Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

39, 354-361 (1982)

Dissolution of Autographa califomica Nuclear Polyhedrosis Polyhedra by the Digestive Fluid of Trichoplusia ni (Lepidoptera:Noctuidae) Larvaele2 DAVIS Department

W. PRITCHETT,~ of Entomology,

S. Y. YOUNG,

University

of Arkansas,

Virus

AND W. C. YEARIAN Fayetteville,

Arkansas

72701

Received April 24, 1981; accepted August 3, 1981 The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished. KEY WORDS: Digestive fluid; Trichoplusia ni: Autographa californica; nuclear polyhedrosis virus; phase contrast microscopy; electron microscopy.

digestive tract (Paschke and Summers, 1975). Following this process in vivo has Baculoviruses are characterized by the been difficult due to the limited number of occlusion of enveloped rod-shaped nu- occlusions ingested, the presence of hetercleocapsids with a DNA genome within a ogeneous material in the digestive tract, proteinaceous matrix (Smith, 1976). The and the rapidity with which the occlusions matrix is surrounded by a dense outer layer are dissolved (Vago and Croissant, 1959). or envelope (Harrap, 1972) thought to be Attempts have been made, however, to proteinaceous in nature (Nordin and Madfollow the fate of the occluded virus from dox, 1971; Smith, 1976). Minion et al. ingestion through dissolution and virion (1979) reported, however, that the envelope release (Vago and Croissant, 1959; Sumwas composed of carbohydrates. mers, 1971; Granados, 1978). The dissoluA baculovirus infection in an insect host tion of baculoviruses by digestive fluid has begins with the ingestion of the occluded been easier to observe in vitro (Vago and virus (Heimpel and Harshbarger, 1965), Croissant, 1959; Faust and Adams, 1966; dissolution of the matrix protein, and re- Watanabe, 1974; Kawarabata et al., 1980). lease of the enveloped nucleocapsids in the Studies on the dissolution of baculoviruses by digestive fluid are limited, and in vitro studies have dealt mainly with Bom’ published with the approval of the Director of the hyx mori nuclear polyhedrosis virus (NPV) Arkansas Agricultural Research Station. and digestive fluid. Observations are presz Mention of a trade name does not imply endorseented here on the in vitro dissolution of ment or guarantee of the product or the exclusion Autographa californica NPV (AcNPV) by of other products of similar nature. digestive fluid collected from Trichoplusia 3 present address: Department of Biology, Northeast Louisiana University, Monroe, La. 71201. ni larvae. INTRODUCTION

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DISSOLUTION

MATERIALS

OF

AND METHODS

Digestive fluid was obtained from fifth-in&u T. ni larvae reared on an artificial diet (Burton, 1969). The fluid was collected by irritating the larvae and causing them to regurgitate. The larva’s head was placed over the lip of a 12-ml centrifuge tube held in ice, gently tapped against the tube, and the regurgitant collected. Digestive fluid from approximately 400-800 larvae was collected and pooled. Particulate material was removed from the digestive fluid by centrifugation at 4°C in two steps. The fluid was centrifuged at 3000g for 5 min and the supernatant was centrifuged again at 26,000g for 15 min. Clarified digestive fluid was either used immediately or stored in a sealed container at -20°C until used. The pH of this digestive fluid was 9.8 (Pritchett et al., 1981). Polyhedra. Polyhedra of AcNPV, isolated by P. Vail (USDA/ARS Western Cotton Research Laboratory, U.S. Department of Agriculture, Phoenix, Ariz.), were used in the study. The virus was propagated in Heliothis virescens larvae reared on an artificial diet. Polyhedra were purified by filtration through organdy and centrifugation from sucrose solutions (Young et al., 1977). Purified polyhedra were lyophilized and stored at room temperature . Phase contrast microscopy. Freeze-dried AcNPV polyhedra were suspended in distilled deionized water at a concentration of 2 x 108 PIB/ml. Polyhedra (5 X lo6 PIB) in 50 pl were placed on a clean microscope slide and air dried at 25°C (Vago and Croissant, 1959; Nordin and Maddox, 1971). After drying, a drop of the clarified digestive fluid was added to the slide, quickly covered with a coverslip, placed under the microscope, and observations begun immediately. The reaction was recorded by both continuous observation and micrographs at timed intervals at 800x witha phase-contrast microscope. A minimum of five observations were made for each preparation. Digestive

fluid

preparation.

AcNPV

POLYHEDRA

355

Electron microscopy. Specimens were prepared for electron microscopy by exposing freeze-dried AcNPV to digestive fluid (2 mg/ml) for timed intervals, followed by the addition (1:20) of 6.25% phosphatebuffered gluteraldehyde, pH 7.2, to stop the dissolution process and fix the samples. After 1 hr in the fixative, samples were centrifuged, washed with distilled water, and postfixed with 1% osmic acid. Samples for scanning electron microscopy were dehydrated through an ethanol series. The pellet was resuspended in absolute alcohol, a drop of the preparation placed on a specimen mount, and allowed to dry. Specimens were coated with gold and observed with an ISIscanning electron microscope at 30 kV. Samples for transmission electron microscopy were prepared through postfixation in osmic acid as stated above. After postfixation, samples were placed in 0.5% uranyl acetate overnight, dehydrated through an ethanol series, and embedded in Spur’s Epon. Embedded specimens were sectioned using a glass knife on an ultramicrotome. Sections were then stained with uranyl acetate followed by lead citrate, and examined with a Joel Jem-100CX electron microscope at 80 kV.

