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Cytopathology of a nucleopolyhedrosis of the beet armyworm, Spodoptera exigua
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
Cytopathology
12, %-97
( 1968)
of a Nucleopolyhedrosis Spodoptera D.K.
Department
HUNTER'~'
of Biological Riuerside, Received
of the Beet Armyworm,
erigua AND I. M. HALL::
Control, California January
University 92502
of California,
16, 1968
The pathology of the fat cells during the course of a nucleopolyhedrosis of the beet armyworm, Spodoptera exigua, is described. Ribosome-filled cytoplasmic rings were observed around nuclei after 3 days of infection. These rings may be the sites of polyhedral protein synthesis. Nuclei in adipose tissue hypertrophied from 8.4 to 25.5 p in diameter after 4 days of infection. The inclusion bodies were irregularly shaped and varied from 1 to 6 + (average 2.05 p) in diameter. Membranelike structures, often discontinuous, were observed around polyhedra; they apparently deVirus particles within the inclusions veloped after protein deposition had ceased. always occurred in bundles of 2-7 rods ( average 3.13 CL).
neberry and Kishaba, 1966) each of which had been inoculated with a droplet of virus suspension ( 2.5 X lO* polyhedra/disc). Controls were fed discs treated with distilled water. After 24 hr of incubation at 27”C, larvae that had consumed all of the medium were placed into ‘/2-pint cartons with diet and reincubated. For sectioning, five larvae were removed at 24-hr intervals for the first 3 days and at 12-hr intervals through the 5th day. The specimens were fixed in alcoholic Bouin’s Solution ( Duboscq-Brasil modification) and embedded in Paraplast. Sections from the midgut region were cut at 7 p and stained according to Hamm ( 1966). Nuclei and polyhedra were measured with an ocular micrometer.
INTRODUCTION The nucleopolyhedrosis of the beet armyworm, Spodoptera exigua, was first reported by Steinhaus ( 1949, 1951, 1957), who determined the dimensions of the virus rods to be approximately 270 X 40 mp. Since that time, little additional information has appeared in the literature on the disease or pathogen. The purpose of this study was to describe the larval cytopathology as observed with light and electron microscopes. Emphasis was placed on infection in the fat body. METHODS AND MATERIALS Light Microscope Studies. Individual fourth-instar larvae from an insectary culture were fed discs of artificial diet a (Hen-
Electron Microscope Studies. Healthy and diseased larvae were collected on the 3rd and 4th days following ingestion of the inoculum. Adipose cubes (2-3 mm square) were dissected from tissue lying laterad to the midgut region and fixed in buffered 2.5% glutaraldehyde. The tissue was postfixed in 1% osmium tetroxide and embedded in Maraglas (Bisalputra and Weier, 1963). Ultrathin sections in the sliver-gray inter-
* Extracted from a doctorate dissertation accepted by the Department of Entomology, University of California, Riverside, July 24, 1967. 2 Present address: Stored-Product Insects Research Laboratory, Department of Agriculture, Fresno, California. 3 Professor of Insect Pathology, Department of Biological Control, University of California, Riverside. 4 Formaldehyde was excluded. 93
94
HUNTER
AND
ference range were cut and stained in saturated uranyl acetate and 1.4% lead citrate. Examination was done on a Hitachi HU-11 electron microscope at 75 kv. RESULTS
Light
Microscope
Studies
Cells of the fat body, tracheal matrix, and hypodermis were susceptible to infection. Effects on the hemocytes were not investigated. There was no apparent difference between healthy (Fig. 1) and infected fat tissue (F) for the first 24 hr following ingestion of the polyhedra. However, the infected fat nuclei began to hypertrophy on the 2nd day and continued to enlarge through the 4th day. Table 1 shows size change as determined by measurement of 50 nuclei/day of infection. Virogenic stromata (VS) of the type reported by Xeros (1956) appeared in about 3.5 days (Fig. 2) followed by polyhedra (P) development on the 4th day (Fig. 3). Most infected nuclei had disintegrated by 4.5 days. On the 5th day after infection, the integrity of the fat body was completely lost and the hemocoel was filled with released polyhedra (Fig. 4). Figure 2 also shows advanced symptoms of infection in the hypodermis (H) and TABLE CHANGE
Days Control
IN DIAMETER FAT CELLS OF
1 OF DISEASED
Spodoptera
of infection ( l-5 1
NUCLEI
Diameter
a.4 8.4 9.5 12.6 17.3 25.5
days )
2 3 3.5 4 4.5 5 a No measurements disintegrated.
