Autoradiographic studies on nucleic acid metabolism in granulosis-infected fat body of larvae of Carpocapsa

JOURNAL

OF INVERTEBRATE

18, 70-80

PITHOLOGY

Autoradiographic

Studies

Granulosis-Infected

on Nucleic

Fat

Body

G. BENZ Department

of Entomology,

Swiss

Acid

of Larvae

Metabolism

in

of Curpocupsa

R. WAGER

AND

Federal

Received

(1971)

Institute

December

of Technology,

Zurich,

Switzerland

21, 1970

Autoradiographs were prepared of larvae of the codling moth C’arpocapsa pomonella injected with tritiated thymidine or uracil at different times after infection wit,h granulosis virus. The first reaction of fat body cells on virus infection consists of a sharp increase in the rate of RNA synthesis (mostly ribosomal RNA). This is followed by the degeneration of the nucleolus and the chromatin, and the concomitant reduction of the rates of RNA and DNA synthesis to normal and subnormal levels, respectively. The subsequent, formation of the “virogenic stromata” involves a second resurgence of RNA synthesis of relatively long duration (three times normal level) and a tremendous increase of DNA synt,hesis with a pronounced maximum (30 times normal level) at 60-70 hr after infection. The results are compared with histochemical data on the same virosis and nuclear polyhedroses of other insects.

membrane (Huger and Iirieg, 1961). Nucleus and cytoplasm can not therefore be distinguished properly in t)he middle and late stages of the disease. On the other hand, the quantitative analysis of t,he incorporat’ion of labeled nucleic acid precursors by means of autoradiographs depends orI the proper measurement of the areas in which the marker has been incorporated and on the proper estimation of t,he volumes of the incorporating units, as described by Benz (1967) for the nuclear polyhedrosis of Diprion hewyuiae. However, since large parts of the cytoplasm of fat body cells are occupied by fat vacuoles, which are not involved in the nucleic acid metabolism, the “nuclear field,” as defined by W%ger and Benz (1971), may be regarded as a funct’ional unit 1vit.h relatively well defined borders. Timing of the biochemical processes, i.e., incorporation of nucleic acid precursors, poses a further problem when dealing with granuloses, because it is not possible to strictly synchronize the pathogenic processes within an individual or groups of larvae. Thus the same stage may be found in sections of larvae which had been killed at different times after infection. Based on the

INTRODUCTION

Studies on the nucleic acid metabolism of virus-infected insects have been reported by several authors. The papers of Gratia et al. (1945), Tarasevich (1952), Yamafuji et al. (1954), Benz (1960,1967), Watanabe (1967), Morris (196Sa,b), and Shigematsu and Xoguchi (1969a,b) deal with nuclear polyhedroses, whereas t,hose of Hayashi and Kawase (1965a,b,c), Kawase (1967), Iiawase and Hayashi (1965), Iiawase and Kawamori (1968), and Morris (196Sb) report on cytoplasmic polyhedroses. Concerning granuloses, only few histochemical st’udies deal with aspects of the nucleic acid metabolism (Wittig, 1959; Huger, 1960; Watanabe and Iiobayashi, 1970; W&ger and Benz, 1971). The present analysis of autoradiographic studies on the nucleic acid metabolism of the granulosis-infected larval fat body of the codling moth Carpocapsa pomonella is based on the last-mentioned paper. Autoradiographs of granulosis-infected cells are more difficult to evaluate than those of polyhedrosis-infected tissues because the development of granulosis viruses usually leads to an early breakdown of the nuclear 70

NJCLEIC

ACID

distribution of t,he stages in different individuals, it is possible to calculate the medium time after infection for each stage. The autoradiographically determined data on the incorporatJion of nucleic acid precursors (for details see Materials and Methods) could then be timed on t,his basis. The method, though perhaps not fully satisfactory, allowed a good estimation of the temporal sequence of nucleic acid synthesis during virogenesis in granulosis-infected fat body cells of C. pomonella. MATERIAL

