JOURNAL

OF INVERTEBRATE

Development Nuclei

PATHOLOGY

25,

359-169

(1975)

of Viruslike Particles in the Crystal-Containing of the Midgut Cells of Tenebrio molitor D. THOMAS

AND

J. GOURANTON

Groupe de Recherches de Biologie Cellulaire, Avenue du G&&al Leclerc, B.P. 25 A, 35031 R ENNES-Cedex, France Received

May

2 I, 1974

Rod-shaped structures appear in some crystal-containing nuclei of the midgut cells of Tenebrio moliror when the intranuclear crystal disintegrates. Mature viruslike particles are formed by these flexuous rods engulfed within dense shells. An autoradiographic study after a tritiated thymidine injection has shown that DNA synthesis occurs in the infected nuclei. An intense incorporation of tritiated lysine is also noted in the infected nuclei a few hours after the injection. Relations between the crystal and the viruslike particles have been investigated by observations on infected nuclei containing previously labeled crystals. The crystal proteins do not appear to be used for the synthesis of the viruslike particles. Finally, no direct relation seems to exist between the intranuclear crystals in Tenebrio midgut cells and the occasional presence of the described viruslike particles.

INTRODUCTION Intranuclear crystals of the midgut cells in Tenebrio molitor were first reported at the end of the last century. Their proteinaceous nature has been established in sections (Gouranton, 1969) and this has been confirmed by our biochemical observations when it became possible to isolate the crystals (Thomas and Gouranton, 1972). Recently we have studied their synthesis utilizing labeled amino acids (Thomas and Gouranton, 1973a). Their significance is not however totally clear. The ability of viruses to induce nuclear inclusions is well known especially in certain insect virus diseases where crystalline-protein inclusion bodies appear within the infected cells. It was therefore of interest to investigate the viruslike particles that we have observed in the midgut cells of T. molitor.

Zeikus and Steinhaus (1969) have described as “viruslike particles” small dense granules which they observed both in crystals and free in the nuclei of the mealworm T. molitor. They have noted that the exact shape of particles was difficult to discern in section. They thought that the

crystals were associated with these viruslike particles since they found the crystals and viruslike particles only in specimens showing the pupal-winged adult teratology. Nevertheless, it is difficult to interpret as viruslike particles the small, dense, and irregular areas which are sometimes observed on the crystals. Moreover, the intranuclear crystals are constantly found in the midgut cells of Tenebrio which show no teratological changes. The presence of complex viruslike particles has been reported in some nuclei of the midgut cells of T. molitor. The particles appeared when the intranuclear crystals disintegrated (Devauchelle, 1970). Since the disintegration of a crystal in an infected cell appears to be a new feature, we thought that it was interesting to determine the nature of these viruslike particles and to understand the significance of the phenomenon observed in the nuclei of infected cells. MATERIALS AND METHODS For electron microscopy, the midguts of T. molitor adults were fixed in 2% osmium tetroxide solution in 0.1 M sodium phosphate buffer atpH 7.2 for 1 hr. Generally the 159

Copyright ,I 1975 by Academic Printed in the United States.

Press, Inc. Ail rights of reproduction

in any form

reserved.

160

THOMAS

AND

GOURANTON

FIG. I. Transverse section of a crystal. The amorphous zone (az) first develops on the inclined faces of the crystal (cr) (nu, nucleolus). x 31,000. FIG. 2. Hexagonal section of a crystal. The crystal (cr) is surrounded by an amorphous zone (az). ~23,000. FIG. 3. Later stage of infection. The amorphous zone (az) completely surrounds the crystal and thisdisintegrates (dcr, disintegrated crystal). x 12,000.

