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Nucleopolyhedrosis of Heliothis: Morphological description of inclusion bodies and virions
JOURNAL
OF
INVERTEBRATE
14,
PATHOLOGY
186-193
(1969)
Nucleopolyhedrosis of Heliothis: Morphological of Inclusion Bodies and Virions 13. G. GREGORY,C. M. IGNOFFO,AND M. International
Minerals
and Chemical Corp., Growth Libertyville, lllinois 6004s Received
February
26,
Description
SHAPIRO
Sciences
Center,
1969
PoIyhedral inclusion bodies of the He&this nucleopolyhedrosis of Heliothk zea average 916.4 k 9.5 mp in diameter with a range from 300 to 2240 rnF. No prominent surface patterns or structures are evident on unpurified inclusions. Purified inclusions can be severely etched revealing virion sockets. The rod-shape virions, ’ which are individually occluded, average 336 -t- 22 X 62 t 4 my.
INTRODUCTION “Wilt disease” of Heliothti was first described by Mally from larvae of Heliothis armigera ( 1891, 1892). Later Lounsbury (1913), and Chapman and Glaser (1915) described similar symptoms in larvae of Heliothis obtectus and Heliothis obsoleta. It was not until 1936 that Parson suggested that a virus caused the Hetiothis “wilt disease.” The first photomicrographs of inclusion bodies and partially occluded virions were published in 1956 (Smith and Rivers, 1956). Additional photomicrographs of vu-ions from Heliothis virescens, Heliothis armigera, and Heliothis zea were later published by Steinhaus ( 1957), Bergold and Ripper (1957) (who proposed the name Borrelina armigera for the virus they observed), and Adams (in Ignoffo, 1968), respectively. The present paper describes the morphology of the inclusion bodies and virions obtained from Heliothis zea larvae infected with nucleopolyhedrosis. MATERIALS AND METHODS Inclusion bodies of the Heliothis nucleopolyhedrosis used herein were collected 186
from dead or living H. zea larvae using a previously described procedure ( Ignoffo, 1965). A saline citrate, tissue-extraction and differential-centrifugation technique modified from Dobrovolska et al. (1965) was used to obtain highly purified inclusion bodies (Shapiro and Ignoffo, 1969). Measurements of virions were made from photographs after either sodium hydroxide (0.01 M; pH 13) or sodium carbonate (0.01 M NazC03-6.05 M NaCl; pH 9.2) treatment of inclusion bodies (Shapiro and Ignoffo, 1968). Virions were stained with 2% phosphotungstic acid (PTA) at pH 7.2. Carbon replicas were prepared by the following procedure: Suspensions of inclusion bodies, dried on freshly cleaved mica strips, were shadowed at 15°C with palladium (20 mg) and then coated with carbon. The carbon-palladium film was stripped from mica with NaOH ( 1 N), and after 3 min was washed in water and then mounted on copper grids for viewing. A grid-treatment procedure was used to obtain the number of virions/inclusion body. Formvar-coated grids, on which a film of inclusion bodies was previously dried, were treated with NaOH for 3 min, flushed with distilled water, and then immediately stained with
NUCLEOPOLYHEDROSIS
2% PTA. The Hitachi electron microscope (HU-llB), vacuum evaporator (HUS-SB), and Zeiss Ultraphot were used for studies reported herein. RESULTS AND DISCUSSION
Inclusion
Body Description
Inclusion bodies are irregular and appear six-sided in outline (Fig. 1). The edges of the polyhedrons are rounded. The mean diameter of inclusion bodies is 916.4 39.5 mp (standard error of the mean) with a range from 300 to 2240 mu. Approximately 90% of the bodies range from 610 to 1200 rnp (Table 1). These measurements
FIG. 1.
