- Email: [email protected]
Infection of leafhopper and its tissues cultivated in vitro with Chilo iridescent virus
432
NOTES TABLE
FUNGI
OCCURRING
ON HELEOMYZIDAE
Total dead insects observed
Insect Heleomyzidae Culex pipiens
493 529
AND
ON
pipiens
IN
CAVES
MARL
IN THE NETHERLANDS
ETLlO?T&Oph-
thora
SPP.
Paecilomyces SP.
SP.
Unknown
15 -
-
15 53
Entomophthora is by far the most important entomophthorous fungus occurring in these caves causing the death of flies and mosquitoes. The fungi may be very important in controlling the population level of the insects. Until January 18, mortality due to the fungi among the flies varied in a number of caves from 56% to 80%. The mortality during February rose to an even higher level, but it was not possible to obtain accurate figures at this time since many flies began to’ leave the caves. The disease incidence in the mosquito population was still higher. By January 18, 83-91% had die,d, and by February 7, 92-97%. On these dead bodies entomophthorous fungi were found. M. H. TEERNSTRA-EEKEN~ A. ENGEL Laboratory University
of Applied
Entomology
of Amsterdam, Netherlands
Received June 2, 1967 1 Thanks are due to Dr. J. A. von Arx, director of the Central Bureau of Fungus Cultures, Baarn, the Netherlands and to his co-workers for their help and advice on the mycological work, and to their guest Mrs. 0. Fassatiovi for checking the identification of the fungi.
its Tissues Iridescent
Iridescent viruses of Tipula paludosa and Sericesthis pruinosa are known to have
Verticillium SPP.
4
-
and Chile
Aspergillus
7
-
of Leafhopper with
Hirsutella SPP.
4
383 486
tenella Delacroix, Paecilomyces farinosus (Dicks ex Fr.), Polycephalomyces formosus Kobayashi, Hirsutella entomophila Pat., H. saussurei ( Cooke), Paecilomyces sp., and Entomophthora spp. The mosquitoes are mainly parasitized by Entomophthora spp. A more detailed report about the mycological part of this study will be published later (M. H. Teernstra-Eeken, in preparation. The occurrence of entomophthorous fungi in overwintering populations of C. pip&w has been reported earlier. Weiser and Batko (Folia Parasitol., Prague, 13, 144-149, 1966) found Entomophthora destruens Weiser and Batko on C. pipiens hibernating in cellars and natural caves in Moravia, Czechoslovakia. More than 85% of the hibernating mosquitoes were killed during the winter. The importance of these diseases to natural insect populations was investigated. In November 1966, large parts of the cave walls were cleared of dead insects and the living insects were counted. Approximately 2 weeks later, the number of dead and living insects was determined. This procedure was repeated regularly until the end of February 1967 when the insects began to leave the caves. Table 1 shows that
Infection
1
Culex
Cultivated
in Vitro
Virus
wide host ranges, and are transmitted experimentally to different orders of insects
NOTES
(K. M. Smith et al., Virology, 13, 233-241, 1961; E. A. Steinhaus and R. Leutenegger, J. Insect Pathol., 5, 266-270, 1963; M. F. Day and E. H. Mercer, Australian J. Biol. Sci., 17, 892-902, 1964). Chile iridescent virus (CIV) also seems to have a wide host range. Attempts were made to infect homopterous insects with CIV, because some plant viruses have been known to multiply in the homopterous vectors, whereas no insect virus proper has been reported in homopterous insects. The green rice leafhopper, Neplzotettix cincticeps, was inoculated perorally with CIV. The inoculum used in this experiment was a suspension of purified CIV in 5% sucrose solution. The fourth-instar nymphs of leafhoppers were allowed to suck the inoculum through a stretched Parafilm membrane (Marathon American Can Co., Menasha, Wisconsin ) . After feeding on the virus suspension, the leafhoppers were reared on rice seedlings in small glass tubes at 25’C. Signs of CIV infection became externally recognizable 7 days after the inoculation. At this time bluish iridescence could be observed through the intersegmental membrane or transparent parts of the cuticle. This bluish tint was found to be due to the prominent iridescence from epidermal cells. Fat-body lobes lying underneath the cuticle on the ventral side also showed prominent iridescence, while those on the dorsal side showed rather faint iridescence. In the alimentary canal, only the foregut showed prominent iridescence, whereas other parts did not show iridescence. Healthy mycetomes are usually greenish-yellow in color, but in the infected leafhoppers some parts became pale purple and prominent iridescence was seen on some parts of the mycetome epithelium. Iridescence was also observed in some parts of muscular tissues. Stomach, midgut, hindgut, Malpighian tubules, gonads, salivary glands, and nervous systems usually did not show iridescence,
433 but sometimes iridescence was seen on the surfaces of these organs. Probably such iridescence was derived from the cells attached to these organs and not from thtb cells which constituted these organs. The infected leafhoppers survived mart’ than 20 days after the inoculation. The\ could molt, and some of them became: adults. Cells cultivated in vitro from the embryos of N. cincticeps were inoculated with CIV. The cells were cultivated according to the method of J. Mitsuhashi (Japan. J. Appl. Entomol. Zool., 9, 107-114, 1965). :Z 50-day-old culture, in which epithelial cell sheets were well formed, was used in this experiment. The inoculum was prepared aseptically from larvae of the rice stem borer, Chile suppressalis, infected with CIV (J. Mitsuhashi, Appl. Entomol. Zool., 1, 130-137, lQ66). Twenty-four hours aftc,l inoculation, granules of various sizes and vacuoles appeared in the cytoplasm of epithelial cells. Iridescence appeared in tllch developed epithelial cell sheets as well :IS in the original explants 7 days after tllct inoculation. Some infected cells began to degenerate at the 10th day and detachrtl themselves from the glass surface. If the infected culture was kept without changing the medium, the medium becamr turbid owing to the virus released from the cli\integrated cells. The tissues cultivated in vitro from small brown planthopper, Lao,delphax striatclla, were also inoculated with CIV. The same histopathological changes were observed :LS those occurring in N. cinctkeps tissue crritures. The electron-microscope observations were carried out on the infected cells cultivated from N. cincticeps embryos. The infected cells were fixed with osmium t+ troxide solution buffered at pH 7.2 and embedded in Epon 812, 14 days after the inoculation. At that time, some cells were found still to be alive, while other cells had
434
NOTES
FIG. 1. An ultrathin section of the CIV-infected cells cultivated N, nucleus; S, symbiotic bacteria; V, CIV particles. 10,000 X.
