Metabolic changes in diseased insects

JOURNAL

OF INVERTEBRATE

PATHOLOGY

Metabolic II.

(1968)

l&476486

Changes

in Diseased

Insects

Radioautographic Studies on DNA and RNA Synthesis in Nuclear-Polyhedrosis and Cytoplasmic-Polyhedrosis Virus Infections OSWALD

Department

of

Forestry

and Victoria,

N.

MORRIS

Rural Development, British Columbia,

Received

December

Forest Canada

Research

Laboratory,

28, 1967

The metabolism of DNA and RNA in Lepidoptera and Hymenoptera tissues infected with a nuclearor cytoplasmic-polyhedrosis virus was studied by radioautography. Glutaraldehyde fixative caused greater tissue shrinkage than did freezesubstitution but was satisfactory for this technique. In Lepidoptera fat body infected with nuclear-polyhedrosis virus, DNA, nuclear RNA, and cytoplasmic RNA synthesis first increased and then decreased. In the midgut of Hymenoptera, DNA synthesis and nuclear swelling were as in the Lepidoptera, but RNA synthesis first decreased and then increased. Large amounts of lysine-, arginine-, and histidine-rich substances accumulated in the nuclei during early stages of infection. Both DNA and RNA syntheses were generally low in the gut of early pupae of Hymenoptera infected as larvae with nuclear virus. Cytoplasmic polyhedrosis in Lepidoptera midgut epithelium produced only a minor effect on DNA synthesis of infected cells. As this disease progressed, nuclear RNA synthesis remained relatively constant, but cytoplasmic RNA synthesis decreased sharply.

INTRODUCTION

Previous in vivo studies on the metabolism of virus-infected Lepidoptera by chemical and histochemical analyses indicate that both the DNA and RNA contents of nuclear-polyhedrosis-infected cells increase until just prior to polyhedron formation and then decrease (Morris, 1962; Benz, 1963; Morris, 1966a, b). Radioautographic studies on DNA synthesis have confirmed this and established that DNA synthesis ceases in nuclei containing mature polyhedra (Morris, 1967; Morris, 1968). The data on Lepidoptera midgut epithebum infected with Smithiauir~s suggest that cytoplasmic infection has only a minor effect on nuclear DNA and RNA metabolism (Benz, 1963). Chemical analysis of midgut cells of Bombyr mori infected with cytoplasmic-polyhedrosis virus showed an 476

increase in RNA and DNA (Kawase and Hayashi, 1965). Watanabe ( 1966) reported increased incorporation of uridine-sH, particularly in the nuclei of midgut cells of the silkworm (B. mori) infected with a cytoplasmic virus. He suggested that the nucleus is the site of viral RNA synthesis during the progress of the cytoplasmic disease. This paper deals with quantitative changes in DNA and RNA syntheses in fat body and midgut cells of lepidopterans and hymenopterans infected with nuclear- or cytoplasmic-polyhedrosis viruses. MATERIAL

AND

METHODS

Metabolism in Lepidoptera Larval Body Infected with a NuclearPolyhedrosis Virus

Fat

RADIOAUTOGRAPHIC

Field-collected second-instar larvae of hemlock looper, Lambdina fiscellaria lugubrosa, were reared individually on western hemlock, Tsuga heterophylla, in sterile glass vials to the fourthinstar as a check for possible latent infection. Forest tent caterpillars, Malacosoma disstria, were reared individually from field-collected eggs to the third-instar on artificial media ( McMorran, 1965). Hemlock loopers were each fed 10’ nuclear polyhedra isolated from the oak looper, L. f. somniaria, in distilled water or were fed distilled water alone (Morris, 1964). Tent-caterpillar larvae were placed individually in vials containing artificial media smeared with a suspension of approximately lo7 homologous nuclear polyhedra per milliliter of distilled water or with distilled water alone. All larvae were reared at 23°C.

