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A cytoplasmic polyhedrosis virus from the larval blackflies Cnephia mutata and Prosimulium mixtum (Diptera: Simuliidae)
JOURNAL
OF INVERTEBRATE
A Cytoplasmic hia mutata
PATHOLOGY
25. 273-274
(1975)
Polyhedrosis Virus from the Larval and Prosimulium mixtum (Diptera
In recent years there has been increased interest in developing pathogens for biological control of blackflies (Anonymous, International Development Research Centre Monograph, IDRC-006e, 1972). Surprisingly, J. Weiser’s (J. Invertebr. Pathol. 12, 36-39, 1968) report of an iridescent virus from a single larval specimen of Simulium ornatum in Czechoslovakia is the only record of any viral disease of simuliids. The present report concerns a cytoplasmic polyhedrosis virus (CPV) discovered in blackflies while collecting larvae for mermithid nematode parasites. In late May, 1973, larval blackflies, Cnephia mutata, sampled from a single stream near Bay Bulls, Newfoundland, Canada, were suspected of being infected with a CPV. A further attempt to obtain more larvae proved futile because rains and swollen stream conditions made collection impossible for two weeks, by which time this univoltine blackfly species had completed larval development. Thus confirmation of the disease agent as a CPV was not made until 1974. During April and May, 1974, at the same stream, samples of several hundred C. mutata larvae were taken and an incidence of CPV infection ranging from 10 to 22% was found. The CPV infected C. mutata were also found in single samples of several hundred larvae from two of four other streams in 1974 at an incidence ca I %. Most larvae collected were in either the penultimate or terminal instar but infected larvae as small as the third-instar were found. Another blackfly, Prosimulium m&turn, was in close association with C. mutata in all streams sampled but CPV was found from only one stream and rate of infection was < 0.1% (three of the several thousand larvae). Incidence of disease was probably
Blackflies Cnep:Simuliidae)
higher in P. mixturn but the darker pigmentation of this species made detection of CPV more difficult than in the lightly pigmented C. mutata. Patently infected larvae were recognized with the unaided eye or with a dissecting microscope, by the opaque, chalky appearance of the midgut (Fig. 1). Generally, the anterior and posterior thirds of the midgut were whitened and more rarely, the entire midgut was grossly infected. Aqueous squash preparation of infected larvae and stained sections of whole larvae revealed inclusion bodies within the epithilial cells of the midgut and free inclusions in the gut lumen released from ruptured infected cells. Only midgut tissues stained positively for polyhedra by the method of J.J. Hamm (J. Invertebr. Pathol. 8, 1255 126, 1966). Most inclusion bodies in wet mounts appeared to be hexagonal or pentagonal in outline using light microscopy. Infected tissues were prepared for electron microscopy by standard techniques using 4% glutaraldehyde and 2% osmium tetroxide in Millonig’s buffer for fixation. The size of 50 inclusion bodies measured from electron micrographs ranged from 0.37 to 1.06 pm (ave 0.64 + 0.02 pm) in diameter. Inclusion bodies (Fig. 2) were polymorphic as viewed in electron micrographs. The irregularities of the inclusion body surfaces and empty sockets indicate that partial disruption may have occurred during histological processing; possibly pH of the buffer was too high. However, the general structure is clearly that associated with previously described cytoplasmic polyhedrosis viruses. Although the usefulness of this particular CPV as a control agent for blackflies is yet to be explored, its discovery emphasizes the need for researchers interested in biological
273 Copyright L 1975 by Academic Printed in the United States.
Press, Inc. All rights
of reproduction
in any form
reserved.
274
NOTES
FIG. I. CPV infected midgut
tissue.
(1) and noninfected
(N)
terminal
instar
Cnephia
mutata
larvae.
Arrow
indicates
infected
x I 1.