Inclusion
bodies
from
virus-killed
OF Heliothis
187
are in good agreement with inclusion measurements from H. armigera which range from 700 to 1200 mu with the “majority measuring about 1100 mu” (Bergold and Rivers, 1957). No definite surface patterns or prominent structures are apparent (Fig. 2). Some polyhedron faces are rhomboids. Faces of highly purified inclusions ( -1 X 10” PIB/mg) were severely etched, revealing virion sockets and probable loss of virions (Fig. 3). A similar observation was reported for the nucleopolyhedrosis of Hemerobius (Hill and Smith, 1959) and the cytoplasmic polyhedrosis of Antheraea (Smith et al., 1959). Loss of virions plus severe clumping could partially explain the
Heliothis
zea
larvae
(4500
X ).
TABLE 1 FREQUENCY DISTRIBUTIONS OF INCLUSION DIAMETER AND NUMBER OF VIRIONS/ INCLUSION BODY FOR POLYHEDRAL INCLUSION BODIES OF Heliothis NUCLEOPOLYHEDROSIS
BODY
Virions Inclusion
2. larvae
FIG.
zea
Palladium-shadowed, (15,000X ).
body
Diameter b.)
Frequency
210409 410-609 610409 810-1009 1010-1209 1210-1409 1410-1609 1610-1809 1810-2009 2010-2209 2210-2409
2 23 225 208 122 24 23 3 3 1 1
carbon
replica
of unpurified
Inclusion body (Number)
Frequency
15-18 19-22 23-26 27-30 31-34 35-38
8 15 17 15 17 7
inclusion
bodies
from
virus-killed
Heliothis
NUCLEOPOLYHEDROSIS
FIG.
3.
Palladium-shadowed,
carbon
replica
reported loss in activity of highly purified inclusions (Ignoffo, 1964, 1965; Magnoler, 1968). Difficulties encountered in dissolving or staining inclusion bodies, retention of polyhedral shape following extreme physical forces, and electron microscopic observations of sectioned and degraded inclusions indicate that the outer layer(s) of inclusions may be different from those of its interior. Denaturation at the inclusion body surface could produce a coating of fibroustype proteins. This external coat should contain active --SH or phenolic -OH groups of tyrosine and a higher isoelectric point than the inner, undenatured, inclusion body protein, No change, however, in the lattice
OF
of highly
Heliothis
purified
189
inclusion
bodies
( 15,000
X ).
pattern of the outer layer is evident nor is a membrane or membranelike structure present to support this speculation.
Virion Description Virions are rod shaped and are probably helically symmetrical (Figs. 4, 5). The genetic material is DNA (Estes and Ignoffo, 1965). Virions are occluded in the cubic-protein lattice of the inclusion body without any apparent disruption of the lattice pattern Bundles of G-ions, as described in other nuclear polyhedroses (Bird, 1959; Bergold, 1963), were never observed. Based upon counts of 79 dissolved inclusion bodies the number of virions/inclusion body, standard
190
GREGORY,
FIG. 4.
Dissolved
inclusion
IGNOFFO,
body
deviation (SX) and standard error of the mean ( SE ) , was 26.4, 5.8, and 0.7, respectively. The range extended from 15 to 38 virionsjinclusion with 82% of the total sample containing between I9 and 34 virions/inclusion body (Table 1). The absolute dimension of the virion was somewhat dependent upon preparation techniques ( Table 2). Differences obtained, however, were not statistically significant at the 5% level. The average length and standard deviation of 599 alkali-treated virions was 336 + 22 rnp. Individual virion lengths ranged from 243 to 378 rnju, The
AND
and
SHAPIRO
released
virions
(25,000
X ).
average diameter was 62 2 4 mu with a range extending from 47 to 98 mu. The average dimension of H. armigera rods with developmental membranes measured 320 ” 10 my X 90 -C 10 rnp (Bergold and Rivers, 1957 ) . Structures similar to those described as virions of silkworm nucleopolyhedrosis by Kozlov and Alexeenko (1967) and as procedural artifacts by Summers and Paschke (1968) were also observed in preparations of Heliothis nucleopolyhedrosis virus (Fig. 6). These structures are quite similar to photomicrographs of bacterial rhapido-
NUCLEOPOLYHEDROSIS
‘IG. 5. Virions ~,ooOX ).
