- Email: [email protected]
A new nuclear polyhedrosis of the cabbage looper, Trichoplusia ni
JOURNAL
OF INVERTEBRATE
340-346 (1966)
PATHOLOGY
8,
A New
Nuclear
Cabbage
Polyhedrosis
Looper,
A. M. HEIMPEL Entomology U.S.
Research Department
Trichoplusia
the ni
AND J. R. ADAMS
Division, of Agriculture,
Accepted
of
February
Agricultural Research Beltsville, Maryland
Service
1, 1966
Studies of the larvae of Trichoplusia ni infected with different samples of nuclearpolyhedrosis virus obtained from several geographical areas indicated that these viral populations contained two morphologically distinct viruses which replicate in separate nuclei of susceptible tissues. Polyhedra, containing bundles of rods enclosed in double membranes, were found in nuclei of cells of the gut, fat body, hypodermis, and tracheal matrix. Smaller polyhedra, containing single rods enclosed in double membranes, were found in nuclei of cells of the fat body, hypodermis, tracheal matrix and, rarely, in the
gut.
California, South Carolina, Texas, and VirFor years it has been generally accepted ginia, showed the gut virus present in matethat the nuclear-polyhedrosis viruses affect- rial from all geographical areas sampled. An electron microscopic examination of the ing Lepidoptera infect some of the tissuesin gut nuclear polyhedra and the nuclear polythe hemocoel, including the fat body, hypohedra from hemocoel tissueswas carried out dermis, cells of the tracheal matrix, and the to determine whether the viruses were morblood cells. These viruses are classed in the phologically similar. We noted that polyhedra genus Borrelinavirus. Another group of nuof two sizes were present in the nuclei of fat, clear-polyhedral virus diseases infect the tracheal, and epidermal cells; these differentnuclei of the gut cells of speciesin the family Tenthredinidae, Hymenoptera. These viruses sized polyhedra were never mixed in a cell have been proposed to the International Bac- but occupied the nuclei of separate cells. This teriological Nomenclature Committee under phenomenon was also investigated and is discussed. the genus name Birdiavims. Mention has been made of polyhedra in METHODS AND MATERIALS the nuclei of lepidopteran gut cells by Bergold (1953), and Tanada (1954) and Cabbage-looper larvae were obtained from Laudeho and Amargier (1963); however, a laboratory stock reared on a semisynthetic they paid no particular attention to this diet described by Ignoffo (1963), modified by phenomenon. Recently, during a routine his- the addiiton of dried, ground alfalfa. The topathological examination, we detected medium was held in quart jars, each conlarge numbers of relatively large polyhedra taining approximately 50 looper larvae. Each in the nuclei of the gut cells of larvae of the jar was inoculated with a water suspension cabbage looper, Tm’ckoplusia ni. Subsequent of approximately 3 X lo7 polyhedra after examination of larvae infected with virus the larvae had reached the third instar. Inmaterial, acquired from New York State, fected larvae, collected on the 3rd and 4th 340
NEW
Fx:s. 1-4. Polyhedral 1. Midgut epithelium.
body 940 X.
NUCLEAR
POLYHEDROSIS
formation in the nuclei of cells of the following tissues of Trichoplusia 2. Fat body. 940 X. 3. Tracheal matrix. 940 X. 4. Epidermis. 940 X.
341
ni.
342
FIG.
(NM,
HEIMPEL
5. Section of a gut epithelial cell nucleus nuclear membrane). 2 1,600 X.
AND
ADAMS
of the cabbage
looper
showing
polyhedral
body
formation
NEW
FIG.
6.
Section
of an epidermal
NUCLEAR
cell
nucleus
343
POLYHEDROSIS
showing
polyhedral
body
formation.
22,2.10 x
FIG. 7. FIG. 8. membranes. FIG, 9. 10,260 X.
Section Section 10,260 Section
of polyhedra containing bundles of virus rods enclosed within a double membrane. 48,150 X. of an epidermal cell nucleus showing enclosure of bundles of virus rods within double X. of an epidermal cell nucleus showing enclosure of single rods within a double membrane. 344
NEW
NUCLEAR
day after contamination with virus, were fixed in Bouin-Duboscq solution, dehydrated in an ethanol or a butyl alcohol series, and embedded according to standard procedures. Sections (2-4 p) were stained with Hamm’s modification of Hubschman’s (1962) triple stain (personal communication). Tissues for electron microscopy were fixed in either Scollidine-buffered (Bennett and Luft, 1959) or phosphate-buffered osmium tetroxide (Millonig, 1962), dehydrated in an ethanol series, and embedded in either methacrylate or Epon-Araldite (Mollenhauer, 1964). Sections cut with an LKB Ultratomel were double-stained in uranyl acetate and lead citrate (Reynolds, 1963) and examined in a Hitachi HC-11’ electron microscope.