the western

At 24-hr intervals, after initial feeding of virus, duplicate ether-anesthetized hemlock-looper larvae were injected with $1 (5p.c) of thymidine-“H (specific activity 3.9 c/m&l, Radiochemical Centre, Amersham, England) or with 2&l (1.25,~~) uridine-“H (S.A. 7.2 C/mM, New England Nuclear Corporation, Boston, Mass.) After 2 hr, and 1 hr, respectively, they were fixed by freeze-substitution, and processed for radioautography as described earlier (Morris, 1968). Tent caterpillars were injected with 2.5~1 (0.625~~) thymidine-“H (S.A. 15c/ rnhq International Nuclear Corporation, City of Industries, California) or with 2.5~1 (1.25 PC) uridine-:iH. After 2 hr and 1 hr, respectively, small sections of the larvae were prefixed for 12-24 hr in cold 5% glutaraldehyde in 0.1 hf phosphate buffer (Gomori) at pH 7.5 and postfixed in 2% osmic acid in phosphate buffer at pH 7.01 for 6 hr. Radioautographs were prepared using Kodak NTB2 nuclear emulsion. Metabolism in Hymenoptera Larval Midgut Infected with a Nuclear-Polyhedrosis Virus

STUDIES

477

Fifth-instar Diprion hercynaae reared at the Insect Pathology Research Institute, Sault Ste. Marie, Ontario were placed (at 23°C) on white spruce, Picea glauca, sprayed with a suspension containing 6 X 10’ homologous midgut nuclear polyhedra per milliliter of distilled water or with distilled water alone. At 24-hr intervals, duplicate larvae were injected with thymidine-“H (I.N.C.) or with uridineJH. After 2 hr and 1 hr, respectively, dissected midguts were fixed with glutaraldehyde and osmic acid and processed for radioautography, In addition, some larvae were injected with radioactive uridine or thymidine 10 days after initial feeding on contaminated foliage and 48 hr before pupation. These were processed within 20 hr folIowing pupation. Metabolism in Lepidoptera Larval Midgut Infected with a Cytoplasmic-Polyhedrosis Virus Third-instar Malacosoma disstria were placed on rearing media smeared with a suspension containing 2 X lo7 homologous cytoplasmic polyhedra in distilled water or with distilled water a1one.l At 24-hr intervals, duplicate larvae were injected with uridine-3H or thymidine-:‘H and processed following glutaraldehyde and osmic acid fixations. All tissue sections (4~) were stained with toluidine blue, aceto-carmine (Hann, 1965) or methyl green pyronin. A few were stained for protein (napthol yellow S or the Millon reaction). DNA and RNA were removed from control sections of diseased insects by enzymatic digestion ( Morris, 1966a, 1968). DNA synthesis was determined in all sections by correlating the percentage of labeled nuclei with nuclear diameters at different stages of infection. In nuclearpolyhedrosis virus infections, the nuclear diameters are known to increase as the 1I\lalacosoma disstria cytoplasmic polyhedra were supplied by Dr. F. T. Bird, Insect Pathology Research Institute, Sault Ste. Marie, Ontario,

0 (Normal) 34 5-6

18 29 29

5 10 15

M.d.

nuclear (b) ZJ

a Average bL (few ~Average

0 (Normal) 1 2-3

diameter grams); number

Time elapsed after initial feeding on virus contaminated foliage ( days )

9 17 31

M.d. 74 74 25

L.f.1. 80 90 15

Percentage of nuclei incorporating thymidine-aH

in 200

2 80 43

and

AND INFECTED

nuclei

nuclei

Percentage of incorporating thymidine-sH

OF THYMIDINE-JH ( HYMENOPTERA)

density). developing

nuclear (p ) 0

of 200 nuclei. H (high grain of silver grams

Average diameter

INCORPORATION

TABLE

1

0 0 96

M.d.

of cells polyhedra

2

and

L H L

M.d.

osmium

corresponding

L H L

Density of thymidine-sH silver grains in nuclei h

cytoplasm.

Percentage containing 0 68 98

of nuclei polyhedra

( HTG.)

26 12 21

C

in

of

12 28 7

8 25 11 tetroxide;

M.d.

in

L.f.1.

Incorporation uridine-sH nuclei

URIDINE-BH INTO GUT OF Diprion hercyniae WITH A NUCLEAR-P• L~HEDROSIS VIRUS

TABLE

L H L

L.f.1.

