Ultrastructural Changes in Midgut of CPV infected Tropical Tasar Silkworm, Antheraea mylitta (D) (Lepidoptera : Saturniidae)

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1 Int. J. Indust. Entomol. Vol. 21, No. 1, 2010, pp. 117~125 International Journal of Industrial Entomology Ultrastructural Changes in Midgut of CPV infected Tropical Tasar Silkworm, Antheraea mylitta (D) (Lepidoptera : Saturniidae) Deepak Deewaji Barsagade and Mangala Nimbaji Kadwey Department of Zoology, RTM Nagpur University Nagpur India, (Received 20 May 2010; Accepted 07 July 2010) The tropical tasar silkworms, Antheraea mylitta (D) produce famous silk Kosa in central part of India. Due to outdoor rearing it became susceptible to viral infection including cytoplasmic polyhedrosis virus (CPV). The common mode of entry of cytoplasmic polyhedrosis virus is per os and cause gresserie disease to the larvae. Histopathological studies elucidated the insect CPV virus produces infective polyhedral inclusion bodies (PIBs) in the midgut cell cytoplasm of virus infected fifth instar larvae. The PIBs multiply enormously in the cytoplasm without invading the nucleus. Ultrastructural studies confirmed the pathological effects of CPV on in midgut cell cytoplasm. The multiplication of polyhedral inclusion bodies took place into the vacuoles and form virogenic stromata in the cytoplasm of cells. However, the encapsulations of polyhedral inclusion bodies into the polyhedrin protein occurred and polyhedra were released into the lumen. At the late stage of infection, cells showed the regressed cytoplasmic organelles with large vacuoles and elongated mitochondria. Hence, the horizontal transmission of CPV causing the midgut cells disintegration in the tasar silkworm, Antheraea mylitta (D) confirmed during infection. Key words: Antheraea mylitta (D), Cytoplasmic polyhedrosis virus, Midgut, Polyhedral inclusion bodies Introduction Among the diseases, virosis (Grasserie) caused by cytoplasmic polyhedrosis virus (CPV) is very common which *To whom the correspondence addressed P.G.D.T. of Zoology, MJF Educational Campus, RTM Nagpur University, Amrawati Road, Nagpur , India dr_ddbars@rediffmail.com accounts for the loss of 25~30% of total cocoon crop of tasar silk industry (Sahay et al., 2000; Singh et al., 2006) and thus pose a serious problem in tasar sericulture. The appearance of polyhedral bodies of CPV was first time observed in the midgut cells of diseased silkworm larvae by Ishimori (1934) and Aruga (1971). Since then, cytoplasmic polyhedrosis has been recognized as one of the most important disease of silkworm infecting substantial economic losses to the sericulture industry in silk producing countries (Aruga, 1971). Although the CPV have very wide range, they are affecting mainly the Lepidopteran insects (Martignoni and Iwai, 1991). The CPV remain in a stable infectious state in the environment even after death of infected host. A large number of progeny of CPV particles released due to liquid oozing from the body and became available as a source for transmission to other susceptible individuals (Richards et al., 1999). The released CPV remain viable to accomplish in part by the polyhedrin protein matrix that surrounds the infective units of the virions and provides some degree of protection against environmental degradation (Rohrmann, 1986a). A. mylitta eco-races showed distinct insusceptibility to the preoral infection of a CPV to subdermal infections and the host resistance to preoral infections depending on inhibitory mechanism in the gut against the invasion of the virus into the midgut cells (Watanabe, 1971). It has been reported that CPV may persist with its host in occult or latent form of infection through various natural stressors, crowding and temperature conditions (Steinhaus, 1958a, b; Tanada et al., 1964). Payne (1981) reviewed the mechanism and frequency of occurrence of such infection. An effective mechanism would ensure virus maintainace in low density population in order to avoid the vertical transmission of diseases. In the lepidopteran insects, vertical transmission was observed in armyworm, Mythimna separata, gypsy moth, Lymantria disper (Shaprio and Robertson, 1987) and silkworm Bombyx mori (Khurad et al., 2004). The vertical transmission of TnCPV in Trichoplusia ni population were observed during