FIG. 2. Electronmicrographs body showing occluded virions
of infected midgut tissue and inclusion bodies. (A) Section through (arrows). ~71,000. (B) Inclusion bodies in midgut epithelial cell. x6000.
control of blackflies to look for their disease agents (viral or other) in nature. This work was supported by Grants D43, A4664 and A9724 from the National Research Council of Canada. We wish to thank Dr. A. Bal, Memorial University of Newfoundland for assistance with the electron microscopy. C. H. BAILEY M. SHAPIRO
an inclusion
Research Unit on Vector Pathology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
R. R. GRANADOS Boyce Thompson Institute for Plant Research, 1086 North Broadway Yonkers, New York 10701 Received July 3, 1974
OF INVERTEBRATE
A Cytoplasmic hia mutata
PATHOLOGY
25. 273-274
(1975)
Polyhedrosis Virus from the Larval and Prosimulium mixtum (Diptera
In recent years there has been increased interest in developing pathogens for biological control of blackflies (Anonymous, International Development Research Centre Monograph, IDRC-006e, 1972). Surprisingly, J. Weiser’s (J. Invertebr. Pathol. 12, 36-39, 1968) report of an iridescent virus from a single larval specimen of Simulium ornatum in Czechoslovakia is the only record of any viral disease of simuliids. The present report concerns a cytoplasmic polyhedrosis virus (CPV) discovered in blackflies while collecting larvae for mermithid nematode parasites. In late May, 1973, larval blackflies, Cnephia mutata, sampled from a single stream near Bay Bulls, Newfoundland, Canada, were suspected of being infected with a CPV. A further attempt to obtain more larvae proved futile because rains and swollen stream conditions made collection impossible for two weeks, by which time this univoltine blackfly species had completed larval development. Thus confirmation of the disease agent as a CPV was not made until 1974. During April and May, 1974, at the same stream, samples of several hundred C. mutata larvae were taken and an incidence of CPV infection ranging from 10 to 22% was found. The CPV infected C. mutata were also found in single samples of several hundred larvae from two of four other streams in 1974 at an incidence ca I %. Most larvae collected were in either the penultimate or terminal instar but infected larvae as small as the third-instar were found. Another blackfly, Prosimulium m&turn, was in close association with C. mutata in all streams sampled but CPV was found from only one stream and rate of infection was < 0.1% (three of the several thousand larvae). Incidence of disease was probably
Blackflies Cnep:Simuliidae)
higher in P. mixturn but the darker pigmentation of this species made detection of CPV more difficult than in the lightly pigmented C. mutata. Patently infected larvae were recognized with the unaided eye or with a dissecting microscope, by the opaque, chalky appearance of the midgut (Fig. 1). Generally, the anterior and posterior thirds of the midgut were whitened and more rarely, the entire midgut was grossly infected. Aqueous squash preparation of infected larvae and stained sections of whole larvae revealed inclusion bodies within the epithilial cells of the midgut and free inclusions in the gut lumen released from ruptured infected cells. Only midgut tissues stained positively for polyhedra by the method of J.J. Hamm (J. Invertebr. Pathol. 8, 1255 126, 1966). Most inclusion bodies in wet mounts appeared to be hexagonal or pentagonal in outline using light microscopy. Infected tissues were prepared for electron microscopy by standard techniques using 4% glutaraldehyde and 2% osmium tetroxide in Millonig’s buffer for fixation. The size of 50 inclusion bodies measured from electron micrographs ranged from 0.37 to 1.06 pm (ave 0.64 + 0.02 pm) in diameter. Inclusion bodies (Fig. 2) were polymorphic as viewed in electron micrographs. The irregularities of the inclusion body surfaces and empty sockets indicate that partial disruption may have occurred during histological processing; possibly pH of the buffer was too high. However, the general structure is clearly that associated with previously described cytoplasmic polyhedrosis viruses. Although the usefulness of this particular CPV as a control agent for blackflies is yet to be explored, its discovery emphasizes the need for researchers interested in biological
273 Copyright L 1975 by Academic Printed in the United States.
Press, Inc. All rights
of reproduction
in any form
reserved.
274
NOTES
FIG. I. CPV infected midgut
tissue.
(1) and noninfected
(N)
terminal
instar
Cnephia
mutata
larvae.
Arrow
indicates
infected
x I 1.
FIG. 2. Electronmicrographs body showing occluded virions
of infected midgut tissue and inclusion bodies. (A) Section through (arrows). ~71,000. (B) Inclusion bodies in midgut epithelial cell. x6000.
control of blackflies to look for their disease agents (viral or other) in nature. This work was supported by Grants D43, A4664 and A9724 from the National Research Council of Canada. We wish to thank Dr. A. Bal, Memorial University of Newfoundland for assistance with the electron microscopy. C. H. BAILEY M. SHAPIRO
an inclusion
Research Unit on Vector Pathology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
R. R. GRANADOS Boyce Thompson Institute for Plant Research, 1086 North Broadway Yonkers, New York 10701 Received July 3, 1974