), fixation by freeze-substitution osmium tetroxide.

0 0 100

L.f.1.

Percentage containing

a

Incorporation uridine-sH nuclei

OF LEPIDOPTERA

Density of thymidine-sH silver grains in nuclei C

OF THYMIDINE-3H AND URIDINE-3H INTO FAT BODY INFECTED WITH NUCLEAR-P• LYHEDROSIS VIRUSES

a Lambdina fi.sceZhiu hcgubrosa ( Hulst ) ( Geometridae) (L.f.1. (Hbn.) (Lasiocampidae) ( M.d. ), fixation by glutaraldehyde and b Average of 100 nuclei. c L (few grams), H (high grain density). d Average number in 100 nuclei and 100 corresponding cytoplasm.

0 (Normal) 4-6 7-8

L.f.1.

L.f.1.

Md.

Average diameter

Time elapsed after initial feeding on virus contaminated foliage ( days )

INCORPORATION

of

10 7 13

Incorporation uridine-sH corresponding cytoplasm

Malacosoma

4 19 9

L.f.1.

Incorporation uridine-sH corresponding cytoplasm

C

in

of

of

disstria

7 14 3

M.d.

d

in

05

FIGS. 1-3. Labeling of fat body of fourth-instar Lambdina ji.sceZZuria Zugubrosa 1 hr after intrahemocoelic injection of uridine-sH at 0 (normal), 5, and 6 days, respectively, post-inoculation with a nuclear-polyhedrosis virus. Labeling is low in nuclei with mature polyhedra (arrow). Fixation by freeze-substitution. Toluidine blue stain. FIG. 4. DiEerential labeling of nuclear-polyhedrosis-virus-infected fat body of third-instar M&COsoma disstriu injected with uridine-aH, 6 days postinoculation. Hypertrophied nuclei without polyhedra are heavily labeled. Prefixation by cold glutaraldehyde and postfixation by osmic acid. Toluidine blue stain. FIGS. 5-7. Labeling of midgut epithelium of fifth-instar Dip&m hercyniae 2 hr after intrahemocoelic injection of thymidine-sH at 0 (normal), 1, and 3 days, respectively, postinoculation with a nuclear-polyhedrosis virus. Note margination of label in nuclei containing mature polyhedra (arrows). Glutaraldehyde fixation. Methyl-green-pyronin stain.

480

MORRIS

FIGS. 8-10. Labeling of midgut epithelium of fifth-instar D. hercyniae 1 hr after intrahemocoelic respectively, postinoculation with a nuclearinjection of uridine-aH at 0 (normal), 1, and 3 days, polyhedrosis virus. Note nuclear hypertrophy accompanying reduction in nuclear labeling (Fig. 9, nucleus with mature polyhedra. arrow). Dense inclusions are nuclear protein. P (Fig. 10) indicates Glutaraldehyde fixation. Methyl-green-pyronin stain. FIG. 11. Primordial mesenteron epithelial cells (P) of D. hercyniae pupa containing deeply stained polyhedra. Unstained polyhedra ( p) are located outside the cells. Glutaraldehyde fixation. Hann’s stain.

RADIOAUTOGRAPHIC

disease progresses (Morris, 1962). One to two hundred nuclei of the midgut or fat body were arbitrarily chosen and measured at each period after initial ingestion of virus. The density of silver grains over nuclei was the criterion for intensity 01 thymidine incorporation and was expressed as low (few grains) or high (numerous grains). RNA synthesis, in uridine-injected insects, was determined by counting silver grains developed in 200 arbitrarily chosen nuclei and the corresponding cytoplasm at each infection stage. RESULTS