2 118 Deepak Deewaji Barsagade et al. rearing (Fuxa et al., 1992, 2002). The virus transmitted in the progeny either may kill the host larvae at an early stage or would latent form and express at late stage with favorable fluctuation in the environmental conditions. Material and Methods Tasar silkworm, Antheraea mylitta (D) is the principle nonmulberry silk producing insect and is cultivated in the tropical forest of India including the region of Bhandara, Chandrapur and Gardchiroli District of Vidarbha in the Maharashtra state. The non dipausing cocoons were brought to the laboratory and rearing was carried out at the insectaries of the Department of Zoology, RTM Nagpur University, Nagpur. Isolation and purification of cytoplasmic polyhedrosis virus (CPV) The grasserie infected fifth instar larvae were collected from the rearing field and were triturated individually. The larvae were crushed in the pestle and mortar and the homogenate was filtered through muslin cloth. The filtrate was observed under the microscope for presence of PIBs in it. The homogenate was contaminated with other microbes and it was purified by repeated centrifugation until clear layer of PIBs was obtained. These isolated PIBs were stored in refrigerator until their use. The serial ten fold dilutions in distilled water were prepared initially. The 60 larvae starved for six hours and distributed in four trays. First two trays containing equal number of larvae were inoculated individually by providing 1 sq. cm piece of Terminalia arjuna leaves coated with 20 µl suspension (about 2000 PIBs) PIBs /ml. The larvae of third and forth tray were also fed with 1 sq. cm piece of leaves dipped in distilled water and thereafter they were provided with fresh leaves. The larvae in the third and forth tray were treated as control and were placed for away from the inoculated ones. Both the inoculated and control larvae were scarified at varying intervals of 24 h, 48 h and 74 h and termed them as early, mid and late stage of infection. Histological methods The larvae from each group were dissected under stereoscopic binocular microscope and the midgut tissue was separated, cleaned and fixed in alcoholic Bouin s. There after, dehydrated in the graded alcohol series, cleared in Xylene and embedded in paraffin wax. The embedded tissues were stained with Azan stain (Aniline blue, Phasphotungstic acid PTA Stain) and haematoxylene eosin (HE) stains (Humason, 1962). Transmission Electron Microscopy (TEM) The transmission electron microscopy was carried out at Fig. 1. Isolated cytoplasmic polyhedrosis virus from midgut of infected fifth instar larvae of A. mylitta (a) and (b) magnified view showing isolated pure polyhedral bodies (Arrow) Azan stain X1000. All India Institute of Medical Sciences, New Delhi. The midgut dissected gently and fixed in cold Karnovasky s fixative for 48 h. The tissues were washed repeatedly in cold phasphate buffer (ph-7.2, 0.1 M) and post fixed for one hour in Osmium tetraoxide. The tissues were stained enbloc with 0.5% uranyl acetate during dehydration in graded alcohol and embedded in Araldite or Styrene- Methacrylate. The ultrathin sections were collected on pioloform coated grid after cutting with glass knife on LKB-4800A Ultramicrotome. At various stages 0.5 µm thick alternate sections were cut and observed at desirable magnification under Morgagni-200 transmission electron microscope (TEM). Results Isolation and purification of polyhedral inclusion bodies (PIBs) The purified PIBs were observed under the light microscope. The PIBs were mostly hexahedral, tetragonal, spherical, icosahedral, colourless and highly retractile bodies measuring about 1.5 to 2.5 µm in diameter (Figs. 1a, b). Histopathological effects of CPV virus on the midgut In the fifth instar larvae of tasar silkworm, Antheraea mylitta (D), the midgut epithelium was greatly folded and rests upon a basement membrane. The cells of midgut epithelium were differentiated into three types, the columnar cells(cec), the goblet cells(gc) and the regenerative cells(rc). The columnar cells were tall and closely associated with each other so that their boundaries were indistinct. They were interspersed apically with goblet and basally with regenerative cells. The columnar cells contain granular cytoplasm and fine brush border facing towards the lumen. The nuclei were large, spherical or elliptical and situated in the middle or apical half of the cells. The chromatin material well distinct and scattered