disintegrated. Ultrathin sections of the infected cells revealed the presence of numerous CIV particles and some empty membranes in the cytoplasm. CIV particles were found neither in nuclei nor in the intracellular symbiotic bacteria, which harbor in cytoplasm as well as in nuclei.
Persistence
of
Cotton
In studies of the effect of surfactants residues of nuclear-polyhedrosis virus
on on
1 In cooperation with the Texas Agricultural Experiment Station, Texas A&M University. ’ Mention of trade names does not necessarily imply endorsement of the product by the U.S. Department of Agriculture.
Nephotettir
cincticeps
JUN
embryos:
M ITSUHASHI
Division of Entomology National Institute of Agricultural Sciences Nishigahara, Kita-ku, Tokyo, Japan Receive#d June 8, 1967
Nuclear-Polyhedrosis
Heliothis
on
from
Virus
Foliagel*’ cotton leaves, most virus on the surface of cotton leaves in the field lost infectivity or disappeared within 3 days. A study -was therefore made to determine more accurately peaks of activity of a commercial viral suspension on cotton leaves. No surfactant was added. Previous assays (Bullock,
NOTES TABLE
FUNGI
OCCURRING
ON HELEOMYZIDAE
Total dead insects observed
Insect Heleomyzidae Culex pipiens
493 529
AND
ON
pipiens
IN
CAVES
MARL
IN THE NETHERLANDS
ETLlO?T&Oph-
thora
SPP.
Paecilomyces SP.
SP.
Unknown
15 -
-
15 53
Entomophthora is by far the most important entomophthorous fungus occurring in these caves causing the death of flies and mosquitoes. The fungi may be very important in controlling the population level of the insects. Until January 18, mortality due to the fungi among the flies varied in a number of caves from 56% to 80%. The mortality during February rose to an even higher level, but it was not possible to obtain accurate figures at this time since many flies began to’ leave the caves. The disease incidence in the mosquito population was still higher. By January 18, 83-91% had die,d, and by February 7, 92-97%. On these dead bodies entomophthorous fungi were found. M. H. TEERNSTRA-EEKEN~ A. ENGEL Laboratory University
of Applied
Entomology
of Amsterdam, Netherlands
Received June 2, 1967 1 Thanks are due to Dr. J. A. von Arx, director of the Central Bureau of Fungus Cultures, Baarn, the Netherlands and to his co-workers for their help and advice on the mycological work, and to their guest Mrs. 0. Fassatiovi for checking the identification of the fungi.
its Tissues Iridescent
Iridescent viruses of Tipula paludosa and Sericesthis pruinosa are known to have
Verticillium SPP.
4
-
and Chile
Aspergillus
7
-
of Leafhopper with
Hirsutella SPP.