taken
since
IN
ex&ua
most
(p)
k 2 2 f 2 2 -n -a
+
c
2.33 2.00 1.55 2.40 4.61 3.30
nuclei
HALL
trachea (T) while the fat cells have not yet Figure 3 begun polyhedron formation. shows the variability in degree of infection in adjacent fat cells. Variability also OCcurred between like tissues of larvae in a sample. Polyhedral size ranged from less than 1 ~1 to about 6 11in diameter with a mean of 2.05 ZL 0.60. Electron
Microscope
Studies
Healthy fat nuclei exhibited several electron-dense nucleoli ( NL) (Fig. 5). Less compact particulate patches of chromatin (C) occurred throughout the nucleoplasm. Nucleoli were not observed on the 3rd day of infection. The chromatin began aggregating into large clumps (CC) and a peculiar ring of cytoplasm (CR) was observed around the nucleus (Figs. 6 and 7). The ring was composed of ribosomes (R) embedded in a less dense homogenous matrix. Peripheral to the ring occurred numerous glycogen granules ( G ) . Polyhedra were irregular in form and the larger mature-appearing inclusions exhibited membranelike structures similar to those reported by Benz ( 1963). The membranes (M ) were discontinuous at points where virus bundles (VB) were not yet completely embedded (Fig. 8). Figure 9 shows the “pericrystalline lattice” of a polyhedron and the double developmental membrane (DM) limiting the virus bundles. Based on 192 observations, numbers of rods/bundle ranged from 2 to 7 with a mean of 3.13 2 1.27. DISCUSSION
had
Nuclei of the tracheal matrix and hypodermis generally showed advanced symptoms of infection before those of the fat body. This observation would suggest that the infective agent either invades susceptible tissue at random, or that cells of the trachea and hypodermis have inherent
CYTOPATHOLOGY
of 3.
FIGS. 1-4. infection. Polyhedra
OF
ARMYWORM
NUCLEOPOLYHEDROSIS
95
296 X. 1. Fat lobes of healthy larva. 2. Virogenic stromata (VS) in fat cells after 3 days Hypodermis (H) and tracheal m atrix (T) showing polyhedra (P) development. in fat cells after 4 days of infection. 4. Polyhedra in the hemocoel (5th day of infection).
96
HUNTER AND HALL
FIGS. 5-9. 5. Healthy fat cell nucleus with electron-dense nucleoli 9,418 X. 6, 7. Fat nuclei after 3 days of infection. Chromatin clumps (CR) composed of ribosomes (R) are demonstrated. Glycogen granules 8. Polyhedra with membranelike 11.,880 X and 22,860 X, respectively. virus bundles (VB). 41,400 X. Polyhedron section showing protein membrane (DM) limiting the virus bundles. 179,400 X.