3 Ultrastructural changes in midgut of CPV infected Antheraea mylitta 119 Fig. 2. Cross section passing through the midgut of healthy fifth instar larvae of A. mylitta showing columnar epithelial cells (CEC), goblet cells (GC), regenerative cells (RC) and muscle layer (ML). HE X400. throughout the nucleus. The goblet cells were large flask or vase shaped, modified epithelial cells. The goblet cells contain centrally the oval nuclei and bulk of cytoplasm in the basal region. They open apically in the lumen of the midgut and excrete the salts. The regenerative cells were small and irregular shaped in the larvae. These cells can be sharply distinguished from rest of the epithelial cells due to their basal location, small size, centrally located large spherical nuclei with granular cytoplasm. They produced columnar cell periodically in the life of the silkworm (Fig. 2). In columnar epithelial cells after 24 h post inoculation (p. i.) the clumping of chromatin material observed in few nuclei which were still present in the middle region of the cells. The PIBs of CPV attached to the brush border membrane of epithelial cells and make their entry (Fig. 3a, b). These PIBs were deposited in the basal region of cell cytoplasm and multiply there. The cell membrane became fragile and cause regression from the basal to apical region of the epithelial cells. The basement membrane and muscle layer do not show any significant changes after 24 h p. i. At the basal region of cytoplasm few goblet cells were filled with PIBs and nuclei were completely destroyed. The regenerative cells were intact without showing any histopathological changes (Figs. 3c, d). Fig. 3. Cross- section of midgut, after early stage of p. i. of CPV in fifth instar showing PIBs in cytoplasm and nuclei remained unaffected in columnar epithelial cells, HE X400 (a), CEC showing PIBs enters from brush border membrane, note nuclei of remained unaffected, Azan stain X400 (b), showing PIBs at basal region of CEC, HE X1000 (c) and the PIBs and vacuoles found in cytoplasm of regenerative cells, Azan stain X1000 (d) BM, Basement membrane; BBM, Brush border membrane; Nu, Nucleus.

4 120 Deepak Deewaji Barsagade et al. Fig. 4. Cross section of midgut after mid stage p. i., PIBs in the cytoplasm of CEC and goblet cells, Azan stain X1000 (a), showing hypertrophy and lysis of CEC (arrow), Azan stain X400 (b), Magnified view of CEC showing cell lysis and release of PIBs into lumen, Azan stain X1000 (c) and showing the PIBs in cytoplasm of regenerative cells, HE X1000 (d). After 48 h p. i., the nuclei remain spherical and very small sized in the basal region of the cells and the chromatin material was found indistinct and mostly disintegrated. The polyhedral bodies of CPV were not found in the nuclei. However, as the infection of virus proceeds from the basal to the apical region of the cells, all PIBs spread throughout the cytoplasm. The intracellular vacuoles became extensively large and filled with polyhedra. The hypertrophy of cytoplasm and cell membrane was observed distinctly in most of the columnar cells. During the progress of infection, the columnar cell contains subsequently the matured polyhedra. The cells became elongated, cone-shaped causing the cell lysis and all polyhedra released into lumen of the midgut. At this stage, columnar epithelial cells remain attached to the basement membrane (Fig. 4a). The large amounts of PIBs of CPV were observed in the cytoplasm of the goblet cells and the cell membrane were broken at several places. The matured polyhedra released from the apical opening or after cells bursting (lysis) (Figs. 4b, c). In the regenerative cells, the PIBs of CPV were first deposited and multiplied in the cytoplasm of cells and subsequently the nuclei were destroyed completely. On the other hand vacuole formation takes place in regenerative cells and filled with PIBs (Fig. 4d). After 72 h p. i., all columnar epithelial cells were severely damaged due to the viral infection while the basal portion of epithelium was still attached to the basement membrane. In the damaged columnar epithelial cells, large empty vacuoles appeared and, the small, spherical nuclei distributed randomly. The matured PIBs released from the cells in the lumen of the midgut. However, due to viral infection, the basement membrane and muscle layer were not found to be much affected. The goblet cells and regenerative were also completely disintegrated and cell debris including PIBs were scattered in lumen of the midgut (Fig. 5). Ultrastructural changes in the midgut TEM studies on the midgut in the fifth instar larvae of silkworm, Antheraea mylitta, show the tall columnar epithelial cells containing well-developed cytoplasmic organelles. The nuclei were filled with dense mass of chromatin and distinct nucleoli. The endoplasmic reticulum and Golgi