AND

DISCUSSION

Metabolism in Fat Body of Lepidoptera Infected with Nuclear-Polyhedrosis Virus DNA labeling of virus-infected fat body (Table 1) followed the pattern already described (Morris, 1967, 1968). The 5-p increase in nuclear diameter during early stages of infection in M. disstria was apparently insufficient to be reflected by a change in the percentage of nuclei labeled. The rise and fall of DNA metabolism during the progress of the disease paralleled approximately the behavior of both nuclear and cytoplasmic RNA (Figs. l-4). The behavior of RNA may be partly explained by the findings of Yamafuji et al. (1965) that nuclear virus inoculations in B. mori cause an activation of ribonuclease. Yamafuji et al. ( 1954), using histochemical methods, observed that the amount of RNA did not change appreciably in the course of development of nuclear polyhedrosis. Gratis et al. ( 1945 ) and Morris ( 1966a, b),

STUDIES

481

however, using similar techniques, observed the rise and fall in RNA metabolism. Metabolism in the Midgut of D. hercyniae (Hymenoptera) Larvae Infected with a Nuclear-Polyhedrosis Virus The data (Table 2, Figs. 5-7) show that DNA synthesis and nuclear swelling during the progress of D. hercyniae midgut polyhedrosis follow the course of polyhedrosis in Lepidoptera fat body (Table 1) but RNA synthesis (Figs. S-10) decreases in early stages of infection and later increases. The observed nuclear hypertrophy is directly related to increased nuclear protein synthesis which, in turn, is related to RNA synthesis (Leuchtenberger and Schrader, 1951). Nuclei in early stages of infection (Fig. 9, arrow) accumulated large amounts of napthol yellow S-stainable materials which were sparse in normal cells, indicating increased synthesis of lysine, arginine, and histidine during the progress of the disease. The amount of basic protein was considerably reduced in nuclei containing mature polyhedra (Fig. 13, P). Healthy nuclei contained faint traces of tyrosine (Millon reaction) which were absent in infected cells. The proteins observed must be used in the construction of viral protein coat and polyhedral crystal since the latter are known to contain varying amounts of these amino acids (Wellington, 1951, 1953; Bergold and Wellington, 1953). The RNA metabolism observed during early infection probably represents a primary host-virus interaction. The twofold decrease in nuclear RNA at 24 hr postinfection may be explained by the transfer

FIG. 12. Same section as in Fig. 11 showing older proliferated pupal cells containing mature polyhedra in nucleus (N). P indicates polyhedra from shed larval gut absorbed on new pupal mesenteron epithelium. Glutaraldehyde fixation. Harm’s stain. FIG. 13. Labeling of midgut regenerative cells (rg) of D. hercyniua, 10 days postinoculation as 48 hr after intrahemocoelic injection of thymidine-sH, and larva with nuclear-polyhedrosis virus, 20 hr approx. after pupation. P indicates polyhedra shed from larval gut; dark staining inclusions Thymidine incorporation in regenerative cells and imaginal are remnants of basic nuclear proteins. discs in hyperdermal regions (rh) is high. Glutaraldehyde fkation. Methyl-green-pyronin stain.

482

FIG. 14. X indicates FIG. 15. uridine-sH. FIG. 16.

MORRIS

Same section as in Fig. 13 showing an area of heavily labeled proliferating hystolyzed larval gut. Glutaraldehyde fixation. Methyl-green-pyronin stain. Labeling of proliferating cells of virus-infected D. hercyniae pupa injected RNA synthesis is low. Glutaraldehyde fixation. Toluidine blue stain. Same section as in Fig. 15 showing heavy labeling of imaginal discs.

cells

(rg).

as larva

with

RADIOAUTOGRAPHIC

phenomenon of ribonucleic acid to the cytoplasm. This transfer (“messenger”) RNA is produced only intermittently and usually has a short life in the cytoplasm before becoming associated with ribosomes. Results from this study are comparable to those of SV-40 virus infections (Bernhard, 1965) of vertebrate cultured cells. At 10 hr postinfection, increased DNA synthesis was accompanied by a pronounced depression of RNA synthesis but RNA synthesis was abnormally high after the 18th hr. Benz’s (lQ60) data on D. hercyniae showed increased nuclear RNA content after a few hours of infection, RNA depression less than 24 hr later, and subsequent nuclear RNA content increase accompanying nuclear hypertrophy. The behavior of cytoplasmic pyroninophilic inclusions paralleled cytoplasmic RNA synthesis in the present study. Metabolism in Pupal Gut of D. hercyniae Infected as Larvae with a NuclearPolyhedrosis Virus In the present study, polyhedra from the shed larval gut appeared to be engulfed by the newly regenerated primordial pupal epithelial cells (Fig. 11) which have long been thought to have amoeboid characteristics (Henneguy, 1904). In older proliferated cells, a few nuclei were heavily infected with polyhedral virus (Fig. 12). DNA synthesis in undifferentiated replacement cells was low (Fig. 13) but a few of the more highly differentiated ones showed high DNA synthesis (Fig. 14). Whether the latter merely reflect the occasional wave of mitotic activity or a