DEVELOPMENT

OF

VIRUSLIKE

PARTICLES

IN

TENEBRlO

161

pieces were prefixed in 2.5% glutaraldehyde epithelial cells of the midgut. They are in the same buffer for 30 min. After washing formed during the migration of cells from and dehydration the tissues were embedded the crypts to the lining of the midgut. The in an Araldite-Epon mixture. Sections were time required for this migration, and consestained with uranyl acetate and lead citrate. quently for the renewal of the whole epitheFor electron microscopical autoradiolium, is about 4 days (Thomas and graphy of DNA each insect received 6 PCi of Gouranton, 1973b). The crystals may be tritiated thymidine with a specific activity of larger or smaller in different insects and 26 Ci/mmole. The insects were dissected their form may sometimes, but rarely, vary. from 3 hr to 5 days after the injection and We have noted lamellar crystals in some anithe midguts were prefixed with 2.5% mals but we have never found insects totally devoided of crystals. glutaraldehyde. For the autoradiographic study of proteins, 5-6 rcCi of tritiated lysine, The viruslike particles were found only in with a specific activity of 40 Ci/mmole, were about 25% of the midguts of those T. injected into other insects. These latter were molitor adults examined. In this 25% the number of infected cells was never nudissected from 1 hr to 4 days after the inmerous. The insects containing viruslike jection. Their midguts were prefixed with particles showed no sign of a disease. The 10% formalin in a phosphate buffer which infected cells were scattered among the contained a IOOO-fold excess of nonrahealthy midgut epithelial cells and generally dioactive lysine. In two cases the postfixation was carried out with 2% osmium te- showed differing stages of infection. The first troxide solution. Ultrathin sections were modification noted in an infected nucleus is the appearance of an amorphous structure treated according to the technique described crystal (Fig. 2). by Larra and Droz (1970). The Ilford L4 around the intranuclear When the crystal is observed in transverse emulsion was used and the exposure time section (Fig. 1), it appears that the amorwas about 2 mo. For enzyme digestion, the midguts were phous zone first develops on the inclined faces of the crystal. No modification is disfixed for 90 min with 10% formalin in 0.1 M phosphate buffer at pH 7.2. The pieces were cernible elsewhere in the cell. At a later then rinsed and embedded in GMA (Leduc stage of infection, the amorphous zone comand Bernhard, 1967). Thin sections were in- pletely surrounds the crystal, and this subsecubated at 37°C in a solution of 0.5% quently disintegrates (Fig. 3). At a more or less advanced stage of the crystal disinpronase (Calbiochem, B grade) in distilled water for 15-30 min. tegration, rod-shaped structures, with a In order to elucidate the nucleolar ap- mean length of 0.5 pm, appear in the nucleus pearance in the infected cells, nucleolar (Fig. 4). From observations of cross sections segregation was experimentally produced in (Fig. 5) it appears that a rod is composed of healthy midgut cells with actinomycin D. an inner core, opaque to the electron beam, with an average width of 250 A surrounded RESULTS by a thin cortical zone which measures 25A Electron microscopical observations of and is less opaque. The rod is enclosed in a infected ceil. In the midgut epithelium of T. unit membrane. The space between the rod molitor adults, as in many other Coleoptera, and this membrane is variable but the rod the replacement cells form crypts projecting surrounded by the membrane has an apthrough the muscular coat, like villi, all over proximate diameter of 600 A. Numerous the outer surface of the midgut. The intranufine fibrils can be seen between the rodclear crystals are never found in the em- shaped structures (Fig. 4). In some nuclei, dense lamellar structures limited by a bryonic cells of the crypts but are constantly observed in most of the differentiated membrane have been observed (Fig. 6).

162

FIG. appear FIG. FIG. FIG.

THOMAS

4. in 5. 6. 7.

At a more or less advanced the nucleus. ~85,000. Cross section of a rod-shaped Dense lamellae (la) and ovoid An ovoid particle observed in

AND

GOURANTON

stage of the crystal

disintegration,

structure. x 200,000. particles are observed section. x 120,000.

These lamellae seem to engulf one or several rods so that ovoid large particles are formed. When observed in section, this particle appears to be composed of a coiled rod surrounded by a dense lamella (Fig. 7). The number of these particles increases in the nucleus while the intranuclear crystal disintegrates and sometimes totally disappears. Fibrils are no longer seen. At a late stage of the cell infection the nucleus is hypertrophied; the nuclear envelope may be

rod-shaped

in this infected

nucleus.

structures

and fine fibrils

(fib)

~95,000.

disrupted and particles are found in the cytoplasm. The infected cells are removed in the gut lumen. Electron microscopical autoradiography of DNA. When healthy insects are dissected

a few hours after a tritiated thymidine injection, labeled midgut nuclei are found only in regeneration crypts. In addition, in infected insects some of the differentiated midgut cells’ nuclei are labeled. These are precisely the nuclei where the particles des-

DEVELOPMENT

FIG. 8 -9. Insects FIG. 8. At

an early

stage

of evolution,

disintegrated crystal). x 12,500. FIG. 9. A nucleus containing

rod-shaped

OF

killed

VIRUSLIKE

few hours

PARTICLES

after

an intense

thymidine

particles

(r) is also

tritiated

IN

thymidine

incorporation densely

labeled.

163

TENEBRIO

injection. is observed x 14,500.

(az,

amorphous

zone;

dcr,

164

THOMAS

AND

GOURANTON

FIG. 10. Insect killed several days after a tritiated thymidine injection. Some ovoid mature nuclear bieb, are labeled. x 11,000. FIG. 1 I. Insect killed few hours after a tritiated lysine injection. A dense label is observed x 13,500.

particles,

present

in the infected

in a

nuclei.

