20 Pathology and Morphogenesis of a Granulosis Virus of the Diamondback Moth Tetsu Asayama Plant Protection Laboratory, Aichi-Ken Agricultural Research Center, Nagakute, Aichi 480-1 1, Japan Abstract In 1970 a granulosis virus (GV) was found to infest diamondback moth (Plutella xylostella (L) larvae in Aichi Prefecture in Japan. GV infection initially causes swelling of each segment and a series of changes in color from the green of healthy larvae to a dark discoloration at the time of GV-induced death. Body weight increases as the disease progresses. The disease occurs in early summer and is practically absent in winter. The incubation period varies from 2 to 16 days depending upon rearing temperature, the higher the temperature the lower the incubation period. GV of diamondback moth was not infectious to several other important lepidopterous pests. The author describes various pathological changes in host cells, the morphology of the virus, and cytopathological details. Multiplication of GV takes place in the cytoplasm of the cells of fat body, Malpighian tubules, brain, and epidermis. Introduction Infestation of crucifers by larvae of the diamondback moth (DBM), Plutella xylostella (L) (Lepidoptera: Yponomeutidae) is a serious problem in Japan. During a survey of infectious disease agents of DBM, larvae infected with granulosis virus (GV) were found in a cabbage field in Aichi Prefecture (Asayama and Osaki 1970). This was the first record of a virus disease of DBM occurring in nature. This paper deals with the progress of studies of GV in DBM. Symptoms of Granulosis Virus Infection The first symptom of GV infection is swelling of each larval segment (Figure 1). At an advanced stage of infection, the larval body color changes to pale green, pale yellow green, or pale yellow, in contrast to the dull green of a healthy larva. During this process, the diseased larva appears larger and body weight increases to 1.5-2.0 times that of a healthy larva (Figure 2). Figure 1. External symptoms of granulosis virus infection of DBM
206 Asayama Figure 2. Comparison larval-body weights between granulosis virus-infected larvae and healthy larvae Late 3rd instar larva Late 4th instar larva The integument of the moribund larva ruptures quite easily and a turbid fluid flows out from the disintegrated integument. Dead larvae become flaccid and the body color is dark. Pathological Properties GV-infected larvae often migrate to the top of the cabbage plant, where they attach themselves to the cabbage leaf and die in a hanging position (Figure 3). Under natural conditions GV infection increases in early summer but decreases in the winter. Pathological changes in GV-infected larval cells show the following process. In the early stage of GV infection, the nuclei of fat body cells swell and clump of the GV capsules appear in the cytoplasm. In the later stages of infection, fat body cells become highly hypertrophic and many vacuoles occur in the cytoplasm. Finally, the boundary of the nuclear membrane becomes obscure, the cytoplasmic constituents disintegrate, and the infected cells become loosely connected with each other by the cell membranes. Figure 3. (left). Granulosis virus-infected larvae hanging from the top of cabbage plant Figure 4. (right). Scanning electron micrograph showing normal shape of capsules isolated from the diseased larvae. Bar = 1
Granulosis Virus of DBM 207 The length of incubation period is influenced by the rearing temperature. Third instar larvae showed typical symptoms of the granulosis 9-16 days after inoculation at 14 "C, 6-7 days at 18 C, 3-6 days at 22 C 2-6 days at 26 C and 2-4 days at 30 C. Granulosis may also be induced by dipping the larvae in 0.2-0.8 µg/ml endrin solution for few minutes. In peroral infection experiments, the DBM GV was not infectious to common cabbageworm, Pieris rapae crucivora; beet worm, Plusia nigrisigna; mulberry caterpillar, Mamestra illoba; tobacco cutworm, Spodoptera litura; common cutworm, Agrotis fucosa; or silkworm, Bombyx mori. Virus Morphology The normal inclusion body (capsule) was ovocylindrical, 411 ± 17 nm long and 240 ± 13 nm wide (Figure 4). The virion (enveloped nucleocapsid) was 260-280 nm long and 70-100 nm wide. Matured capsules were surrounded by an epicapsular layer. Abnormally shaped capsules were classified under the following categories (Asayama 1976a): (1) Capsule shape abnormal but inner structure of virion normal. Cubic capsules (Figure 5) and agglomerated capsules (Figure 6) belong to this group. (2) Outer form of capsule is normal, but no nucleocapsid is occluded in the capsule (Figure 7 d). (3) Elongate capsules. There are four types of elongate capsules occluding; (a) no nucleocapsid (Figure 7 e), (b) long nucleocapsid (Figure 7 f), (c) excessively encapsulated virion but of normal size (Figure 7 g), and (d) two capsules connected to each other (Figure 7 h,i). Figure 5. Formation of cubic type capsules in the Malpighian tubule cells infected with granulosis virus. Bar = 0.5 µm Figure 6. right a. Formation of agglomerated inclusion (ai) bodies in fat body cells infected with granulosis virus. b. Enlarged view of agglomerated inclusion body. Bar = 0.5 µm Multiplication sites and Morphogenesis Multiplication of DBM GV occurs in the cytoplasm of fat body cells, Mulpighian tubules, brain, and epidermal cells. Virus multiplication was not observed in the cuticle
208 Asayama f Figure 7. Inner structures of capsules of different shapes. a-c; two nucleocapsids (nc) arrayed side by side within a capsule. d; vesicular inclusion body (vi). e-i; elongate capsule. j, long nucleocapsid (nc) and epicapsular layer (el). Bar=0.5 layer of the integument, nor in goblet and columnar cells of the mid gut, tracheal matrix, muscules, or silk glands (Asayama and Inagaki 1975b, Asayama 1976b). The sequence of morphogenesis of the GV in fat body cells of DBM larva was as follows (Asayama 1975a): (1) Appearance of nucleocapsids associated with the endoplasmic reticulum (Figure 8). (2) Regular stacking array of nucleocapsids (3) Dispersal of nucleocapsids from the cluster to the cytoplasmic matrix. (4) Envelopment of nucleocapsids by a membrane which originates from de novo membrane morphogenesis in the cytoplasmic matrix (Figure 9). (5) Encapsulation and (6) Completion of capsule formation with epicapsular layer (Figure 10). Figure 11 shows a diagrammatic representation of sequence of morphogenesis of DBM GV. There was no difference in the sequence of virus morphogenesis between GV and nuclear polyhedrosis virus (NPV). The sequence was as follows: (1) Protrusion of nucleocapsids, (2) morphology of stacking array of nucleocapsids, (3) dispersal of nucleocapsids mass, (4) envelopment, (5) incipient deposition of the inclusion body protein on the enveloped nucleocapsid, (6) formation of inclusion body (capsule or polyhedron), and (7) formation of inclusion body membrane (epicapsular layer or polyhedral membrane) (Asayama 1982). The enveloped nucleocapsid of DBM GV was distinguishable by the presence of a spicular structure at the posterior end of enveloped nucleocapsid. However, no marked structure was observed at the anterior end of enveloped nucleocapsid. Deposition of capsule protein started from the anterior part of the enveloped nucleocapsid.
