department of Entomology Wageningen, The Netherlands
FIG. n. Device dish with engraved
for monospore circles. Photo
Multiplication
cultures; petri by G. Eimers.
Received March 9, 1967
of
Tipula
and
Cells
of
Antheraea
The continuous line of Antheraea eucdypti ovarian cells which was established by T. D. C. Grace (Nature, 195, 788-789, 1962) is susceptible to infection by Sericesthb iridescent virus (A. J. D. Bellett and E. H. Mercer, Virology, 24, 645-653, 1964). This note reports the successful infection of this cell line with Tip&a iridescent virus (TIV), and with Chile iridescent virus (CIV) described by M. Fukaya and S. Nasu (Appl. Entomol. Zool., 1, 69-72, 1966). The suspensionsof the two viruses were prepared as follows: Larvae of the greater Gallerk mellonella, which had wax moth,
Chile
Iridescent
Viruses
in
eucalypti
been infected with either TIV or CIV, were surface-sterilized with 1% hyamine. Their abdominal legs were clipped and the oozing hemolymph, containing fragments of fat-body tissue, was collected in capillary tubes. The hemolymph was added to a modification of the Grace’s culture medium described previously (T. Hukuhara, J. Sericult. Sci. Japan, 35, 349-354, 1966). The cell cultures were incubated overnight with the virus suspension (1.5-2.0 ml) at 20-21°C. After this period the cultures were supplied with fresh culture medium. For light- and electron-microscope examinations the TIV-infected cells were fixed
NOTES
279
FIG. 1. Antheraea eucalypti cells fixed 21 days after inoculation with TIV. A. Light micrograph of infected cells showing intracytoplasmic inclusions. The arrow points to one of them. 1100 X. B. Electron rnicrograph of an ultrathin section of an infected cell showing groups of virus particles in the cytoplasm. Uranyl acetate stain. 10,000 X. C. Infected cell similar to B at a higher magnification. 40,000 >;. S, nucleus: P51, plasma membrane; V, virus particle.
280
NOTES
FIG. 2. Electron micrographs of ultrathin sections of Anthernea eucalypti cells fixed 6 days after inoculation with CIV. Uranyl acetate and lead nitrate stain. A. Infected cell showing the virus particles in the cytoplasm and protrusions of the plasma membrane. 10,000 X. B. Spherical body filled with large numbers of virus particles which is presumed to be an infected cell or its debris. 10,000 x. C. Infected cell showing protrusions in various stages of their development. 20,000 X. E, extracellular body; N, nucleus; PM, plasma membrane; V, virus particle.
281
NOTES
WI the 21st day after inoculation, and the CIV-infected cells on the 6th day in the following manner: The cells adhering to the coverslips in the culture vessels were fixed with Bouin’s fluid and stained with Harris hematoxylin; the cells, free-floating in the medium, were centrifuged for 15 minutes at 1500 rev/min, fixed with glutaraldehyde-OsOq, embedded in Epon. and sectioned. Light-microscope studies of the cells from TIV- and CIV-infected cultures showed that most of them contained, in their cytoplasm, one to several spherical inclusions, which stained intensely with hematoxylin, and were surrounded by a clear halo (Fig. 1A). Such bodies were not found in the cells from uninfected cultures. Ultrathin sections of TIV- and (XV-infected cells revealed the presence of virus particles in the cytoplasm but never in the nucleus ( Fig. 1B and Fig. 2A). These virus particles are identical in appearance with those noted in infected cells in susceptible insects. Sections of CIV-infected cells showed a number of protrusions of the plasma membrane, varying in size (Fig. 2A, C). Some appeared as a light bulging of the mcmhrane where a single virus particle
Mycotoxin
Production
by
\I>-cotosins from entomogenous fungi Ilnve been demonstrated and investigated hv marry workrrs ( C. Toumanoff, Coml,t. filWll. icacl. sci.. 187, :3913$X3, 192s; c. E. Burnside, c7.S. Dept. Agr. Tech. Bull. 149, 13 pp., I930; Y. Kodaira, Res. Rep. Fat. Textile Sericult. Shinshti IyniL-., 4, 4749, 1954; and D. W. Roberts, J. Zmertebrate Pa&d., 8, 212-221, 222-227, 1966). In the cease of entomophthoraceous fungi, the present note is the first to report the demonstration of the presence of a mvcotoxin in the culture filtrate.
was seen. Others had protruded farther, and were assuming a fingerlike shape, in which several virus particles were arranged in a row. Still others seemed to be breaking away from the membrane. Small extracellular bodies consisting of one to three virus particles enveloped by a membrane were often seen near the surface of CIVinfected cells. These bodies were apparently formed by the budding process in the cell membrane. Also, in CIV-infected cultures, spherical bodies (25 p) filled with large numbers of virus particles were sometimes found ( Fig. 2B). Presumably, these represent infected cells or their debris. TOSIHIKO HUKUHARA~ YOKO HASHIMOTO
Laboratory Faculty of Unioersit y Bunkyo-ku,
of Sericulture Agriculture of Tokyo Tokyo, Japan
Received March 14, 1967 ’ The authors are grateful to Dr. hl. Fukayn of the Faculty of Agriculture, Tokyo University of Education, for providing the larvae of the greater wax moth infected with Chile iridescent viros.
Species
of
Entomoph.thora
The entomophthoraceous fungi protluced mycotoxins in both stationary and shake cultures. The standard culture filtratc~ of Elm?zop/fthora was obtained &rr iuoculating species of Entomophthom in 50 ml of a modified peptone medium (Bactopeptone 10 g; dipotassium phosphate 4.3 g; potassium biphosphate 3.4 g; and distilled water added to make 1000 ml) held in a 2%~ml Erlenmeyer flask. The inoculated flask was shaken ( 160 oscillations/min ) at room temperature ( 22”27°C ) for 96 hr. The fungus mvcelia were