DEVELOPMENT

OF

VIRUSLIKE

FIGS. I2 14. Insect dissected 4 days after a tritiated lysine viously labeled crystals are observed. Neither the amorphous tegrated crystal). x 14,000.

PARTICLES

IN

TENEBRfO

165

injection. Several stages of the disintegration of pretone (az) nor the particles are labeled (dcr. disin-

166

THOMAS

AND

cribed above and nuclear modifications are found. An intense thymidine incorporation is observed in these nuclei at an early stage of evolution when the crystal begins to disintegrate (Fig. 8). The nucleus is also densely labeled in the next stage where the rodshaped structures appear (Fig. 9). The ovoid mature particles which are found in the nuclei at a late stage of infection are not labeled after a short incubation with the radioactive precursor. However, they may be labeled on sections in insects dissected several days after the tritiated thymidine injection (Fig. 10). Autoradiographic study after tritiated lysine injection. A few hours after a tritiated

lysine injection in an insect, a dense label is observed in the infected nuclei (Fig. 11). We know that several days after a tritiated lysine injection most of the intranuclear crystals of the midgut cells in Tenebrio are labeled, while most of the free radioactive precursor has disappeared (Thomas and Gouranton, 1973a). Some of these labeled crystals may subsequently disintegrate if the cell becomes infected. In an insect dissected 4 days after a tritiated lysine injection we have observed several stages of the disintegration of labeled crystals in nuclei where particles are formed (Figs. 12-14); neither the amorphous zone which develops around the labeled crystal nor the particles which are formed in the nucleoplasm are labeled. Enzyme digestion of G&IA-embedded tissues. Ultrathin sections of formalin-fixed

and GMA-embedded midguts were incubated in a solution of 0.5% pronase at 37” C. After 15 min the crystals were partially digested while the surrounding amorphous zones became less dense. After 30 min the crystals were totally digested (Fig. 15) and the surrounding zones were also completely digested in most of the sections. On the sections incubated for the same time in an enzyme-free solution, no modification was dbserved (Fig. 16). Actinomycin D treafment. In those nuclei containing particles, large opaque structures may be observed. They are composed of two

GOURANTON

constituent parts: One is granular; the other is very opaque (Fig. 17). Actinomycin D treatment of insects results in nucleolar segregation in the midgut cells. Two nucleolar fractions similar to the above have been observed (Fig. 18). DISCUSSION Nature of particles. In cross sections the rod-shaped structures described in the present paper resemble the rod-shaped virus particles observed in nuclear-polyhedrosis viruses and granulosis viruses of insects (Hughes, 1972; Arnott and Smith, 1968) or nonoccluded rod-shaped viruses (Gouranton, 1972). Nevertheless, the rod-shaped structures observed in the present study differ from all these viruses by their flexuous nature and also by their engulfment within a dense shell limited by a unit membrane in the mature particle. After a tritiated thymidine injection the only structures which were labeled, besides some dividing nuclei of renewal cells, were the infected nuclei. This indicates that most of unincorporated tritiated thymidine has been removed and that the label can be considered as specific of the incorporated thymidine. The autoradiographic study after the tritiated thymidine injection shows that a DNA synthesis begins in the nucleus at an early stage of infection and still continues as the rods appear. The DNA synthesized in the infected nuclei is very probably viral DNA. The fibrils observed in the infected nuclei are similar to DNA fibrils. They probably represent the viral DNA and they disappear as the viral particles are formed. Mature viral particles may be labeled in insects dissected several days after a tritiated thymidine injection. These particles contain labeled DNA. A few hours after a tritiated lysine injection a more dense label is noted in the infected nuclei than in the healthy nuclei. This intense incorporation of lysine is probably in relation with the presence of developing virus particles. On the basis of his morphological study

FIGS. IS- 16. Enzyme digestion of GMA embedded tissues. FIG. 15. After 30 min of incubation in a solution of pronase, the crystal (cr) is totally digested but the amorphous zone (az) of this section is not still completely digested. x 13,000. FIG. 16. Section incubated in an enzyme-free solution (az, amorphous zone; cr, crystal). x 14,000. FIG. 17. Large opaque structures observed in nuclei containing particles. They are composed of a granular part (nu 1) and a very opaque part (nu 2). x 21,000. FIG. 18. Actinomycin D treatment of a healthy insect. Two nucleolar fractions are observed, a granular fraction (nu 1) and an opaque fraction (nu 2)(cr, crystal). x 11,000.