of
Heliothis
OF
nucleopolyhedrosis
after
TABLE
DIMENSIONS OF THE Heliothis
zea
Heliothis
concentration
from
Number measured
dissolved
inclusion
13odies
2
NUCLEOPOLYHEDROSIS VIFUONAFTER ALAKALINE TREATMENT OF INCLUSION BODIES Size
Treatment
191
in millimicrons
Average length
SDB
Average diameter
SD
Sodium
carbonate
115
373
16
75
7
Sodium
hydroxide
484
298
28
50
2
Q SD = Standard
deviation.
192
FIG.
GREGORY,
6.
Virionlike
structures
found
IGNOFFO,
in preparations
somes (Pate et al., 1967) and/or bacterial cell-wall fragments (Marsh and Walker, 1968 ) , REFERENCES BERGOLD, G. H. 1963. The nature of nuclear polyhedrosis. In “Insect Pathology-An Advanced Treatise” (E. A. Steinhaus, ed. ), Vol. 1, 413-456. Academic Press, New York. BERGOLD, G. H., AND RIPPER, W. E. 1957. The polyhedral virus of Heliothis armigeru ( Hbn. ) ( Lepidoptera: Noctuidae). Nature, 180, 764765. BIRD, F. T. 1959. Polyhedrosis and granulosis viruses causing single and double infections in the spruce budworm, Choristoneura fumiferana Clemens. I. Insect Pathol., 1, 406-430.
AND
SHAPIRO
of Heliothis
nucleopolyhedroris
virus
( 40,000
X ).
CHAPMAN, J. W., AND GLAYER, R. W. 1915. A preliminary list of insects which have wilt, with a comparative study of their polyhedra. J. Econ. Entomol., 8, 140-150. DOBROVQL~YA, H. M., KOK, I. P., SMIRNOVA, I. A., ANU CHISTYAIZOVA, A. V. 1965. Biological activity of DNA preparations isolated from the tissues of silkworms infected with nuclear polyhedrosis virus. Mikrobiol. Zhurnal., 27, 73-75. ESTES, Z. E., AND 1~x0~~0, C. M. 1965. The nucleic acid composition of nuclear polyhedral bodies affecting Heliothis zea (Boddie). J. Invertebrate Pathol., 7, 25&259. HILL, G. J., AND SMITH, K. M. 1959. Further studies on the isolation and crystallization of insect cytoplasmic viruses. J. Insect Pathol., 1, 121-128. IGNOFFO, C. M. 1964. Production and virulence
NUCLEOPOLYHEDROSIS
.