AND

?VIETHODS

The same strain of C. pomoflella was used and the same methods of rearing, infecbing, and killing the larvae were applied as by WSiger and Benz (1971). Normal and infected larvae were narcotized with CO2 and injected via an abdominal proleg wit’h either 4 ~1 of methyl-3H-thymidine (specific activity = 3 Ci/mmole) or 1 ~1 of 3H-uridine (specific activity = 2 Ci/mmole), the radioactivity being 1 &i/p1 in both series. Batches of 5-10 larvae were injected at 12 hr intervals from 0 to 72 hr and at 96 and 120 hr after infection. Zero controls, i.e., healthy larvae, were injected at different times from the beginning t,o t,he end of the experiment. Larvae injected with thymidine were sacrificed exactly 4 hr after injection whereas those injected with uridine were sacrificed 2 hr after inject,ion. The killed larvae were fixed in Bouin’s fluid. The autoradiographic technique was the one described by Benz (1967) except that Kodak Nuclear Track Emulsion NTB 3 was used. Only some sections on which Feulgen’s reaction was performed were stained prior to coat,ing with the photographic emulsion. After coating, the dry preparations labeled with 3H-thymidine or 3H-uridine were exposed for 10 and 20 days at 2”C, respectively. Aft(er photographic processing, the autoradiographs were stained with toluidine blue or hematoxylin after Mayer or, if

METABOLISM

71

previously stained by Feulgen’s method, with light green. For each stage of the disease (see WBiger and Benz, 1971) 50-60 typical cells from 3 or 4 individuals were selected at random. Cells with extremely small or large nuclei were not considered. The rate of incorporation of the markers was estimated after the method of Benz (1967). At a magnificat’ion of 800 X the squares of the ocular grid in the microscope enclosed a standard area (SA) of 56.25 p2 (side length, 7.5 P). Depending on the stage of the disease,t,he areas of sections of nuclei or “nuclear fields” were measured and the silver grains above the areas were counted. The background above sections of larvae injected with water was negligible (0.12 grains/SA). Since the soft P-rays of tritium have only a weak potency to penetrate tissues, but nonetheless in a certain small range (1.5 P, after Lajtha and Oliver, 1959) the number of silver grains per SA (density) represents the radioactivity of a certain volume. This standard volume (SV) may be estimat,ed as approximately 1.5 X SA p3. The density of the silver grains thus gives a measure for the specific incorporation (SI) of a la,beled precursor (Benz, 1967). The total rate of incorporation was calculated by multiplying the value SI with the quotient V/SV, V being the volume of the corresponding nucleus OI “nuclear field.” For calculating t*he volumes, the nuclei or “nuclear fields” were regarded as spheres with the radius r’ = (area,/3.14)1’2. Since many of the nuclei are ovoid, the calculated volumes represent only approximate values. An additional source of error comes from the fact that the sections did not always run through the equator of the nuclei. Therefore some nuclei were probably larger than estimated. However, the differences between the true and the calculated volumes were probably negligible. A Fortran VI computer program was formulated which allowed the calculation at

72

BENZ

AND

each stage of: (1) the mean area of the nuclei or “nuclear fields,” (2) the mean number of silver grains per SA, (3) the number of silver grains per mean nuclear area, (4) the mean volume of the nuclei or “nuclear fields,” and (5) the medium rate of incorporation per cell. Each mean was calculated with the corresponding standard error, the standard deviation and, except for (3), each stage was compared nith each other by the Student’s t test. The input consisted of: stage, area of nucleus or equivalent in SA, and number of silver grains of area. RESULTS Incorporation of Thymidine Healthy Larvae

and Uricline in

Thymidine is incorporated exclusively into DNA. In healthy larvae only the chromatin of the nuclei is therefore labeled. The highest incorporation of 3H-thymidine was found in the midgut epithelium, followed by the fat body, the hypodermis, and the silk glands. Not all nuclei of the fat body were labeled, and the density of the silver grains varied considerably above different nuclei. Some especially large nuclei, distributed irregularly in the fat body, incorporated distinctly more thymidine t’han ordinary nuclei and were therefore not evaluated. Variation in the rate of incorporation ~vas especially large between nuclei of different individuals. Since the density of silver grains above different sections of the same nucleus shows only minor variation, differences in the density of silver grains above different nuclei indicate true metabolic differences. Autoradiographs of healthy larvae (zero controls) injected and killed at different times did not show differences in the rate of thymidine incorporation, indicating that one control value was sufficient for comparison with the different stages of diseased nuclei. The RNA marker 3H-uridine is incorpo-