345
POLYHEDROSIS
hypodermis, tracheal matrix, and fat body (Figs. 6 and 9), and appeared rarely in the nuclei of the gut cells. CONCLUSIONS
There is no alternative but to conclude that populations of the nuclear-polyhedrosis virus specific for T. ni (from the geographical areas mentioned) are mixed. One virus with single rods embedded in the polyhedron appears to affect all susceptible tissues of the body, but mainly the tissues of the hemocoel. A second virus that forms bundles of rods in the polyhedron is frequently found in the nuclei of the gut cells; however, it also infects tissues of the hemocoel. The implication of this fact upon previously conducted bioassays and field trials is obRESULTS vious. We are taking immediate steps to try to separate the viruses and test them inIn the gut the nuclear-polyhedrosis virus dividually. appears to form polyhedra in nuclei of coThis discovery stimulated the histopatholumnar cells of midgut epithelium on about logical investigation of the corn earworm. the 2nd to 3rd day after infection, whereas Heliothis zea, with “specific nuclear-polyin the fat body and other tissues in the hedrosis virus” of that insect. At this time hemocoel the polyhedra form slightly later. we can only report that polyhedra have been Figure 1 shows gut tissue with a few polydetected in the nuclei of the gut of this inhedra in the nuclei. Figures 2-4 show fat sect. The possibility that this virus is again body, tracheal matrix, and epidermal tissues a different virus is being investigated. One from an infected insect taken 4 days after is also tempted to speculate whether these infection. viruses that can propagate in gut-cell nuclei Thin sections of the infected gut, trachea, might not be nonspecific viruses that might epidermis, and fat body clearly show the readily cross-infect other species since they formation of bundles of virus rods containare apparently more common than imagined ing numbers of virus rods surrounded by a heretofore. double membrane. Figure 5 shows these .hCKNOWLEDGMENT bundles as they are being incorporated into the polyhedra. The virus that forms bundles We are grateful for the technical help given us Wilcox and Mr. Richard M. of rods enclosed in a double membrane in- by Mr. Theodore fected all susceptible tissuesincluding nuclei Lipscomb. of the gut, tracheal matrix, hypodermis, and REFEREXCES fat body (Figs. 5, 7, and 8). The virus that BENNETT, H. S., AND LUFT, J. H. 1939. S-Collicontains single rods, exclusively, embedded dine as a basis for buffering fixatives. J. Biopkys. in the polyhedron, was most prevalent in the Biockem. Cytol., 6, 113. G. H. 1953. Insect viruses. In “.4dvances in Virus Research,” (K. M. Smith and M. A. Lauffer, eds.), Vol. 1, pp. 91-139. Academic Press, New York.
BERGOLD,
1 Mention of a proprietary product not necessarily imply endorsement of by the U.S. Department of Agriculture.
herein does this product
346
HEIMPEL
HUBSCHMAN, J. H. 1962. A simplified azan process well suited for crustacean tissue. Stain. Technol., 37, 379-380. IGNOFFO, C. M. 1963. A successful technique for mass-rearing cabbage loopers on a semi-synthetic diet. Ann. Entomol. Sot. Am., 56, 178-182. LAUDEHO, Y., AND AMARGIE~, A. 1963. Virose a poly&&es nuclCaires & localisation inhabituelle chez un lCpidopt&re. Rev. Pathol. Vtgetale Entontol. Agr. France, 52, 207-210. MILLONIG, G. 1962. Further observations on a phosphate buffer for osmium solutions. Proc.
AND
ADAMS
5th Intern. Congr. Electron Microscopy, 2, P-8. (Academic Press, New York). MOLLENHAUER, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol., 99, 113-114. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell&r Biol., 17, 208-212. TANADA, Y. 1954. A polyhedrosis virus of the imported cabbage worm and its relation to a polyhedrosis virus of the alfalfa caterpillar. Ann. Entomol. Sot. Am., 47, 553-5’14.