4
383 486
tenella Delacroix, Paecilomyces farinosus (Dicks ex Fr.), Polycephalomyces formosus Kobayashi, Hirsutella entomophila Pat., H. saussurei ( Cooke), Paecilomyces sp., and Entomophthora spp. The mosquitoes are mainly parasitized by Entomophthora spp. A more detailed report about the mycological part of this study will be published later (M. H. Teernstra-Eeken, in preparation. The occurrence of entomophthorous fungi in overwintering populations of C. pip&w has been reported earlier. Weiser and Batko (Folia Parasitol., Prague, 13, 144-149, 1966) found Entomophthora destruens Weiser and Batko on C. pipiens hibernating in cellars and natural caves in Moravia, Czechoslovakia. More than 85% of the hibernating mosquitoes were killed during the winter. The importance of these diseases to natural insect populations was investigated. In November 1966, large parts of the cave walls were cleared of dead insects and the living insects were counted. Approximately 2 weeks later, the number of dead and living insects was determined. This procedure was repeated regularly until the end of February 1967 when the insects began to leave the caves. Table 1 shows that
Infection
1
Culex
Cultivated
in Vitro
Virus
wide host ranges, and are transmitted experimentally to different orders of insects
NOTES
(K. M. Smith et al., Virology, 13, 233-241, 1961; E. A. Steinhaus and R. Leutenegger, J. Insect Pathol., 5, 266-270, 1963; M. F. Day and E. H. Mercer, Australian J. Biol. Sci., 17, 892-902, 1964). Chile iridescent virus (CIV) also seems to have a wide host range. Attempts were made to infect homopterous insects with CIV, because some plant viruses have been known to multiply in the homopterous vectors, whereas no insect virus proper has been reported in homopterous insects. The green rice leafhopper, Neplzotettix cincticeps, was inoculated perorally with CIV. The inoculum used in this experiment was a suspension of purified CIV in 5% sucrose solution. The fourth-instar nymphs of leafhoppers were allowed to suck the inoculum through a stretched Parafilm membrane (Marathon American Can Co., Menasha, Wisconsin ) . After feeding on the virus suspension, the leafhoppers were reared on rice seedlings in small glass tubes at 25’C. Signs of CIV infection became externally recognizable 7 days after the inoculation. At this time bluish iridescence could be observed through the intersegmental membrane or transparent parts of the cuticle. This bluish tint was found to be due to the prominent iridescence from epidermal cells. Fat-body lobes lying underneath the cuticle on the ventral side also showed prominent iridescence, while those on the dorsal side showed rather faint iridescence. In the alimentary canal, only the foregut showed prominent iridescence, whereas other parts did not show iridescence. Healthy mycetomes are usually greenish-yellow in color, but in the infected leafhoppers some parts became pale purple and prominent iridescence was seen on some parts of the mycetome epithelium. Iridescence was also observed in some parts of muscular tissues. Stomach, midgut, hindgut, Malpighian tubules, gonads, salivary glands, and nervous systems usually did not show iridescence,
433 but sometimes iridescence was seen on the surfaces of these organs. Probably such iridescence was derived from the cells attached to these organs and not from thtb cells which constituted these organs. The infected leafhoppers survived mart’ than 20 days after the inoculation. The\ could molt, and some of them became: adults. Cells cultivated in vitro from the embryos of N. cincticeps were inoculated with CIV. The cells were cultivated according to the method of J. Mitsuhashi (Japan. J. Appl. Entomol. Zool., 9, 107-114, 1965). :Z 50-day-old culture, in which epithelial cell sheets were well formed, was used in this experiment. The inoculum was prepared aseptically from larvae of the rice stem borer, Chile suppressalis, infected with CIV (J. Mitsuhashi, Appl. Entomol. Zool., 1, 130-137, lQ66). Twenty-four hours aftc,l inoculation, granules of various sizes and vacuoles appeared in the cytoplasm of epithelial cells. Iridescence appeared in tllch developed epithelial cell sheets as well :IS in the original explants 7 days after tllct inoculation. Some infected cells began to degenerate at the 10th day and detachrtl themselves from the glass surface. If the infected culture was kept without changing the medium, the medium becamr turbid owing to the virus released from the cli\integrated cells. The tissues cultivated in vitro from small brown planthopper, Lao,delphax striatclla, were also inoculated with CIV. The same histopathological changes were observed :LS those occurring in N. cinctkeps tissue crritures. The electron-microscope observations were carried out on the infected cells cultivated from N. cincticeps embryos. The infected cells were fixed with osmium t+ troxide solution buffered at pH 7.2 and embedded in Epon 812, 14 days after the inoculation. At that time, some cells were found still to be alive, while other cells had
434
NOTES
FIG. 1. An ultrathin section of the CIV-infected cells cultivated N, nucleus; S, symbiotic bacteria; V, CIV particles. 10,000 X.
disintegrated. Ultrathin sections of the infected cells revealed the presence of numerous CIV particles and some empty membranes in the cytoplasm. CIV particles were found neither in nuclei nor in the intracellular symbiotic bacteria, which harbor in cytoplasm as well as in nuclei.
Persistence
of
Cotton
In studies of the effect of surfactants residues of nuclear-polyhedrosis virus
on on
1 In cooperation with the Texas Agricultural Experiment Station, Texas A&M University. ’ Mention of trade names does not necessarily imply endorsement of the product by the U.S. Department of Agriculture.
Nephotettir
cincticeps
JUN
embryos:
M ITSUHASHI
Division of Entomology National Institute of Agricultural Sciences Nishigahara, Kita-ku, Tokyo, Japan Receive#d June 8, 1967
Nuclear-Polyhedrosis
Heliothis
on
from
Virus
Foliagel*’ cotton leaves, most virus on the surface of cotton leaves in the field lost infectivity or disappeared within 3 days. A study -was therefore made to determine more accurately peaks of activity of a commercial viral suspension on cotton leaves. No surfactant was added. Previous assays (Bullock,