5 Ultrastructural changes in midgut of CPV infected Antheraea mylitta 121 Fig. 5. Cross section of midgut after late stage p. i., the columnar epithelial cells showing complete disruptions and nuclei were randomly distributed with cell debris (arrow), Azan stain X1000. Fig. 7. TEM of midgut after 24 h p. i., of CPV showing elongated nucleus and few matured polyhedral inclusion bodies in the cytoplasm. Fig. 6. Transmission electron photo micrograph (TEM) of midgut columnar epithelial cells of fifth instar larvae showing nucleus(nu), rough endoplasmic reticulum (RER), Golgi bodies (Gb), mitochondria (Mt), Lysosomal bodies (Lys), protein granules and lipid droplets. Magnified view of mitochondria and lysosomes (Inset). bodies were well distinct. Several mitochondria were found throughout the cytoplasm. The large number of small protein as well as few lipid droplets and few lysosomal bodies are scattered in the cytoplasm (Fig. 6). After the 24 h p. i., the midgut epithelial columnar cells showed the basement membrane attached with cells. The disorganized endoplasmic reticulum and few small, elongated mitochondria were distributed sparsely throughout the cytoplasm. At few places the mitochondria were concentrated around the empty vacuoles. The partially filled protein and lipid droplets were observed in the cytoplasm. The large autophagic lysosomal bodies and vacuoles were widely distributed throughout the cytoplasm (Fig. 7). After 48 h p. i., the endoplasmic reticulum and Golgi complex were almost indistinct. As a result of infection, the virogenic stromata were randomly distributed and matured polyhedral bodies arranged in single or several parallel rows (Fig. 8a). The large vacuoles were surrounded by the thick layers containing fine pore-like structures (Fig. 8b). The polyhedral inclusion bodies (PIBs) enter into the vacuoles and form the virogenic stromata (Fig. 8c). In the vacuoles PIBs get multiplied and simultaneously encapsulated in the protein coat assembly and formed a cord. These cords were observed attached to the layers of the vacuoles (Fig. 8d). As the polyhedra attain the maturity, the cord material around the virus particles became disrupted and modified into large pore complex on the vacuole wall. The mature polyhedra released thereafter into cytoplasm through the pore complex. The asymmetrical morphogenesis and encapsulation of PIBs became evident in the vacuoles (Figs. 8e-g). After 72 h p. i., the midgut cells were internally destructed and at few places, interconnected cellular membranes were also ruptured. The nuclei were partially filled with chromatin material and distinct nucleoli (Fig. 9a). The cytoplasm was devoid of endoplasmic reticulum and Golgi complexes. The shape of mitochondria was changed into extensively elongated form but few were still small and elliptical. The numerous large empty vacuoles were distributed throughout the cytoplasm. The secretory protein and lipid granules were also visible. The autophagic lysosomal bodies were found in large number adjacent to the mitochondria (Fig. 9b). The fully-matured polyhedra and cell debris were well evident in the cytoplasm (Fig.10).

6 122 Deepak Deewaji Barsagade et al. Fig. 8. TEM of midgut of 48 h p.i. showing nucleus, formation of virogenic stromata and matured PIBs in cytoplasm (a). Magnified view of Fig. 8a virogenic stroma showing the morphogenesis of virus particle (arrow), surrounded by thick layers (arrow head) and vacuoles in cytoplasm (b). Virogenic stroma (Vs) showing pore complex in the layers of virogenic stroma (arrow) and interstitial space showing matured PIBs (c). Magnified view of Fig. c, showing inside the virogenic stroma (Vs), the encapsulation of PIBs in protein coat assembly in cord form and matured virions arises apically (d). Showing asymmetrical process of morphogenesis and encapsulation of PIBs inside virogenic stroma (arrow) and release of matured virus particles in the cytoplasm and form PIBs (arrow head) (e). Showing virogenic stromata filled with PIBs and matured PIBs in cytoplasm (arrow) (f). Magnified view of Fig. 8f showing virogenic stroma filled with different size of PIBs (arrow) (g).