M’;iGER

rated in all organs of healt,hy larvae, above all in the midgut epithelium, the fat body, the silk glands, and the hypodermis. Contrary to the findings wit’h the DXA marker, the RNA precursor was incorportlted in practically all cells, although a four times lower dose had been injected. As with thymidine, incorporation of uridine in zero controls gave no variations at different times. During the 2 hr of incorporation, healthy fat body cells incorporated uridine mainly int,o bhe nuclei, especially into t,he nucleoli, although incorporation int,o the cytoplasm was also noticed. The rate of uridine incorporat)ion of different fat body cells both lvithin an individual and between individuals varied much less than in the case of thymidine incorporation. This fact indicat’es that the nuclei of these cells synthesize RNA more or less continuously, whereas DNA synthesis is discontinuous. The DNA Cells

Metabolism

of G~ar~ulosis-il?fected

Specific &corporation of 3H-thymidine. The densit’y of silver grains as a measure of the specific incorporation of the DNA precursor (i.e., incorporation per standard volume) varies considerably at different stages of the disease. Such values are computed in Table 1. It is evident that the specific incorporation of 3H-thymidine is normal up t’o 25 hr after infection. However, Ivhereas in healthy insects only part of the nuclei become labeled with tritium, most nuclei of the fat body are labeled in infected larvae. Morphologically the nuclei still resemble normal nuclei up to about 20 hr after infect*ion (stage 1 of WBgar and Benz, 1971; for more detailed informabion on morphological features, this paper should be consulted). Later the nucleoli begin to swell (stage a), reaching their maximal volume at 24-30 hr after infection. At that time the chromatin condenses along the nuclear periphery (stage 3) and degenerates there. At the same t>ime the specific incorporation of 3H-thymidine decreases.

NUCLEIC

ACID

1

TABLE SPECIFIC

INCORPORATION

OF 3H-THYMIDINE

IN SECTIONS

Stage

73

METABOLISM

GRAINS CELLS

(SILVER OF FAT

BODY

PER STANDARD

Areab

Time after infection

AREA

Silvergrains

SA)

per SA

P2

SA

2

&

Sig.”

48.7 53.1 61.7 65.8 79.3 169.3 207.9

0.9

6.78

f

0.42

a

20 25 30 35 60 75

60 50 60 60 60 50 51 50 50

300.9 238.8

5.94 5.17 3.20 2.88 33.93 22.65 6.70 2.87

f f f f f f f f

0.41 0.30 0.28 0.23 2.69 1.43 0.43 0.28

a,b b c c

90 100

0.9 1.1 1.2 1.4 3.0 3.7 5.4 4.3

-1 Normal 1 1 2 3 5 6 7 8

-

0 N = number of nuclei counted. b Area = area of section of nucleus or nuclear field. c Sig. = significance of differences. Same letters indicate at 5% level. All other differences are significant (P < 0.01).

It is not,eworthy that up to this stage of the disease nearly all silver grains are found above the chromatin. The bulk of the peripheral chromatin disappears at 42-48 hr after infection. The nuclei swell considerably (Fig. 1) and the nuclear membrane is disrupted (stage 4). Distinctly Feulgen-positive strands appear de novo in the “nuclear field,” and the specific incorporation of 3H-thymidine increases dramatically to a level which is about five times higher than in healthy nuclei. The Feulgen-positive strands are considered to represent “virogenic stromata.” They condense and appea,r on histochemical preparations as intensely Feulgen-positive masses (st,age 5) heavily labeled, especially along their margins. The stromata then degenerate or, at least, lose Feulgen-positive material. Protein islets and pseudonucleoli are formed in the still expanding “nuclear field” (stage 6). The specific incorporation of 3Hthymidine decreases but is still more than three times higher than in normal nuclei. When the pseudonucleoli disappear and the proteinaceous masses almost fill the nuclear field (stage 7)) specific incorporation of 3Ht,hymidine is reduced to the level of normal

that

corresponding

values

a& c

are not

different

nuclei, the silver grains being evenly distributed over the whole nuclear field. Later on (stage 8) specific incorporation of 3Hthymidine is reduced to subnormal values. Since the “nuclear fields” expand continuously (Fig. I), the specific incorporation of 3H-thymidine, which gives information on the rate of incorporation per unit volume only, does not indicate whether the reduced incorporation found in stages 6 to 8 represents a true decrease or merely a dilution effect. Incorporation of 3H-thymidirze per nucleus. In order to obtain better information on the real rate of incorporation per nucleus or nucleus equivalent, we estimated the volumes of the nuclei or “nuclear fields” (see methods) at different stages of granulosis (Fig. 1) and, on the basis of these values, calculated the approximate rates of incorporation per unit. The values are computed in Table 2 and shown in Fig. 2. Figure 1 shows that the volumes of the nuclei increase at stage 2. However, since the specific incorporation is reduced at this sta.ge the rate of incorporabion per nucleus appears normal for some time, reaching a subnormal level only at stage 3. Afterward