STUDIES

483

metabolic response to the presence of infectious nucleic acid is unknown. Both nuclear and cytoplasmic RNA syntheses in replacement cells were low (Fig. 15). Imaginal tissues in the epidermal regions incorporated high amounts of radioactive thymidine (Fig. 13) and uridine (Fig. 16). Berry et al. (1967) recently found that after pupation RNA synthesis in most insect tissues decreases but increases later at the pupal-adult transformation. The rather low overall level of nucleic acid metabolism in the gut during pupal development probably explains Birds (1953) and Stairs’ (lQ65) observations that embryonic regenerative cells in Hymenoptera are resistant to virus infection but the new tissues formed from such cells may be infected. Metabolism in Gut of Malacosoma disstria Infected with a Cytoplasmic-Polyhedrosis Virus The results (Table 3, Figs. 17, 18) indicate that infection by cytoplasmic (RNAcontaining) virus had only a minor effect on DNA synthesis. The slight decrease in nuclear diameter suggests that cytoplasmic RNA synthesis is not completely independent of DNA synthesis and may reflect a loss of nuclear protein through the nuclear membrane into the cytoplasm. Goldstein ( 1958) has shown that labeled protein, unlike RNA, moves into the nucleus as well as out of it. In general, the replication of RNA-containing viruses has little or no effect on the nuclear integrity (Doi and Spiegelman, 1962; Salzman, 1962; Sanders, 1964; Bernhard, 1965; Erikson and Franklin, 1966).

FIGS. 17, 18. Labeling of normal (Fig. 17) and cytoplasmic-polyhedrosis-virus-diseased (Fig. 18) midgut epithelium of M. disstriu larva 2 hr after intrahemocoelic injection of thymidine-“H. Glutaraldehyde fixation. Methyl-green-pyronin and toluidine blue stains, respectively. FIGS. 19-21. Labeling of midgut epithelium of M. disstria larva 1 hr after intrahemocoelic injection of uridine-3H at O(normal), 3, and 4 days postinoculation with cytoplasmic-polyhedrosis virus. Nuclear labeling is only slightly reduced. Cytoplasmic labeling is greatly reduced, Glutaraldehyde fixation. Methyl-green-pyronin stain (Fig. 19). Toluidine blue stain (Figs. 20, 21).

0 0 d 98

Percentage of cells containing cytoplasmic polyhedra Average cliameter 14 13 12

AND INFECTED

nuclear (p) n

OF THYMIDINEJH

(Lrl~l0cAMPlD.4~)

INTO

GUT

OF Malacosomn

at this

30 39 26

stage

of infection.

H H H

Density of thymidine-aH silver grains in nuclei 7~

A CYTOPLASMIC-POLYHEDROSS

3

Percentage of nuclei incorporating thymidine-sH

WITH

URIDINE-SH

(1 Average of 200 randomly chosen nuclei. 0 H (high grain density). D Average number in 200 nuclei and 200 corresponding cytoplasm. d Cytoplasmic polyhedra are probably too small to be identified definitely

0 (Normal) 2-3 4-5

Time elapsed after initial feeding on virus contaminated foliage (days)

INCORPORATION

TABLE

( HRN.)