OF INVERTEBRATE
340-346 (1966)
PATHOLOGY
8,
A New
Nuclear
Cabbage
Polyhedrosis
Looper,
A. M. HEIMPEL Entomology U.S.
Research Department
Trichoplusia
the ni
AND J. R. ADAMS
Division, of Agriculture,
Accepted
of
February
Agricultural Research Beltsville, Maryland
Service
1, 1966
Studies of the larvae of Trichoplusia ni infected with different samples of nuclearpolyhedrosis virus obtained from several geographical areas indicated that these viral populations contained two morphologically distinct viruses which replicate in separate nuclei of susceptible tissues. Polyhedra, containing bundles of rods enclosed in double membranes, were found in nuclei of cells of the gut, fat body, hypodermis, and tracheal matrix. Smaller polyhedra, containing single rods enclosed in double membranes, were found in nuclei of cells of the fat body, hypodermis, tracheal matrix and, rarely, in the
gut.
California, South Carolina, Texas, and VirFor years it has been generally accepted ginia, showed the gut virus present in matethat the nuclear-polyhedrosis viruses affect- rial from all geographical areas sampled. An electron microscopic examination of the ing Lepidoptera infect some of the tissuesin gut nuclear polyhedra and the nuclear polythe hemocoel, including the fat body, hypohedra from hemocoel tissueswas carried out dermis, cells of the tracheal matrix, and the to determine whether the viruses were morblood cells. These viruses are classed in the phologically similar. We noted that polyhedra genus Borrelinavirus. Another group of nuof two sizes were present in the nuclei of fat, clear-polyhedral virus diseases infect the tracheal, and epidermal cells; these differentnuclei of the gut cells of speciesin the family Tenthredinidae, Hymenoptera. These viruses sized polyhedra were never mixed in a cell have been proposed to the International Bac- but occupied the nuclei of separate cells. This teriological Nomenclature Committee under phenomenon was also investigated and is discussed. the genus name Birdiavims. Mention has been made of polyhedra in METHODS AND MATERIALS the nuclei of lepidopteran gut cells by Bergold (1953), and Tanada (1954) and Cabbage-looper larvae were obtained from Laudeho and Amargier (1963); however, a laboratory stock reared on a semisynthetic they paid no particular attention to this diet described by Ignoffo (1963), modified by phenomenon. Recently, during a routine his- the addiiton of dried, ground alfalfa. The topathological examination, we detected medium was held in quart jars, each conlarge numbers of relatively large polyhedra taining approximately 50 looper larvae. Each in the nuclei of the gut cells of larvae of the jar was inoculated with a water suspension cabbage looper, Tm’ckoplusia ni. Subsequent of approximately 3 X lo7 polyhedra after examination of larvae infected with virus the larvae had reached the third instar. Inmaterial, acquired from New York State, fected larvae, collected on the 3rd and 4th 340
NEW
Fx:s. 1-4. Polyhedral 1. Midgut epithelium.
body 940 X.
NUCLEAR
POLYHEDROSIS
formation in the nuclei of cells of the following tissues of Trichoplusia 2. Fat body. 940 X. 3. Tracheal matrix. 940 X. 4. Epidermis. 940 X.
341
ni.
342
FIG.
(NM,
HEIMPEL
5. Section of a gut epithelial cell nucleus nuclear membrane). 2 1,600 X.
AND
ADAMS
of the cabbage
looper
showing
polyhedral
body
formation
NEW
FIG.
6.
Section
of an epidermal
NUCLEAR
cell
nucleus
343
POLYHEDROSIS
showing
polyhedral
body
formation.
22,2.10 x
FIG. 7. FIG. 8. membranes. FIG, 9. 10,260 X.
Section Section 10,260 Section
of polyhedra containing bundles of virus rods enclosed within a double membrane. 48,150 X. of an epidermal cell nucleus showing enclosure of bundles of virus rods within double X. of an epidermal cell nucleus showing enclosure of single rods within a double membrane. 344
NEW
NUCLEAR
day after contamination with virus, were fixed in Bouin-Duboscq solution, dehydrated in an ethanol or a butyl alcohol series, and embedded according to standard procedures. Sections (2-4 p) were stained with Hamm’s modification of Hubschman’s (1962) triple stain (personal communication). Tissues for electron microscopy were fixed in either Scollidine-buffered (Bennett and Luft, 1959) or phosphate-buffered osmium tetroxide (Millonig, 1962), dehydrated in an ethanol series, and embedded in either methacrylate or Epon-Araldite (Mollenhauer, 1964). Sections cut with an LKB Ultratomel were double-stained in uranyl acetate and lead citrate (Reynolds, 1963) and examined in a Hitachi HC-11’ electron microscope.