Cl

t)he rate of thymidine incorporation, i.e., DSA synthesis, increases drastically. The maximum reached at’ stage 5 (under our conditions at about 60 hr after infection) indicates that, at this stage of the disease, the rate of DNA synthesis is more than 30 times higher than in normal nuclei. It also shows that the decrease in the ratme of specific incorporat’ion of 3H-thymidine found at

0 .

0. i

FIG. 1. Increase of volume of fat body nuclei and nuclear equivalents (i.e., “nuclear fields”) at different stages after infection with granulosis virus. Different. symbols indicate different independent series.

FIG. 2. Rate of thymidine body cell at different stages

TABLE VOLUMES

OF

Stage

Normal 1 1 2 3 5 6 7 8 a Calculated volumes (SA

20 25 30 35 60 75 90 100

Volumes

N 9 60 50 60 60 60 50 51 50 50

on the assumption X 1.5 = 84.37 $).

263 297 370 418 555 1759 2367 4121 2891

per fat

2

NIJCLEIORNUCLEARFIELDS (ABSOLUTEIN@A~YD INCORPORATION OF 3H-THYMIDINE PER CELL Time after infection

incorporation of granulosis.

REL.4TIVE, i.e., NORMAL = 1) ANDTOTAL (i.e., NUCLEAR EQUIVALENT)~

of nuclei Sig.

Incorporation/cell

Relative

2

1 1.1 1.4 1.6 2.1 6.7 9.0 15.7 11.0

20.6 20.7 21.8 15.4 18.1 697.7 583.2 309.8 103.2

a it b

C C

that the specific activities Otherwise same as Table 1.

in Table

1 represent

& f f f f f f f f f

1.6 1.7 1.3 1.6 1.9 91.1 49.2 30.1 18.4

activities

Sig. a a it x,h c c

of standard

NUCLEIC

ACID

75

METABOLISM

TABLE

3

SPECIFIC INCORPORATION OF 3H-URIDINP Stage

Normal 1 1 2 3 5 G 7 8 Q Otherwise

Area

Time after infection

N

10 20 25 35 50 70 90 100

50 50 50 51 51 49 49 50 50

same

as Table

EL2 G5.3 63.2 59.7 87.2 91.7 113.3 215.2 280.1 284.1

f f f f f f f f f

Silvergrains/SA SA

27

2.1 1.1 1.1 l.D 1.0 2.0 3.8 5.0 5.1

7.44 7.97 13.43 18.35 3.09 9.23 3.91 3.01 0.88

f f f f i f f f f

SE

Sig.

0.62 0.43 0.80 0.99 0.37 0.85 0.22 0.18 0.09

a

b % b

1.

stages 6 to 8 corresponds with a real decrease eifie incorporation drops t.o a subnormal level. The distribution of t,he silver grains is in the rate of DNA synthesis. still uneven. They are found above the pseuThe RNA Metabolism in Granulosis-Illfected donucleoli and the rests of the FeulgenCells positive skomaba. Later on (stages 7 to 8) SpeciJic irzcorporation of 3H-uridine. Soon specific incorporat’ion drops to very low aft,er infect‘ion the fat body cells incorporat,e levels and the silver grains are evenly dismore 3H-uridine than normal cells (Table 3). tributed over the “nuclear field.” Incorporation of 3H-uridine per nucleus. The silver grains are primarily found above the cytoplasm, the chromatin, and the Incorporation of 3H-uridine per nucleus or nucleolus. When t,he nucleoli begin to swell “nuclear field” was calculated as for 3H(stage l-2), most grains are found above the thymidine. The values are computed in nucleoli. Specific incorporation of 3H-uridine Table 4. More than in the case of thymidine reaches a first maximum at stage 2 when ineorporat.ion, t,he values of specific incorpo3H-tjhymidine incorporation is still normal. ration become drastically changed when At st#age8, when the nucleolus begins to calculat’ed per nuclear unit, though only after stage 3. The graphic representation in Fig. 3 degenerate, specific incorporation of 3Hshows that the first maximum at st#age2 is uridine drops to subnormal values. After followed by a depression to about normal the nucleolus has disappeared, only the level. However, the second neak maximum condensed chromatin at the nuclear periphof specific incorporation at stage 5 is but the ery is weakly labeled. beginning of a second maximum with a As soon as t’he “virogenic stroma” apbroad shoulder up to stage 7. The ca.lculated pears, the number of the silver grains above values indicate increasing rates of incorpothe central region of the “nuclear field” ration between 50 and 90 hr after infection. increases (stage 4). The specific incorpora- But since these values are not significantly tion of 3H-uridine then reaches a second different at. the 5% level, they might as well (though relatively low) maximum at stage 5. be regarded as indicating a high plateau. The Silver grains are mainly found above the incorporation of 3H-uridine per unit reaches strands of the “virogenic stroma.” At about the same high value as in stage 2. It stage 6, n-hen the stromata disappear, spe- decreasesonly at stage 8, when most of the