of a nuclear polyhedrosis virus from Trichoplusia ni (Hubner) reared on a semisynthetic diet. J. Insect Pathol., 6, 318-326. IGNOFFO, C. M. 1965. The nuclear polyhedrosis virus of Heliothis zeu (Boddie) and Heliothis uirescens (Fabricius). Part I. Virus propaand its virulence, J. Invertebrate gation Pathol., 7, 209-216. IGNOFFO, C. M. 1968. Viruses-Living Insecticides. Current Topics Microbial. Immunol. ( K. Maramorosch, ed. ), 42, 129-167. KOZLOV, E. A., AND ALEXEENKO, I. P. 1967. Electron-microscope investigations of the structure of the nuclear-polyhedrosis virus of the silkworm, Bombyx mod. J. Invertebrate Pathol., 9, 413419. LOUNSBURY, C. P. 1913. Caterpillar wilt disease. 1. Agr. South Africa, 5, 448-452, MAGNOLER, A. 1968. The differing effectiveness of purified and nonpurified suspensions of the nuclear-polyhedrosis virus of Porthetria &par. I. Invertebrate Pathol., 11, 326328. MALLY, F. W. 1891. The bollworm of cotton. U.S. Bur. Entomol. Bull., 24, 48-50. MALLY, F. W. 1892. Report of progress in the investigation of the cotton bollworm. U.S. Bur. Entomol. Bull., 26, 54-56. MARSH, D. G., AND WALKER, P. D. 1968. Free endotoxin and non-toxic material from gramnegative bacteria: Electron microscopy of
OF
Heliothis
193
fractions from Escherichia coli. J. Gen. MicrobioZ., 52, 125-130. Prog. Rept. Expt. Stas. PARSONS, F. S. 1936. Empire Cotton Growing Corp., Barberton, South Africa, 1934-1935, 24-31. PATE,
J. L., JOHNSON, J. L., AND ORDAL, E. J. 1967. The fine structure of Chondrococcus column&s. II. Structure and formation of rhapidosomes. J. Cell Biol., 35, 15-35. SHAPIRO, M., AND IGNOFFO, C. M. 1968. Characterization of Heliothis nucleopolyhedrosis virus. International Minerals and Chemical Quarterly Report, July-Sept. 1968. SHAPIRO, M., AND IGNOF'FO, c. M. 1969. Nuclear polyhedrosis of Heliothis: Stability and relative infectivity of virions. I. Invertebrate Pathol., 14, 130-134. SMITH, K. M., AND RIVERS, C. F. 1956. Some viruses affecting insects of economic importance. Parasitology, 46, 235-242. SMITH, K. M., HILL, G. J., AND RNERS, C. F. 1959. Polyhedroses in neuropterous insects. J. Insect Pathol., 1, 431437. 1957. New records of insectSTEINHAUS, E. A. virus diseases. Hilgardia, 26, 417-430. SUMXERS, M. D., AND PASCHKE, J. D. 1968. Observations on an artifact present in Ttichop&a ni granulosis-virus preparations. J. Invertebrate Pathol., 11, 520-523.
OF
INVERTEBRATE
14,
PATHOLOGY
186-193
(1969)
Nucleopolyhedrosis of Heliothis: Morphological of Inclusion Bodies and Virions 13. G. GREGORY,C. M. IGNOFFO,AND M. International
Minerals
and Chemical Corp., Growth Libertyville, lllinois 6004s Received
February
26,
Description
SHAPIRO
Sciences
Center,
1969
PoIyhedral inclusion bodies of the He&this nucleopolyhedrosis of Heliothk zea average 916.4 k 9.5 mp in diameter with a range from 300 to 2240 rnF. No prominent surface patterns or structures are evident on unpurified inclusions. Purified inclusions can be severely etched revealing virion sockets. The rod-shape virions, ’ which are individually occluded, average 336 -t- 22 X 62 t 4 my.