13 11 14

Incorporation uridine-sH nuclei

disstria \7IRPS

(

in

of

44 14 14

Incorporation u&line-sH corresponding cytoplasm

0

in

of

RADIOAUTOGFtAPHIC

As the disease progressed, RNA synthesis (indicated by incorporation of uridine-3H) remained relatively constant in the nucleus but decreased sharply in the cytoplasm (Table 3, Figs. 19-21). This indicates that replication of the cytoplasmic-polyhedrosis virus does not significantly affect the metabolism of nuclear RNA but has a profound effect on cytoplasmic RNA synthesis. A depression of cytoplasmic RNA synthesis in insect cells infected with RNA-containing viruses has also been reported by Hayashi and Kawase ( 1965). The present data support the view (Franklin and Baltimore, 1962; Hinz et al., 1962; Kjellkn, 1962; Simon, 1961), that replication of RNA viruses is almost independent of nuclear DNA and RNA syntheses and that the cytoplasm is the primary site of viral RNA synthesis. ACKNOWLEDGMENTS The author thanks A. Ficq, Universitk Bruxelles, Belgium, for reviewing the C. Watson for technical assistance, Chatelle for preparing the photographic

Libre manuscript, and E. prints.

do J.

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BERNHARD, W. 1965. Ultrastructural aspects of the normal and pathological nucleolus in mammalian cells. In “The Nucleolus. Its Structure and Functions.” Intern. Symp., Monograph 23,

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STUDIES

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BERRY, S. J., KRISHNAWMARAN, A., OBERLANDER, H., AND SCHNIEDERMAN, H. A. 1967. Effects of hormones and injury on RNA synthesis in Satumiid moths. j. Insect Physiol., 13, 15111537. BIRD, F. T. 1953. The effect of metamorphosis on the multiplication of an insect virus. Can. 1. zooz., 31, 300-303. DOI, R. H., AND SPIEGELMAN, S. 1962. Homology test between the nucleic acid of an RNA virus and the DNA in the cell host. Science, 138, 1270-1272. ERIKSON, R. L., AND FRANKLIN, R. M. 1966. Symposium on Replication of viral nucleic acids. 1. Formation and properties of a replicative intermediate in the biosynthesis of viral ribonucleic acid. Bacterial. Rev., 30, 267-278. FRANKLIN, R. M., AND BALTIMORE, D. 1962. Patterns of macromolecular synthesis in normal and virus-infected mammalian cells. In “Basic Mechanisms in Animal Virus Biology.” Cold Spring Hurb. Symp., 27, 175198. GOLDSTEIN, L. 1958. Localization of nucleus-specific protein as shown by transplantation experiments in Amoeba proteus. Exptl. Cell Res., 15, 635-637. GRATIA, A., BRACHET, J., AND JEENER, R. 1945. Etude histochimique et microchimique des acides nucleiques au tours de la grasserie du ver 1 soie. BuEl. Acad. Roy. Med., 10, 72-81. HANN, J. J. 1965. A modified azan staining technique for inclusion body viruses. .I. Inuertebrute Pathol., 8, 125-126. HAYASHI, Y., AND KAWASE, S. 1935. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombylc mori L. (IV) Subcellular distribution. J. Sericult Sci. Japan, 34, 171-176. HENNEGUY, L. F. 1904. “Les Insectes.” Masson et Cie., Paris. HINZ, R. W., BARSKI, G., AND BERNHARD, W. 1962. An electron microscopic stucly of the development of the encephalomyocarditis (EMC) virus propagated in vitro. Exptl. Cell Res., 26, 571586. KAWASE, S., AND HAYASBI, Y. 1965. Nucleic acid and protein changes in blood and midgut of the silkworm, Bombyx mori ( Linnaeus), during the course of cytoplasmic polyhedrosis. J. Invertebrate Pathol., 7, 49-54. KJELLI~N, L. 1962. Effect of 5-halogenated pyrimidines on cell proliferation and aclenovirus multiplication. Virology, 18, 64-70.

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in

11,

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comtheir Bio-

YAMAFUJI, K., SHIMAMURA, M., AND YOSHIHARA, F. 1954. Behaviour of nucleic acids in formation process of silkworm virus. Enzymologia, 16, 337-342. YAMAFUJI,

K.,

YOSHIHARA,

M. 1956. Action to the formation mologia,

of of

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F.,

AND

ribonuclease silkworm

SHIMAMURA, in virus.

relation Enzy-