76

BENZ

AND

WiiGER

TABLE INCORPORATION

Stage

Time after infection

Normal 1 1 2 3 5 6

10 20 25 35 50 70

7 8

90 100

0 Otherwise

as in Table

Volumes

N

50 50 50 51 51 49 49 50 50

4 OF 3H-U~~~~~~” of nuclei

Incorporation/cell

d

Sig.

relative

407 388 356 628 690 931 2433

a a

1 1 0.9 1.5 1.7 2.3 6.0

3640 3709

c c

E b

2

si.

36.2 35.5 55.6 127.6 30.9 96.4 111.8 123.4 34.5

8.9 9.1

f f i f f i f f i

3.4 2.5 4.1 7.3 3.8 9.4 9.2 10.3 3.5

a a b a ;,e b,c a

2.

0

,o

20

30 2

FIG, 3. Rate of uridine incorporation body nucleus or nuclear equivalent.

per

fat

-

virus rods are enclosed by the capsule protein.

40 3

Swelling

-

so 4

60 5

70 6

80

90 7

100 8

110

h stage

of nucleoli

Pyroninophilic reaction of CytoplaSm Clumping of host chromatin Disruption of nuclear membrane b-1 Virogenic stmmata t-1 Pseudonucleoli Capsule

Synopsis

s1g. .

protein

islets

of the Results

Figure 4 gives a synopsis of the incorporation ra.tes for 3H-thymidine and 3H-uridine, together with an indication of the most important cytologic features at different stages of granulosis. The first reaction of the

FIG. 4. Synopsis of results: Curves indicate rate of DNA synthesis (solid line) and RNA synthesis (broken line) at different stages of granulosis. Horizontal lines beneath time scale indicate times at which certain cytopat,hologic features can be observed.

NUCLEIC

ACID

nucleus on infection with the virus consists in a tremendous increase in the rate of RNA synthesis, which obviously concerns nucleolar RNA, i.e., ribosomal RNA. This first upsurge of RNA synthesis is followed by the degeneration of the nucleolus and the chromatin, and a significant reduction of DNA synthesis to a subnormal rate and of RNA synthesis to a normal level. The nuclear membrane then becomes disrupted and the “virogenic stromata” are formed. This process involves a great increase in the rate of RNA (3X normal level) and DNA synthesis (30X normal level) and is obviously related with the formation of virus particles. The rate of DNA synthesis reaches a maximum at 6(t70 hr after infection and decreases afterward. The rate of RNA synthesis, on the other hand, increases further (Fig. 3) or at least continues at the same high level (Fig. 4) for another 24 hr. The so-called pseudonucleoli are formed during this time. The rate of RNA synthesis decreases rapidly, i.e., shortly before the majority of the capsules become fully mature and refractive to staining. Simultaneously, the pseudonucleoli disappear. It seems logical to correlate these processes with the synthesis of capsule protein. DISCUSSION

The results of our investigation show that the most important biochemical steps involved in the development of the granulosis virus of C. pomonella begin in the nucleus and, after the rupture of the nuclear membrane, continue in a restricted area of the cell which is intimately connected with the former nucleus. This is true not only for the nucleic acid metabolism. Unpublished data on the incorporation of 3H-lysine eonfirm that protein synthesis follows the same rule. It is, therefore, not astonishing that the first fully mature virus rods are found within the nuclear membrane (Huger and Krieg, 1961), although the latter is probably