(NL) and chromatin patches (C). (CC) and the cytoplasmic ring (G) lie peripheral to the ring. structure ( M ) and embedded lattice (PL) and developmental
CYTOPATHOLOGY
OF
ARMYWORM
NUCLEOPOLYHEDROSIS
97
REFERENCES properties which enable the infection process to progress at a faster rate. BENZ, G. 1963. A nuckar polyhedrosis of MalaThe presence of the cytoplasmic ring cosoma alpicola (Stdinger). J. Insect PathoE., demonstrates a very close relationship 5, 215-241. between rihosomes and the nuclear en- BISALPUTRA, T., AND \VEIEH, T. E. 1963. The cell wall of Scencdesnws quadricada. Am. J. velope prior to polyhedral development. Botany, 50, 1001-1019. Krywienczyk ( 1963) suggested that polyHAAM, J. J. 1966. A modified aznn staining techhedral protein in Bondn~~ tori was synthenique for inclusion body viruses. J. Inuertesized in the cytoplasm and later concenbrute Pathol., 8, 125-U& trated in the nucleus. Therefore, it is HENXEHEHRY, T. J., AND KISHAUA, A. N. 1966. possible that in S. exigua, the site of polyhlass rearing cabbage loopers. In “Insect hedral protein synthesis is the cytoplasmic Colonization and hlass Rearing” (C. N. ring. The absence of nucleoli when the Smith ea.). Pp. 461-477. Academic Press, ring was observed may be a result of an N. Y. accelerated demand for nucleoIar RNA for KFZWIENCZYK, J. 1963. Demonstration of nuclear polyhedrosis in Bonlb~x mori (Linnaeus) by protein synthesis. This demand may refluorescent antibody technique. J. Inoertebrate quire RNA at a rate too rapid for immediate Pathol., 5, 309-317. replenishment, thereby causing the breakSTEINHAUS, E. A. 1949. Nomenclature and classidown of these bodies. fication of insect viruses. Bacterial. Reu., 13, Membranelike structures were seen 203-223. around “mature” polyhedra. Their disSTEINHAUS, E. A. 1951. Report on diagnoses of continuous development suggests that they diseased insects 1944-1950. Hilgardia, 20, form after virus bundles are completely en629-678. closed in polyhedral protein. Thus, the STEINHAUS, E. A. 1957. New records of insect presence of a continuous “membrane” may virus diseases. Ifilgndiu, 26, 417-430. be indicative of a polyhedron in which XEROS, N. 1956. The virogenic stroma in nuclear peripheral areas have stabilized and protein and cytoplasmic polyhedroses. Nature, 173, 412-413. deposition has ceased.
OF
INVERTEBRATE
PATHOLOGY
Cytopathology
12, %-97
( 1968)
of a Nucleopolyhedrosis Spodoptera D.K.
Department
HUNTER'~'
of Biological Riuerside, Received
of the Beet Armyworm,
erigua AND I. M. HALL::
Control, California January
University 92502
of California,
16, 1968
The pathology of the fat cells during the course of a nucleopolyhedrosis of the beet armyworm, Spodoptera exigua, is described. Ribosome-filled cytoplasmic rings were observed around nuclei after 3 days of infection. These rings may be the sites of polyhedral protein synthesis. Nuclei in adipose tissue hypertrophied from 8.4 to 25.5 p in diameter after 4 days of infection. The inclusion bodies were irregularly shaped and varied from 1 to 6 + (average 2.05 p) in diameter. Membranelike structures, often discontinuous, were observed around polyhedra; they apparently deVirus particles within the inclusions veloped after protein deposition had ceased. always occurred in bundles of 2-7 rods ( average 3.13 CL).
neberry and Kishaba, 1966) each of which had been inoculated with a droplet of virus suspension ( 2.5 X lO* polyhedra/disc). Controls were fed discs treated with distilled water. After 24 hr of incubation at 27”C, larvae that had consumed all of the medium were placed into ‘/2-pint cartons with diet and reincubated. For sectioning, five larvae were removed at 24-hr intervals for the first 3 days and at 12-hr intervals through the 5th day. The specimens were fixed in alcoholic Bouin’s Solution ( Duboscq-Brasil modification) and embedded in Paraplast. Sections from the midgut region were cut at 7 p and stained according to Hamm ( 1966). Nuclei and polyhedra were measured with an ocular micrometer.