167

168

THOMAS

AND GOURANTON

Devauchelle (1970) thought that the particles were viral in nature. Our observations based on the demonstration of DNA synthesis and of tritiated lysine incorporation in the infected nuclei bring new data in favor of this hypothesis. Modtjications induced in infected cell. At an early stage of infection, before the appearance of the rods, the presence of the amorphous zone around the intranuclear crystal is the only sign of the cell infection. Devauchelle (1972) described in the infected midgut cells of Tenebrio heterogeneous structures but could not ascertain their origin. We have also observed such structures in some infected nuclei. Changes in the nucleolar ultrastructure in virusinfected cells have been reported for several viruses; similar alterations may be induced in healthy cells by actinomycin D (Weiss and Meyer, 1972). In Tenebrio, after an actinomycin D injection, we have observed a nucleolar segregation which has morphological similarities with the dense heterogeneous structures sometimes seen in the infected nuclei. These structures would therefore be the alterated nucleolus of the infected cell. Relations between crystal and virus particles. In digestion experiments with pronase the crystal is rapidly digested. The amorphous zone is also digested and is consequently proteinaceous in nature. In an infected cell the intranuclear crystals always disintegrate; Devauchelle (1972) reported that the amorphous zone could be formed from the crystal. We have seen that when a crystal previously labeled with tritiated lysine disintegrates, the amorphous zone is not labeled. Consequently this zone does not result from an alteration of the crystal. The significance of this amorphous zone is not clear. The inclined faces of the crystal are the surfaces where the crystal growth is the most rapid (Thomas and Gouranton, 1973b). The amorphous zone first develops on these faces; hence, does this zone represent newly synthesized crystal proteins

which cannot crystallize in the infected nucleus? An immunological study will perhaps give the answer. Our autoradiographic study after a tritiated lysine injection shows a dense label in the infected nuclei a few hours after the injection. The developing particles are probably responsible for this tritiated lysine incorporation. But when an infection is noted in a nucleus containing a crystal previously labeled with tritiated lysine, the virus particles which are formed are not labeled. The tritiated lysine of the labeled crystal which disintegrates therefore does not appear to be utilized by the developing particles. The crystal proteins would not be used for the synthesis of the virus particles. This is confirmed by the observation that the particles may appear in the nucleus at a more or less advanced stage of the disintegration of the crystal. It is likely that the appearance of virus particles alters the hostcell metabolism. Changes in physicochemical conditions of the infected nuclei would induce the crystals to disintegrate. As already mentioned, the infection was observed only in about 25% of the insects and, for this 25%, only some cells were infected. Finally, it does not appear that any direct relation exists between the intranuclear crystals in Tenebrio midgut cells and the occasional presence of these particles. It is possible that virus particles also develop in midgut cells devoided of intranuclear crystals; however, it is difficult to assume that an infected cell had no crystal even if the crystal is not observed since it may have disintegrated. ACKNOWLEDGMENTS The authors thank Mrs. Cavalier, Miss de Sallier Dupin, and Mr. Morille for technical assistance.

REFERENCES AND SMITH, K. M. 1968. An ultrastructural study of the development of a granulosis virus in the cells of the moth PIodiu interpunctella (Hbn.). .I. Ultrastruct. Res., 21, 251268. DEVAUCHELLE, G. 1970. Inclusions cristallines et parARNOTT,

H.

J.,

DEVELOPMENT ticules I’intestin

d’allure moyen

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farine scope THOMAS,

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methacrylate

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du

Recent embedding

169

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D., AND GOURANTON, J. 1972. Isolement des intranu&aires de I’intestin moyen du ver de

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molifor L. et observations au microJ. Microsc. (Paris), 14, 125-128. GOURANTON,

de

molitor

L. J. Microsc.

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D.,

J.

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de

(Paris),

au

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AND GOURANTON, des cristaux protiiques

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&de,

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molilor.

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de de

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in some midgut cells nafator L. (Coleoptera).

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LEDUC,

et de L. C.

virus

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Microsc.

2948-295

Development

rod-shaped

Ulrrastrucr.

PARTICLES THOMAS, cristaux

Sri. Paris. 2f&

nonoccluded an adult

VIRUSLIKE

virale dans les noyaux des ceilules de du ColeoptZre Tenebrio molifor (L.).

GOURANTON, J. ultrastructurales I’intestin moyen GOURANTON,

OF

Cells. J. Ultrastrucrure ZEIKUS,

J. 1972.

of Coxsackievirus A9 Nucleolar Ultrastructure R. D., AND

and

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of

the

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E. A.

l-419. 1969.

Teratology

of the beetle Tenebrio molitor. V. Ultrastructural changes and viruslike particles in the foregut epithehum of pupalwinged adults. J. Inverlebr. Parhol., 14, 115-121.

Development of viruslike particles in the crystal-containing nuclei of the midgut cells of tenebrio molitor.

JOURNAL OF INVERTEBRATE Development Nuclei PATHOLOGY 25, 359-169 (1975) of Viruslike Particles in the Crystal-Containing of the Midgut Cells of...
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