RESULTS Under phase contrast, AcNPV polyhedra suspended in distilled water were cuboidal with typical brilliance (Fig. I a). The addition of a drop of digestive fluid to polyhedra resulted in immediate changes in their appearance. After 15 set, many polyhedra had lost their cuboidal shape and appeared rounded. By 0.5 min, most polyhedra were actively involved in the dissolution process. Solubilization of the matrix protein was evidenced by loss of refringence as they cleared internally (Fig. lb). The rounded polyhedra appeared swollen, and Brownian movement increased as the matrix dissolved. With clearing, an irregularity occurred on the surface of polyhedra and small protuberances were observed on some dissolving polyhedra (Fig. lb). These

PRITCHETT,

YOUNG,

AND

YEARIAN

FIG. 1. Phase contrast micrographs of AcNPV polyhedra following treatment with distilled water or 2’. ni digestive fluid. (a) Distilled water; (b) OS-min exposure to digestive fluid; (c) l.O-min exposure to digestive fluid. f, polyhedral envelope fragment; P, protuberance; r, rupture. x6000.

appeared to represent weakened areas in the surface structure and ruptures in the envelopes of some polyhedra were observed (Fig. lb). Some polyhedra dissolved more rapidly than others, but within 0.75 1.0 min all the polyhedra were involved in the process. When the polyhedral envelope ruptured, particles that had been within the polyhedra began to flow out of the opening. Rupture of the polyhedral envelope was quickly followed by its fragmentation with the fragments becoming increasingly smaller with time (Fig. lc). Brownian movement increased in the area immediately adjacent to the disrupted

polyhedra and spread outward as the polyhedral envelope disintegrated. By 2 min all of the polyhedra had undergone dissolution of the matrix and disintegration of the polyhedral envelope. No identifiable structures remained at this time. As viewed by scanning electron microscopy, polyhedra appeared cuboidal and were approximately 1.5 km on a side. Some polyhedra were marked with pitted surface irregularities (Fig. 2a). After exposure to digestive fluid for 0.5 min, the polyhedra lost their cuboidal shape. They became more irregular in shape, roughened in appearance, and protuberances similar to

DISSOLUTION

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AcNPV

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FIG. 2. Scanning electron micrographs of AC?rTPV polyhedra following treatment with distilled water or T. ni digestive fluid. (a) Distilled water (.arrows indicate surface irregularities); (b) OS-min exposure to digestive fluid; (c, d) l.O-min exposure to digestive fluid. Bar = 1 pm. P, protuberance.

those observed under phase were common (Figs. 2b, c). Polyhedral remains were often observed with only a shell-like structure or fragments of a shell remaining (Figs. 2c, d). When observed under higher magnification, cracks and crevices were observed in the shells. Many of these appear to have been left when virus bundles were dislodged (Fig. 2d). Exposure of polyhedra to diges-

tive juice by the procedure used in the electron microscope study resulted in aLreduced rate of dissolution. Even after 1 I nin, a few polyhedra appeared somewhat cu boidal. This appeared to be due to both the and increase in polyhedra concentration clumping of polyhedra so that a longer wriod of time was required to wet those in the middle. Longer exposure (3 min) was re-

358

PRITCHETT,

YOUNG,

quired for complete dissolution of all polyhedra. Ultrastructural changes in AcNPV polyhedra following exposure to digestive fluid were observed in more detail by transmission electron microscopy. In cross sections of untreated AcNPV polyhedra, virions were embedded randomly within a crystalline protein matrix. Surrounding the polyhedral matrix was a denser outer layer or envelope (Fig. 3a). After exposure to digestive fluid for 1 min, polyhedra were seen in various stages of dissolution (Figs. 3b-g). The initial changes in polyhedra observed by transmission electron microscopy were small fissures in the matrix and dissolution of the matrix at sites on the surface. The polyhedral envelope over these sites often ruptured (Fig. 3b). Fissures became more extensive and were most often linked directly with or between virions (Figs. 3b, c). Some nucleocapsids

AND YEARIAN

were observed which appeared to be devoid of their contents (Fig. 3c, arrows) as well as others which were still intact. As the dissolution continued, solubilization of matrix protein resulted in loss of structural integrity. This was most rapid at the surface of polyhedra and in the central area. When solubilization became more extensive, the polyhedral envelope separated from the matrix and only fragments of the matrix remained (Fig. 3d). The matrix of some polyhedra were completely solubilized and appeared to remain intact with virion within the envelope (Fig. 3e). Usually there were ruptures in the envelope and these increased (Fig. 3f) and fragments of the envelope became smaller and less recognizable as it disintegrated (Fig. 3g). By 3 min, only intact virions were scattered about the heterogeneous material present (Fig. 3h). No unenveloped or empty nucleocapsids were observed at 3 min.