INTRODUCTION “Wilt disease” of Heliothti was first described by Mally from larvae of Heliothis armigera ( 1891, 1892). Later Lounsbury (1913), and Chapman and Glaser (1915) described similar symptoms in larvae of Heliothis obtectus and Heliothis obsoleta. It was not until 1936 that Parson suggested that a virus caused the Hetiothis “wilt disease.” The first photomicrographs of inclusion bodies and partially occluded virions were published in 1956 (Smith and Rivers, 1956). Additional photomicrographs of vu-ions from Heliothis virescens, Heliothis armigera, and Heliothis zea were later published by Steinhaus ( 1957), Bergold and Ripper (1957) (who proposed the name Borrelina armigera for the virus they observed), and Adams (in Ignoffo, 1968), respectively. The present paper describes the morphology of the inclusion bodies and virions obtained from Heliothis zea larvae infected with nucleopolyhedrosis. MATERIALS AND METHODS Inclusion bodies of the Heliothis nucleopolyhedrosis used herein were collected 186
from dead or living H. zea larvae using a previously described procedure ( Ignoffo, 1965). A saline citrate, tissue-extraction and differential-centrifugation technique modified from Dobrovolska et al. (1965) was used to obtain highly purified inclusion bodies (Shapiro and Ignoffo, 1969). Measurements of virions were made from photographs after either sodium hydroxide (0.01 M; pH 13) or sodium carbonate (0.01 M NazC03-6.05 M NaCl; pH 9.2) treatment of inclusion bodies (Shapiro and Ignoffo, 1968). Virions were stained with 2% phosphotungstic acid (PTA) at pH 7.2. Carbon replicas were prepared by the following procedure: Suspensions of inclusion bodies, dried on freshly cleaved mica strips, were shadowed at 15°C with palladium (20 mg) and then coated with carbon. The carbon-palladium film was stripped from mica with NaOH ( 1 N), and after 3 min was washed in water and then mounted on copper grids for viewing. A grid-treatment procedure was used to obtain the number of virions/inclusion body. Formvar-coated grids, on which a film of inclusion bodies was previously dried, were treated with NaOH for 3 min, flushed with distilled water, and then immediately stained with
NUCLEOPOLYHEDROSIS
2% PTA. The Hitachi electron microscope (HU-llB), vacuum evaporator (HUS-SB), and Zeiss Ultraphot were used for studies reported herein. RESULTS AND DISCUSSION
Inclusion
Body Description
Inclusion bodies are irregular and appear six-sided in outline (Fig. 1). The edges of the polyhedrons are rounded. The mean diameter of inclusion bodies is 916.4 39.5 mp (standard error of the mean) with a range from 300 to 2240 mu. Approximately 90% of the bodies range from 610 to 1200 rnp (Table 1). These measurements
FIG. 1.
Inclusion
bodies
from
virus-killed
OF Heliothis
187
are in good agreement with inclusion measurements from H. armigera which range from 700 to 1200 mu with the “majority measuring about 1100 mu” (Bergold and Rivers, 1957). No definite surface patterns or prominent structures are apparent (Fig. 2). Some polyhedron faces are rhomboids. Faces of highly purified inclusions ( -1 X 10” PIB/mg) were severely etched, revealing virion sockets and probable loss of virions (Fig. 3). A similar observation was reported for the nucleopolyhedrosis of Hemerobius (Hill and Smith, 1959) and the cytoplasmic polyhedrosis of Antheraea (Smith et al., 1959). Loss of virions plus severe clumping could partially explain the
Heliothis
zea
larvae
(4500
X ).
TABLE 1 FREQUENCY DISTRIBUTIONS OF INCLUSION DIAMETER AND NUMBER OF VIRIONS/ INCLUSION BODY FOR POLYHEDRAL INCLUSION BODIES OF Heliothis NUCLEOPOLYHEDROSIS
BODY
Virions Inclusion
2. larvae
FIG.
zea
Palladium-shadowed, (15,000X ).
body
Diameter b.)
Frequency
210409 410-609 610409 810-1009 1010-1209 1210-1409 1410-1609 1610-1809 1810-2009 2010-2209 2210-2409
2 23 225 208 122 24 23 3 3 1 1
carbon
replica
of unpurified
Inclusion body (Number)
Frequency
15-18 19-22 23-26 27-30 31-34 35-38
8 15 17 15 17 7
inclusion
bodies
from
virus-killed
Heliothis
NUCLEOPOLYHEDROSIS
FIG.
3.
Palladium-shadowed,
carbon
replica
reported loss in activity of highly purified inclusions (Ignoffo, 1964, 1965; Magnoler, 1968). Difficulties encountered in dissolving or staining inclusion bodies, retention of polyhedral shape following extreme physical forces, and electron microscopic observations of sectioned and degraded inclusions indicate that the outer layer(s) of inclusions may be different from those of its interior. Denaturation at the inclusion body surface could produce a coating of fibroustype proteins. This external coat should contain active --SH or phenolic -OH groups of tyrosine and a higher isoelectric point than the inner, undenatured, inclusion body protein, No change, however, in the lattice
OF
of highly
Heliothis
purified
189
inclusion
bodies
( 15,000
X ).
pattern of the outer layer is evident nor is a membrane or membranelike structure present to support this speculation.