7 Ultrastructural changes in midgut of CPV infected Antheraea mylitta 123 Fig. 9. TEM of midgut after 72 h p.i., showing randomly distributed nuclei (arrow) and matured polyhedra and many vacuoles in cytoplasm (a). Magnified view of Fig. 9a showing nucleus, large empty vacuoles, elongated mitochondria, lipid droplets and lysosomal bodies (b). Fig. 10. TEM of midgut after 72 h p.i. showing varying shape of matured polyhedra (arrow). Discussion In the present study the infected larvae were sorted out from the field, shortly before anticipated time of death although the yields of virus from the dying larvae may be more than that from the dead larvae (Ignoff and Shaprio, 1977; Jolly et al., 1979). According to Hunter et al., (1984) the purification of PIBs however, easier and quality of the final product would be better if the larvae were not putrefied or melonized. The PIBs residue were observed containing white and highly retractile bodies after purification in the infected larvae of A.mylitta. Steps in the virus infection process include the entrance of the virions into the gut lumen and dissolution of PIBs into digestive fluid after two to three hours incubation only (Kobayashi, 1971; Shields, 1984a). Apparently, the peritrophic membrane could not prevent the invasion of virions. The intracellular virus particles were located only in the midgut cells in the form of viroplasm (Xeros, 1956) or virogenic stroma (Sohi et al., 1971) often, represented by small micronet structure, characterizing the early stage of viral infection in A. mytillta. The present histological observations confirm the formation of polyhedra in the midgut epithelial cells of A. mylitta larvae within 24 h infection and thus support the earlier findings. The viroplasm was initially found near the brush border of the columnar epithelial cells and thereafter at the base of infected cells. Perhaps the polyhedra are present in almost every cell of the midgut. The virions replicating in the midgut epithelial cells, attacking the integrity of midgut tissue and ultimately they loose their identity (Dhaliwal and Arora, 1998; Prasad and Wadhwani, 2006). Later on, entire cells filled with polyhedra and nuclei begin to disintegrate. At the end of viral infection the shape and location of the polyhedra in midgut tissue were also variable (Aruga et.al., 1963; Miyajima and Kawase, 1971). In the present histological study the midgut cells of the diseased larvae revealed marked hypertrophy in columnar, goblet and regenerative cells containing numerous polyhedra. Similarly, disintegration of nuclei was observed in most of the CPV infected cells. In A. mylitta, at the end of CPV infection the cytoplasm was completely filled with variable polyhedra. After assuming a triangular shape, cells finally burst and polyhedra were discharged into the midgut lumen. The pathological changes were normally confined to the midgut columnar cells, while the goblet and regenerative cells were also found susceptible to the viral infection in tasar silkworm, A. mylitta (D). The viral polyhedra often contribute the horizontal propogation of viral infection as found by earlier worker (Kobayashi, 1971; Smith, 1967). The Ultrastructural studies on the midgut of control silk-

8 124 Deepak Deewaji Barsagade et al. worms showed the tall columnar epithelial cells well equipped with rough endoplasmic reticulum, Golgi bodies, mitochondria and numerous long microvilli, projecting into lumen suggesting their active role in the process of digestion and absorption (Chapman, 1998). In CPV infected silkworms a large number of virions were observed in the cytoplasm of the columnar epithelial cells and the cytopmorphology of the virions revealed their identification belonging to the genus Cypovirus in the family Reoviridae in accordance with criteria given by Field et al., (2001) and Coulibaly et al., (2007). The CPV capsid showed characteristic crystalline inclusion bodies (Lewandowski and Traynor, 1972; Sohi et al., 1971; Xia et al The virogenic stroma and first progeny of virions showed successive growth and formation of polyhedra, so that the free virus particles became occluded in polyhedra in the A. mylitta. Although, some workers were reported the infection of CPV in goblet and regenerative cells (Ishiwata and Seki, 1973; Noguchi, 1995; Zhang et al., 1999, 2008). However, in A. mylitta the presence of virogenic stromata in the infected columnar epithelial cells, subsequent occurrence of micronet structure in the cords, formation of pores in the layers, liberation of particles from the newly formed pore and crystallization of the polyhedra in the midgut cells, exhibited a typical process of multiplication and formation of matured polyhedra was similar to that reported in Lepidoptera species by earlier workers (Arnott et al., 1994; Quiot and Belloncik 1977; Smith,1967; Sohi et al., 1971; Tamashiro and Huang 1963; Tyler and Fields, 1985; Xeros 1956). In A. mylitta, formation of clumpy mass of debris due to extensive destruction of the epithelium was observed during the late stage of infection causing turbid appearance of the larvae similar that in other infected Lepidopteran larvae (Dhaliwal and Arora, 1998; Prasad and Wadhwani 2006). Smith (1967) reported the variable cytopathological changes in midgut cells than that in the cells of other tissues of B. mori. During the present study, cytomorphological and ultrastructural changes were observed in the cytoplasm of midgut cells during the cytoplasmic polyhedrosis virus infection while, no PIBs were found in the nucleus. This result confirmed the alteration in cellular metabolic activity and horizontal transmission of CPV in A. mylitta. Perhaps this is the first ultrastructural report on CPV infection in the tasar silkworm, A. mylitta. Acknowledgment The authors are thankful to UGC New Delhi for their support and Dr. D. 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