FIG. 3. Transmission electron micrographs of AcNPV polyhedra following treatment with distilled water or 7’richoplusia ni digestive fluid. (a) Distilled water; (b, d-g), l.O-min exposure to digestive fluid; (c) enlargement of b (arrows indicate apparent empty capsids); (h) 3.0-min exposure to digestive fluid. a, b, d-h, ~20,000; c, ~40,000.

DISSOLUTION

OF

FIG.

AcNPV

3-Continued.

POLYHEDRA

359

360

PRITCHETT,

YOUNG,

FIG.

AND

YEARIAN

3-Confinued.

DISCUSSION

Observations presented demonstrate in vitro dissolution of AcNPV polyhedra by fifth stage T. ni larval digestive fluid. Although the process appeared to be more rapid in T. ni, it was similar to accounts of in vitro and in vivo dissolution of polyhedra from B. mori NPV by B. mori digestive fluid (Vago and Croissant, 1959). Watanabe (1974) reported dissolution of B. mori NPV in vitro to be somewhat slower with some intact polyhedra remaining after 20 min of exposure. The slower rate of polyhedral dissolution in that study could have been due to the use of digestive fluid collected from larvae which had been starved for 48 hr prior to collection. Kawarabata et al. (1980) reported that prolonged starvation caused a significant decrease in the polyhedra-dissolving activity of the digestive fluid of B. mori larvae. We have also found that the pH and proteolytic activity of digestive fluid collected from T. ni larvae starved for 24 hr was lower than that from

larvae not subjected to starvation (Pritchett et al., unpubl.). Dissolution of the AcNPV polyhedra occurred in clearly identifiable stages. In the first stage the matrix fissured and dissolved, resulting in polyhedra becoming rounded. Summers (1971) reported a similar occurrence with T. ni granulosis virus inelusion bodies. He noted that dissolution occurred initially adjacent to the enveloped virion and suggested that bonding sites of the adjacent protein subunits were not as precise as those of subunits farther removed from the virion. We also observed fissuring and dissolution to begin adjacent to the virion in AcNPV. There were also sites adjacent to the polyhedral envelope where dissolution was rapid. Dissolution of the matrix was followed immediately by the rupturing, fragmentation, and disintegration of the polyhedral envelope, thereby releasing the vu-ion. The protrusions observed on many polyhedra during the course of dissolution suggest the envelope is weakened in these areas. Since

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Y. 1980. Purification and properties of the Bombyx these structures were not observed after the mori nuclear polyhedrosis virus liberated from polyhedral envelope had ruptured, they polyhedra by dissolution with silkworm gut juice. Z. may be the areas of initial envelope disrupZnvertebr. Pathol., 35, 34-42. tion. MINION, F. C., COONS, L. B., AND BROOME, J. R. Only intact virions were observed dis1979. Characterization of the polyhedral envelope of the nuclear polyhedrosis virus of Heliothis virespersed in the microscopic field after a 3-min tens. J. Znvertebr. Pathol., 34, 303-307. exposure to digestive fluid; however, at 1 NORDIN, G. L., AND MADDOX, J. V. 1971. Observamin there were some capsids within tions on the nature of the carbonate dissolution propolyhedra which appeared to be devoid of cess on inclusion bodies of a nuclear polyhedrosis nucleic acid. The absence of empty capsids virus of Pseudaletia unipuncta. J. Znvertebr. Pathol., 18, 316-321. at 3 min could be due to their rapid degraM. D. 1975. Early dation in the digestive fluid. Other inves- PASCHKE, J. D., AND SUMMERS, events in the infection of the arthropod gut by tigators have indicated that only enveloped pathogenic insect viruses. In “Invertebrate Immunvirions are routinely observed following ity” (K. Maramorosch and R. E. Shope, eds.), pp. exposure to digestive fluid both in vivo and 75- 112. Academic Press, New York. J. 1978. Alkaline in vitro (Summers, 1971; Granados, 1978; PAYNE, C. C., AND KALMAKOFF, protease associated with virus particles of a nuclear Kawarabata et al., 1980). This suggested to polyhedrosis virus: Assay, purification, and propSummers (1971) and Granados (1978) that erties. J. Virol., 26, 84-92. the enveloped virions were stable in the en- PRITCHETT, D. W., YOUNG, S. Y., AND GEREN, C. R. vironment of the midgut lumen. More re1981. F’roteolytic activity in the digestive fluid of Trichoplusia ni larvae. Insect Biochem. 11, 523-526. cent investigations have found, however, SMITH, K. M. 1976. “Virus-Insect Relationships,” that in vitro, prolonged exposure results in pp. 3-33. Longmann, New York. a reduction in the virions (Vail et al., 1979; SUMMERS, M. D. 1971. Electron microscopic obserKawarabata et al., 1980). vations on granulosis virus entry, uncoating and

ACKNOWLEDGMENTS The authors wish to thank Elizabeth Martin for her valuable assistance with electron microscopy.

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