METABOLISM

77

disrupted at several places when the first rods appear (Bird, 1963; Allenspach and Benz, unpubl.). The first reaction of the infected cell, i.e., increased synthesis of ribosomal RNA, may be regarded as the first step to an increase in protein synthesis (enzymes and/or structural proteins). An increase of the size and/or number of nucleoli, followed by an increase of RNA in the cytoplasm, are also characteristic of nuclear polyhedroses (Gratia et al., 1945; Benz, 1960, 1963, 1967; Schnyder, 1967). In the case of the nuclear polyhedrosis of Dipprion hercyniae, it leads to a transitory increase of the RNA concentration in the infected midgut (Benz, 1967). Since this increase in RNA is followed by an increase in DNA, Benz has concluded that infection of a cell by a nuclear polyhedrosis virus induces all those biochemical processes which eventually could lead to cell division and dilution of the virus. Thus the first steps in the pathogenesis of nuclear polyhedrosis might be interpreted as an abortive defense reaction of the cell, leading to the synthesis of all enzymes necessary for the multiplication of the virus. Similar mechanisms are probably at work in the granulosis-infected fat body of Choristoneura murinana, where some cells are even stimulated to divide mitotically (Wittig, 1959). However, no mitoses can be observed in the granulosis-infected fat body of C. pomonella. Furthermore, contrary to the situation in D. hercyniae, the increase in the rate of RNA synthesis is not succeeded by an increase in the rate of DNA synthesis; it is followed directly by a reduction of the rate of DNA synthesis to a subnormal level. The histochemical data suggest also that the host chromatin begins to degenerate and that DNA is broken down. However, it is well known that a reduced Feulgen reaction does not always indicate a loss of DNA. As long as quantitative determinations of the DNA contents of the infected cells are missing,

this point is open to discussion. The rupture of the nuclear membrane, which follows the disappearance of the nucleolus and most of the visible chromat’in, might be interpreted as a step of mitosis (1at.e prophase) which, however, would be abortive. The reduction of thymidine incorporation to a subnormal level does not contradict this hypothesis. Thus the first stages of granulosis are not necessarily entirely different from those of t,he nuclear polyhedrosis of D. hercy)Gae. The differences in appearance and in the early synthesis of DNA might be a consequence of t’he cytological characteristics of the affected tissues rat’her than of the different, species of viruses and/or hosts. The large nuclei of t’he midgut of D. hercyniae, with their multiple number of nucleoli, are obviously polyploid, whereas t,he relatively small nuclei of the fat body cells of C. powonella with, as a rule, only one nucleolus, are certainly of a lower degree of ploidy. Since the polyploid cells of D. hercyniae cannot divide mitotically, but seem to be ready to increase their ploidy, the infecting virus may stimulate the synthesis of host’ DNA. In C. pomonella, on the other hand, the virus may stimulate t,he (diploid) cells to divide mitotically, thus inducing the synthesis of all the enzymes and building stones necessary for this step. As in t’he case of the nuclear polyhedrosis virus this might provide the granulosis virus with the biochemical mechanisms which it needs for replication. This mechanism would then be directed by the virus in a way which leads to the destruction of t,he host chromatin and to a halt of any mitotic processeswhen the nuclear membrane is disrupted. As mentioned above: the first mature virus rods appear at’ this stage, indicating that the replication of virus DNA and virus protein begins earlier, probably at the stage when t’he rate of thymidine incorporation reaches the lowest values. After the rate of thymidine incorporation changesfrom decreasing t,o increasing values,