INTRODUCTION The nucleopolyhedrosis of the beet armyworm, Spodoptera exigua, was first reported by Steinhaus ( 1949, 1951, 1957), who determined the dimensions of the virus rods to be approximately 270 X 40 mp. Since that time, little additional information has appeared in the literature on the disease or pathogen. The purpose of this study was to describe the larval cytopathology as observed with light and electron microscopes. Emphasis was placed on infection in the fat body. METHODS AND MATERIALS Light Microscope Studies. Individual fourth-instar larvae from an insectary culture were fed discs of artificial diet a (Hen-
Electron Microscope Studies. Healthy and diseased larvae were collected on the 3rd and 4th days following ingestion of the inoculum. Adipose cubes (2-3 mm square) were dissected from tissue lying laterad to the midgut region and fixed in buffered 2.5% glutaraldehyde. The tissue was postfixed in 1% osmium tetroxide and embedded in Maraglas (Bisalputra and Weier, 1963). Ultrathin sections in the sliver-gray inter-
* Extracted from a doctorate dissertation accepted by the Department of Entomology, University of California, Riverside, July 24, 1967. 2 Present address: Stored-Product Insects Research Laboratory, Department of Agriculture, Fresno, California. 3 Professor of Insect Pathology, Department of Biological Control, University of California, Riverside. 4 Formaldehyde was excluded. 93
94
HUNTER
AND
ference range were cut and stained in saturated uranyl acetate and 1.4% lead citrate. Examination was done on a Hitachi HU-11 electron microscope at 75 kv. RESULTS
Light
Microscope
Studies
Cells of the fat body, tracheal matrix, and hypodermis were susceptible to infection. Effects on the hemocytes were not investigated. There was no apparent difference between healthy (Fig. 1) and infected fat tissue (F) for the first 24 hr following ingestion of the polyhedra. However, the infected fat nuclei began to hypertrophy on the 2nd day and continued to enlarge through the 4th day. Table 1 shows size change as determined by measurement of 50 nuclei/day of infection. Virogenic stromata (VS) of the type reported by Xeros (1956) appeared in about 3.5 days (Fig. 2) followed by polyhedra (P) development on the 4th day (Fig. 3). Most infected nuclei had disintegrated by 4.5 days. On the 5th day after infection, the integrity of the fat body was completely lost and the hemocoel was filled with released polyhedra (Fig. 4). Figure 2 also shows advanced symptoms of infection in the hypodermis (H) and TABLE CHANGE
Days Control
IN DIAMETER FAT CELLS OF
1 OF DISEASED
Spodoptera
of infection ( l-5 1
NUCLEI
Diameter
a.4 8.4 9.5 12.6 17.3 25.5
days )
2 3 3.5 4 4.5 5 a No measurements disintegrated.
taken
since
IN
ex&ua
most
(p)
k 2 2 f 2 2 -n -a
+
c
2.33 2.00 1.55 2.40 4.61 3.30
nuclei
HALL
trachea (T) while the fat cells have not yet Figure 3 begun polyhedron formation. shows the variability in degree of infection in adjacent fat cells. Variability also OCcurred between like tissues of larvae in a sample. Polyhedral size ranged from less than 1 ~1 to about 6 11in diameter with a mean of 2.05 ZL 0.60. Electron
Microscope
Studies
Healthy fat nuclei exhibited several electron-dense nucleoli ( NL) (Fig. 5). Less compact particulate patches of chromatin (C) occurred throughout the nucleoplasm. Nucleoli were not observed on the 3rd day of infection. The chromatin began aggregating into large clumps (CC) and a peculiar ring of cytoplasm (CR) was observed around the nucleus (Figs. 6 and 7). The ring was composed of ribosomes (R) embedded in a less dense homogenous matrix. Peripheral to the ring occurred numerous glycogen granules ( G ) . Polyhedra were irregular in form and the larger mature-appearing inclusions exhibited membranelike structures similar to those reported by Benz ( 1963). The membranes (M ) were discontinuous at points where virus bundles (VB) were not yet completely embedded (Fig. 8). Figure 9 shows the “pericrystalline lattice” of a polyhedron and the double developmental membrane (DM) limiting the virus bundles. Based on 192 observations, numbers of rods/bundle ranged from 2 to 7 with a mean of 3.13 2 1.27. DISCUSSION
had
Nuclei of the tracheal matrix and hypodermis generally showed advanced symptoms of infection before those of the fat body. This observation would suggest that the infective agent either invades susceptible tissue at random, or that cells of the trachea and hypodermis have inherent
CYTOPATHOLOGY
of 3.