Virion Description Virions are rod shaped and are probably helically symmetrical (Figs. 4, 5). The genetic material is DNA (Estes and Ignoffo, 1965). Virions are occluded in the cubic-protein lattice of the inclusion body without any apparent disruption of the lattice pattern Bundles of G-ions, as described in other nuclear polyhedroses (Bird, 1959; Bergold, 1963), were never observed. Based upon counts of 79 dissolved inclusion bodies the number of virions/inclusion body, standard
190
GREGORY,
FIG. 4.
Dissolved
inclusion
IGNOFFO,
body
deviation (SX) and standard error of the mean ( SE ) , was 26.4, 5.8, and 0.7, respectively. The range extended from 15 to 38 virionsjinclusion with 82% of the total sample containing between I9 and 34 virions/inclusion body (Table 1). The absolute dimension of the virion was somewhat dependent upon preparation techniques ( Table 2). Differences obtained, however, were not statistically significant at the 5% level. The average length and standard deviation of 599 alkali-treated virions was 336 + 22 rnp. Individual virion lengths ranged from 243 to 378 rnju, The
AND
and
SHAPIRO
released
virions
(25,000
X ).
average diameter was 62 2 4 mu with a range extending from 47 to 98 mu. The average dimension of H. armigera rods with developmental membranes measured 320 ” 10 my X 90 -C 10 rnp (Bergold and Rivers, 1957 ) . Structures similar to those described as virions of silkworm nucleopolyhedrosis by Kozlov and Alexeenko (1967) and as procedural artifacts by Summers and Paschke (1968) were also observed in preparations of Heliothis nucleopolyhedrosis virus (Fig. 6). These structures are quite similar to photomicrographs of bacterial rhapido-
NUCLEOPOLYHEDROSIS
‘IG. 5. Virions ~,ooOX ).
of
Heliothis
OF
nucleopolyhedrosis
after
TABLE
DIMENSIONS OF THE Heliothis
zea
Heliothis
concentration
from
Number measured
dissolved
inclusion
13odies
2
NUCLEOPOLYHEDROSIS VIFUONAFTER ALAKALINE TREATMENT OF INCLUSION BODIES Size
Treatment
191
in millimicrons
Average length
SDB
Average diameter
SD
Sodium
carbonate
115
373
16
75
7
Sodium
hydroxide
484
298
28
50
2
Q SD = Standard
deviation.
192
FIG.
GREGORY,
6.
Virionlike
structures
found
IGNOFFO,
in preparations
somes (Pate et al., 1967) and/or bacterial cell-wall fragments (Marsh and Walker, 1968 ) , REFERENCES BERGOLD, G. H. 1963. The nature of nuclear polyhedrosis. In “Insect Pathology-An Advanced Treatise” (E. A. Steinhaus, ed. ), Vol. 1, 413-456. Academic Press, New York. BERGOLD, G. H., AND RIPPER, W. E. 1957. The polyhedral virus of Heliothis armigeru ( Hbn. ) ( Lepidoptera: Noctuidae). Nature, 180, 764765. BIRD, F. T. 1959. Polyhedrosis and granulosis viruses causing single and double infections in the spruce budworm, Choristoneura fumiferana Clemens. I. Insect Pathol., 1, 406-430.
AND
SHAPIRO
of Heliothis
nucleopolyhedroris
virus
( 40,000
X ).