the rate of DKA synthesis increases tremendously for about, 30 hr and large a.mounts of new DNA accumulate in the intensely Feulgen-positive “virogenic stromata.” Although the DNA contents of the fat body cells has not been measured the cytochemical observations indicate that stage 5 cells contain much more DNA t,han normal cells (compare Figs. 1 and 11 by W%ger and Benz, 1971). Similar observations on a. histochemical basis have been reported by Gratia et’ al. (1945) and Benz (1963) for nuclear polyhedrosis-infected t,issues of Lepidoptera. The observations of Gratia et al. in Bombyz ntori have been verified by Tarasevich (1958), who measured a net, increase by 50% of the DNA contents in jaundiced silkworms. Benz (1960, 1967), on bhe other hand, found no significant increase of DXA in late stages of nuclear polyhedrosis-infected midgut cells of the hymenopterous D. hercyniae. However, since the polyploid cells of the latter already contain relatively large amounts of Dn’A, the difference might rather depend on cytological than on systematic criteria. As menOioned before, the nuclei of the fat-body cells of C. pomonella are relatively small. An increase of DNA might therefore be necessary for the production of large amounts of virus particles (about 10” capsules,/larva). In D. hercyniae a doubling of the rate of thymidine incorporation during the primary phase of nuclear polyhedrosis led to a net increase of DnTA (probably host DNA) in the midgut’ cells, whereas a 6 times normal rate during the phase of virus multiplicat,ion and simultaneous breakdown of host DNA was sufficient to maintain or slightly increase t)he DSA contents of the cells (Benz, 1967). In the granulosis-infected fat body of C. powLo)lebla thymidine incorporation reaches a 30 times higher rate than in normal cells and may thus lead to an increase in the DNA content’s. Concerning the synthesis of RNA in the granulosis-infected fat body two features

NUCLEIC

ACID

are remarkable: (1) the occurrence of two disGnct phases of synthesis, which are well separated by a short phase of normal uridine incorporation and (2) the long duration of the second phase which transgresses the time of maximal DXA synthesis by 24 hr. The first phase of RNA synthesis falls together with the strong gr0wt.h of t,he nucleolus and thus indicates an intensive production of ribosomal RNA, as mentioned before. The second phase falls together with the formation of the “virogenic stroma,” the “pseudonucleoli,” and the capsules. All evidence available indicates that the “pseudonucleoli” contain RNA: they are pyroninophilic (Wliger and Benz, 1971) and seem to consist of aggregated ribosomes or similar particles (Allenspach and Benz, unpubl.). However, it is not yet clear whether or not the “pseudonucleoli” are synthesized de novo or merely represent, aggregations of ribosomes derived from the former nucleolus. In the latter case the long duration of the second phase of RNA synt.hesis might indicate that, mRKA is synthesized up to the very end of capsule formation. The synthesis of capsule protein begins early. Some fully formed capsules may be found already 6 hr after the rupture of the nuclear membrane (Allenspach and Eenz, unpubl.). We feel that several questions concerning the nucleic acid met.abolism in granulosis infected fat body cells remain open. Furt.her work to clarify these points is under way. REFERENCES BENZ,

G. 1960. Histopathological changes and histochemical studies on the nucleic acid metabolism in the polyhedrosis-infected gut of Diprion hercyniae (Hartig). J. Insect Pathol., 2, 259-273. BENZ, G. 1963. A nuclear polyhedrosis of Malacosoma alpicola (Standinger). J. Znsect Pathol., 5, 215-241. BENZ, G. 1967. Untersuchungen iiber den Nucleinslurestoffwechsel gesunder und virusinfizierter Larven der Fichtenblattwespe Diprion hercyniae (Hartig). Vierfeljahrschr. Naturjorsch. Ges. Zurich, 112, 29-70.

METABOLISM

79

BIRD,

F. T. 1963. On the development of granulosis viruses. J. Insect Pathol., 5, 368376. GRATIA, A., BIXACHET, J., BND JEENER, R. 1945. Etude histochimique et. microchimique des acides nuclbiques au tours de la grasserie du ver b soie. Bull. Acad. Roy. Med. Belg., 10, 72-81. H~YASHI, Y., AND Iiawvas~, S. 1965a. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyx mori L. I. Specific RNA extracted from cytoplasmic polyhedra. J. Sericzelt. Sci. Japan, 34, 83-89. HAYASHI, Y., AND K~~AsE, S. 1965b. St,udies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyx mori L. III. Subcellular distribution. J. Sericdt. &i. Japan, 34, 171175.