FIGS. 1-4. infection. Polyhedra
OF
ARMYWORM
NUCLEOPOLYHEDROSIS
95
296 X. 1. Fat lobes of healthy larva. 2. Virogenic stromata (VS) in fat cells after 3 days Hypodermis (H) and tracheal m atrix (T) showing polyhedra (P) development. in fat cells after 4 days of infection. 4. Polyhedra in the hemocoel (5th day of infection).
96
HUNTER AND HALL
FIGS. 5-9. 5. Healthy fat cell nucleus with electron-dense nucleoli 9,418 X. 6, 7. Fat nuclei after 3 days of infection. Chromatin clumps (CR) composed of ribosomes (R) are demonstrated. Glycogen granules 8. Polyhedra with membranelike 11.,880 X and 22,860 X, respectively. virus bundles (VB). 41,400 X. Polyhedron section showing protein membrane (DM) limiting the virus bundles. 179,400 X.
(NL) and chromatin patches (C). (CC) and the cytoplasmic ring (G) lie peripheral to the ring. structure ( M ) and embedded lattice (PL) and developmental
CYTOPATHOLOGY
OF
ARMYWORM
NUCLEOPOLYHEDROSIS
97
REFERENCES properties which enable the infection process to progress at a faster rate. BENZ, G. 1963. A nuckar polyhedrosis of MalaThe presence of the cytoplasmic ring cosoma alpicola (Stdinger). J. Insect PathoE., demonstrates a very close relationship 5, 215-241. between rihosomes and the nuclear en- BISALPUTRA, T., AND \VEIEH, T. E. 1963. The cell wall of Scencdesnws quadricada. Am. J. velope prior to polyhedral development. Botany, 50, 1001-1019. Krywienczyk ( 1963) suggested that polyHAAM, J. J. 1966. A modified aznn staining techhedral protein in Bondn~~ tori was synthenique for inclusion body viruses. J. Inuertesized in the cytoplasm and later concenbrute Pathol., 8, 125-U& trated in the nucleus. Therefore, it is HENXEHEHRY, T. J., AND KISHAUA, A. N. 1966. possible that in S. exigua, the site of polyhlass rearing cabbage loopers. In “Insect hedral protein synthesis is the cytoplasmic Colonization and hlass Rearing” (C. N. ring. The absence of nucleoli when the Smith ea.). Pp. 461-477. Academic Press, ring was observed may be a result of an N. Y. accelerated demand for nucleoIar RNA for KFZWIENCZYK, J. 1963. Demonstration of nuclear polyhedrosis in Bonlb~x mori (Linnaeus) by protein synthesis. This demand may refluorescent antibody technique. J. Inoertebrate quire RNA at a rate too rapid for immediate Pathol., 5, 309-317. replenishment, thereby causing the breakSTEINHAUS, E. A. 1949. Nomenclature and classidown of these bodies. fication of insect viruses. Bacterial. Reu., 13, Membranelike structures were seen 203-223. around “mature” polyhedra. Their disSTEINHAUS, E. A. 1951. Report on diagnoses of continuous development suggests that they diseased insects 1944-1950. Hilgardia, 20, form after virus bundles are completely en629-678. closed in polyhedral protein. Thus, the STEINHAUS, E. A. 1957. New records of insect presence of a continuous “membrane” may virus diseases. Ifilgndiu, 26, 417-430. be indicative of a polyhedron in which XEROS, N. 1956. The virogenic stroma in nuclear peripheral areas have stabilized and protein and cytoplasmic polyhedroses. Nature, 173, 412-413. deposition has ceased.