Granulosis Virus of DBM 209 Figure 8. Protrusion of nucleocapsids (nc) associated with the endoplasmic reticulum (epm) within the cytoplasm. ib = inclusion body. Bar=0.5 Figure 9. Formation of envelope (en) and envelopment process (1-4) of nucleocapsid within the cytoplasm. Bar = 0.25
210 Asayama Figure 10. Encapsulation process (1-5) of the virions (v) within the cytoplasm. el = epicapsular layer. Bar=0.25 4 Figure 11. Diagrammatic representation of sequence of GV morphogenesis in fat body cells of DBM larva. (1) Appearance of nucleocapsids, (2) regular stacking array of nucleocapsids, (3) dispersal of nucleocapsids, (4) envelopment, (5) encapsulation, and (6) completion of capsule formation Cytopathology Fat body cells of GV infected DBM larvae showed hypertrophy of nucleus, protrusion, subsidence, and partial disappearance of nuclear membrane. However,
Granulosis Virus of DBM 21 1 appearance of virions within the nuclear matrix was not confirmed. Mitochondria of such cells changed into balloon-shaped structures with fragmented cristae. The endoplasmic reticulum exhibited multilayered and whorl shaped figures (Asayama and Inagaki 1975a). In advanced stages of GV infection, these disintegrated cell organelles dispersed in the cytoplasmic matrix (Figure 12). Homogeneous fine granules appeared inside the curved endoplasmic reticulum. Ring-shaped structures associated with the endoplasmic reticulum were also observed in the cytoplasm of GV-infected cells. However, these structures disappeared due to the degeneration of endoplasmic reticulum. Compact and clumped masses appeared in the area of sequestration of cell organelles. Characteristic tubular structures were also observed in the cytoplasm of GV infected cells (Asayama 1975b). These structures were 35-90 nm in width and were randomly branched in two to four directions (Figure 13). These structures proliferated at the site of GV capsule formation. However, they were not connected with virions or capsules. These tubular structures aggregated to form clusters of 6-8 µm in length and 3-4 µm in width in the GV infected cells. Figure 12. GV-infected cells showing the cytopathological changes in cell organelle. Bar=2 µm Figure 13. Proliferation of the tubular structures (ts) in the GV-infected cells. gip = granular inclusion body protein. Bar = 0.25 µm Literature Cited Asayama, T. and N. Osaki. 1970. A granulosis of the diamondback moth, Plutella xylostella. J. Invertebr. Pathol. 15:284-286. Asayama, T. and I. Inagaki. 1975a. Cell alternations caused by the infection with the granulosis virus in the diamondback moth, Plutella xylostella, and the site of appearance of nucleocapsid. (In Japanese with English summary). Jpn. J. Appl. Entomol. Zool. 19:79-84. Asayama, T. and I. Inagaki. 1975b. Multiplication of a granulosis virus of the Plutella xylsotella in the Malpighian tubule. (In Japanese). Jpn. J. Appl. Entomol. Zool. 19:115-116.
212 Asayama Asayama, T. 1975a. Maturation process of the granulosis virus of the diamondback moth, Plutella xylostella. (in Japanese with English summary). Jpn. J. Appl. Entomol. Zool. 19:149-156. Asayama, T. 1975b. Proliferation of the tubular structure in fat-body cells of the diamondback moth infected with a granulosis virus. (In Japanese). Jpn. J. Appl. Entomol. Zool. 19:216-218. Asayama, T. 1976a. Morphology of the inclusion body of Plutella xylostella granulosis virus. (In Japanese). Jpn. J. Appl. Entomol. Zool. 20:44-46. Asayama, T. 1976b. Histopathology of a granulosis in the larva of the diamondback moth, Plutella xylostella. (In Japanese with English summary). Proc. Kansai. Plant Prot. Soc. 18:41-46. Asayama, T. 1982. Studies on the pathology and morphogenesis of the baculoviruses of lepidopterous insects. (In Japanese with English summary). Special Res. Bull. Aichi Agric. Res. Cen. 3:1-85.