HAYASHI, Y., AND K~WASE, S. 1965c. Studies on the RNA in cytoplasmic polyhedra of the silkworm, Bombyx mori 1,. V. Changes in fractionated RNA in the midgut. J. Sericult. Sci. Japan, 34, 244-250. HUGER, A. 1960. Ueber die Natur des Fadenwerkes bei der Granulose von Choristoneura murinana (Hiibner). -~rafzlrzuissensch~jten, 47, 358-359. HUGER, A., AND KRIEG, A. 1961. Electron microscope investigations on t.he virogenesis of the granulosis of Choriafoneztra m~rt%nana (Hiibner). J. Insecf Pafhol., 3, 183-196. KAWASE, S. 1967. Ribonucleic acid extracted from the cytoplasmic-polyhedrosis virus of the silkworm, Bombyr n.ori L. J. Inverfebr. Pufhol., 9, 136-138. K~WASE, S., -END H.iy.4~~1, Y. 1965. Nucleicacid and protein changes in blood and midgut, of the silkworm, Bombyx mori (Linnaeus), during t.he course of cytoplasmie polyhedrosis. J. Inverfebr. Pathol.. 7, 49-54. K’awvass, S., AND K.ZW.~MOILI, I. 1968. Chromatographic studies on RNA synthesis in the midgut of the silkworm, Bombyx mori L. infected with a cytoplasmic-polyhedrosis virus. J. Invertebr. Pathol., 12, 3g5-204. LAJTH~, L. G., AND OLIVER, lb. 1959. The application of autoradiography in the study of nucleic acid metabolism. Lab. Invest., 8, 214224. MORRKS, 0. N. 1968a. Metabolic changes in diseased insects. I. Autoradiographic studies on DNA synthesis in normal and in polyhedrosis-virus infected Lepidoptera. J. Invertebr. Pafhol., 10, 28-38. MORRIS, 0. N. 1968b. Metabolic changes in diseased insects: II. Radioautographic studies

on DNA and RNA synthesis in nuclearpolyhedrosis and cytoplasmic-polyhedrosie virus infections. J. Znvertebr. Palhol., 11, 4i6486. SCHNTDER, U. 1967. Untersuchung einer Kernpolyedrose von Sterrha seriafa Schrk. (= Ptychopoda seriata Schrk. = Acidalia virgularia Hb.) (Geometridae, Lepidoptera) und deren Beeinflussbarkeit durch Hunger, DDT, DNOC und Farnesyl-methyl-aether. Ph.D.Thesis, Swiss Federal Inst,itute of Technology, Zurich, 60 pp. SHIGEMATSU, H., .4lrj~ NOGIJCHI, A. 1969a. Biochemical studies on the multiplication of a nuclear-polyhedrosis virus in the silkworm, Bombgx n~ori L. I. Nucleic acid synthesis in larval tissues after infection. J. Znverfebr. Pdhol., 14, 143-149. SHIGEMATSU, H., SND NOGUCHI, A. 1969b. Biochemical studies 011 the multiplication of a nuclear-polyhedrosis virus in the silkworm, Bombyx mori. III. Functional changes in infected cells, with reference to the synthesis of nucleic acids and proteins. J. Z,nvertebr. Pathol., 14, 308-315. TARASEVICH, 1,. M. 1952. Formen des Phosphors und des Rtickstoffs in gesunden und gelbsiichtigen Maulbeer-Seidenraupen (Bom-

byx mori). Biochimiya, 17, 282-287 (Russian with German summary). T.LR.ISICVICH, L. M. 1958. Der Nucleotidmetabolismus von Polyedrosis infiaierten Seidenraupen und einige Inhibit,oren der Polyedrose. Trans. Z Znt. Congr. Insect Pathol., lst, Prague, 1958, pp. 255-263 (Russian with German summary). WAGER, R., AND BENZ, G. 1971. Histochemical studies on the nucleic acid metabolism in granulosis-infected Carpocapsa pomonella. J. Znvertebr. Pathol., 17, 107-115. WATANABE, H. 1967. Protein synthesis in the tissues of the silkworm, Bombyx mori, infected with nuclear-polyhedrosis virus. J. Znvertebr. Pathol., 9, 428429. WATaNABE, H., AND KOBAYASHI, M. 1970. Histopathology of a granulosis in the larva of the fall webworm, Hyphantria cunea. J. Znvertebr. Pathol., 16, 71-79. WITTIG, G. 1959. Untersuchungen iiber den Verlauf der Granulose bei Raupen von Chorisfoneura murinana (Hb). Arch. Ges. Virusforsch., 9, 365-395. YAMAFUJI, F., SHIMAMURA, M., BND YOSHIHARA, F. 1954. Behaviour of nucleic acids in formation processes of silkworm virus. Enzymologia, 16, 337-342.