345
POLYHEDROSIS
hypodermis, tracheal matrix, and fat body (Figs. 6 and 9), and appeared rarely in the nuclei of the gut cells. CONCLUSIONS
There is no alternative but to conclude that populations of the nuclear-polyhedrosis virus specific for T. ni (from the geographical areas mentioned) are mixed. One virus with single rods embedded in the polyhedron appears to affect all susceptible tissues of the body, but mainly the tissues of the hemocoel. A second virus that forms bundles of rods in the polyhedron is frequently found in the nuclei of the gut cells; however, it also infects tissues of the hemocoel. The implication of this fact upon previously conducted bioassays and field trials is obRESULTS vious. We are taking immediate steps to try to separate the viruses and test them inIn the gut the nuclear-polyhedrosis virus dividually. appears to form polyhedra in nuclei of coThis discovery stimulated the histopatholumnar cells of midgut epithelium on about logical investigation of the corn earworm. the 2nd to 3rd day after infection, whereas Heliothis zea, with “specific nuclear-polyin the fat body and other tissues in the hedrosis virus” of that insect. At this time hemocoel the polyhedra form slightly later. we can only report that polyhedra have been Figure 1 shows gut tissue with a few polydetected in the nuclei of the gut of this inhedra in the nuclei. Figures 2-4 show fat sect. The possibility that this virus is again body, tracheal matrix, and epidermal tissues a different virus is being investigated. One from an infected insect taken 4 days after is also tempted to speculate whether these infection. viruses that can propagate in gut-cell nuclei Thin sections of the infected gut, trachea, might not be nonspecific viruses that might epidermis, and fat body clearly show the readily cross-infect other species since they formation of bundles of virus rods containare apparently more common than imagined ing numbers of virus rods surrounded by a heretofore. double membrane. Figure 5 shows these .hCKNOWLEDGMENT bundles as they are being incorporated into the polyhedra. The virus that forms bundles We are grateful for the technical help given us Wilcox and Mr. Richard M. of rods enclosed in a double membrane in- by Mr. Theodore fected all susceptible tissuesincluding nuclei Lipscomb. of the gut, tracheal matrix, hypodermis, and REFEREXCES fat body (Figs. 5, 7, and 8). The virus that BENNETT, H. S., AND LUFT, J. H. 1939. S-Collicontains single rods, exclusively, embedded dine as a basis for buffering fixatives. J. Biopkys. in the polyhedron, was most prevalent in the Biockem. Cytol., 6, 113. G. H. 1953. Insect viruses. In “.4dvances in Virus Research,” (K. M. Smith and M. A. Lauffer, eds.), Vol. 1, pp. 91-139. Academic Press, New York.
BERGOLD,
1 Mention of a proprietary product not necessarily imply endorsement of by the U.S. Department of Agriculture.
herein does this product
346
HEIMPEL
HUBSCHMAN, J. H. 1962. A simplified azan process well suited for crustacean tissue. Stain. Technol., 37, 379-380. IGNOFFO, C. M. 1963. A successful technique for mass-rearing cabbage loopers on a semi-synthetic diet. Ann. Entomol. Sot. Am., 56, 178-182. LAUDEHO, Y., AND AMARGIE~, A. 1963. Virose a poly&&es nuclCaires & localisation inhabituelle chez un lCpidopt&re. Rev. Pathol. Vtgetale Entontol. Agr. France, 52, 207-210. MILLONIG, G. 1962. Further observations on a phosphate buffer for osmium solutions. Proc.
AND
ADAMS
5th Intern. Congr. Electron Microscopy, 2, P-8. (Academic Press, New York). MOLLENHAUER, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol., 99, 113-114. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell&r Biol., 17, 208-212. TANADA, Y. 1954. A polyhedrosis virus of the imported cabbage worm and its relation to a polyhedrosis virus of the alfalfa caterpillar. Ann. Entomol. Sot. Am., 47, 553-5’14.