CHAPMAN, J. W., AND GLAYER, R. W. 1915. A preliminary list of insects which have wilt, with a comparative study of their polyhedra. J. Econ. Entomol., 8, 140-150. DOBROVQL~YA, H. M., KOK, I. P., SMIRNOVA, I. A., ANU CHISTYAIZOVA, A. V. 1965. Biological activity of DNA preparations isolated from the tissues of silkworms infected with nuclear polyhedrosis virus. Mikrobiol. Zhurnal., 27, 73-75. ESTES, Z. E., AND 1~x0~~0, C. M. 1965. The nucleic acid composition of nuclear polyhedral bodies affecting Heliothis zea (Boddie). J. Invertebrate Pathol., 7, 25&259. HILL, G. J., AND SMITH, K. M. 1959. Further studies on the isolation and crystallization of insect cytoplasmic viruses. J. Insect Pathol., 1, 121-128. IGNOFFO, C. M. 1964. Production and virulence
NUCLEOPOLYHEDROSIS
.
of a nuclear polyhedrosis virus from Trichoplusia ni (Hubner) reared on a semisynthetic diet. J. Insect Pathol., 6, 318-326. IGNOFFO, C. M. 1965. The nuclear polyhedrosis virus of Heliothis zeu (Boddie) and Heliothis uirescens (Fabricius). Part I. Virus propaand its virulence, J. Invertebrate gation Pathol., 7, 209-216. IGNOFFO, C. M. 1968. Viruses-Living Insecticides. Current Topics Microbial. Immunol. ( K. Maramorosch, ed. ), 42, 129-167. KOZLOV, E. A., AND ALEXEENKO, I. P. 1967. Electron-microscope investigations of the structure of the nuclear-polyhedrosis virus of the silkworm, Bombyx mod. J. Invertebrate Pathol., 9, 413419. LOUNSBURY, C. P. 1913. Caterpillar wilt disease. 1. Agr. South Africa, 5, 448-452, MAGNOLER, A. 1968. The differing effectiveness of purified and nonpurified suspensions of the nuclear-polyhedrosis virus of Porthetria &par. I. Invertebrate Pathol., 11, 326328. MALLY, F. W. 1891. The bollworm of cotton. U.S. Bur. Entomol. Bull., 24, 48-50. MALLY, F. W. 1892. Report of progress in the investigation of the cotton bollworm. U.S. Bur. Entomol. Bull., 26, 54-56. MARSH, D. G., AND WALKER, P. D. 1968. Free endotoxin and non-toxic material from gramnegative bacteria: Electron microscopy of
OF
Heliothis
193
fractions from Escherichia coli. J. Gen. MicrobioZ., 52, 125-130. Prog. Rept. Expt. Stas. PARSONS, F. S. 1936. Empire Cotton Growing Corp., Barberton, South Africa, 1934-1935, 24-31. PATE,
J. L., JOHNSON, J. L., AND ORDAL, E. J. 1967. The fine structure of Chondrococcus column&s. II. Structure and formation of rhapidosomes. J. Cell Biol., 35, 15-35. SHAPIRO, M., AND IGNOFFO, C. M. 1968. Characterization of Heliothis nucleopolyhedrosis virus. International Minerals and Chemical Quarterly Report, July-Sept. 1968. SHAPIRO, M., AND IGNOF'FO, c. M. 1969. Nuclear polyhedrosis of Heliothis: Stability and relative infectivity of virions. I. Invertebrate Pathol., 14, 130-134. SMITH, K. M., AND RIVERS, C. F. 1956. Some viruses affecting insects of economic importance. Parasitology, 46, 235-242. SMITH, K. M., HILL, G. J., AND RNERS, C. F. 1959. Polyhedroses in neuropterous insects. J. Insect Pathol., 1, 431437. 1957. New records of insectSTEINHAUS, E. A. virus diseases. Hilgardia, 26, 417-430. SUMXERS, M. D., AND PASCHKE, J. D. 1968. Observations on an artifact present in Ttichop&a ni granulosis-virus preparations. J. Invertebrate Pathol., 11, 520-523.