Development of a nuclear polyhedrosis in cells of Rhynchosciara angelae (Diptera, Sciaridae) and patterns of DNA synthesis in the infected cells

JOURNAL

OF INVERTEBRATE

24,

PATHOLOGY

93-105

(1974)

Development of a Nuclear Polyhedrosis in Cells of Rhynchosciara angelae (Diptera, Sciaridae) and Patterns of DNA Synthesis in the J. S.

Infected

Cells1

A. B.

MORGANTE,

DA CUNHA

Departmento de Biologia, Imtituto Universidade de &io Paula, C.P. 11.461,

C, PAVAN, J. J. Department

of Zoology,

The

AND Departamento

de Biologia,

Institute Received

R. W.

BIESELE,

University

de Bioci&eius, S&o Paulo, 06421,

of Texas

M. C.

Bra&l

RIES~

at Austin,

Austin,

Texas

7871g

de &io

Paula

GARRIDO

da BiocGncias, October

Univemidade

10, 1973

An ultrastructural and autoradiographic study of the infection of cells of Rhy~~chosciura angelae by a nuclear polyhedrosis virus (RPV) is presented. RPV is a DNA virus and causes a dramatic increase in the volume of the infected cells and in the sizes of chromosomes and their DNA contents. The structure of the nucleoli changes with the infection and the changes are mainly related to an increase of DNA synthesis. The concentration of ribosomes increases in the cytoplasm of the infected cells. Autoradiographic study of the DNA synthesis showed that it varies with the infective process. Four patterns of DNA synthesis, in relation to the host chromosomes and the virus, were disclosed by means of tritiated thymidinc incorporation in the infected nuclei. The patterns are: (1) incorporation mainly in the chromosomes, (2) incorporation in the chromosomes and in the nucleoplasm, (3) incorporation only in the nucleoplasm, and (4) incorporation mainly in the chromosomes in dissociation. There is indication of a succession l+ 2 and 3 -+ 4. The succession of patterns indicates that the virus induces first the increase of synthesis of host cell DNA and RNA. The bulk of the synthesis of viral DNA is evident only after the host cell DNA and RNA machinery is amplified. The aspects of the formation of viral membrane indicate that it is a de novo process in which the membrane material is capable of self-assembly.

studies of the relationships between virus and infected cells. The virus develops within the nuclei of cells of the intestinal caeca and of the posterior part of the midgut. These cells have polytcne chromosomes, which are very much enlarged due to the effects of the virus. Constrictions may appear in some specific regions on the enlarged chromosomes. The location of these constrictions seems to vary with the viral strains (Pavan et al., 1971). The giant sizes of the chromosomes in the infected cells permit a

INTRODUCTION

The nuclear polyhedrosis of Rhynchosciara angelae presents some characteristics that make it especially favorable for ‘This study was supported by grants from the National Institutes of Health (NIGMS-RGB, GM-17590-02, and GM-15769-05), University of Texas Graduate School BSSG Grant, Conselho National de Pesquisas and FundaoLo de Amparo B Pesquisa do Estado de S&o Paulo. J. J. Biesele is the recipient of a research career award 5-K06-CA 18366 from the National Cancer Institute. 93 Copyright All rights

@ 1974 by Acrtdemie Press, Inc. of reproduction in any form reserved.

94

MORGANTE

ET AL.

detailed analysis of the virus-chromosome ing to Reynolds (1963.). Micrographs were made with a Siemens Elmiskop I electron relationships. The caecal and the intestinal cells, like other cells with polytene chromo- microscope. The larvae for autoradiographical studies somes,do not divide and become huge with the increase of materials caused by the were dissected in saline solution (NaCl 5.55 g, KC1 0.22 g, and CaCl, 0.44 g in 1 infection. liter of distilled water). The caeca were Analysis of the viral effects in Rhynchoscinra have been carried out by means of separated and placed in vials with 0.25 ml light microscopy (Diaz and Pavan, 1965; of culture medium prepared according to Pavan and Basile, 1966; Pavan and da Terra (1972). Five caeca were put in each Cunha, 1968; Pavan et al., 1971). Ultraculture vial and 10 ~1 of 3H-thymidine (1.9 structural studies of the infected cells, spe- Ci/mmole, 1 mCi/l ml Schwarz BioRecially of the nuclear changes, and of the search Inc., New York), were added. The viral development have also been made (da caeca were incubated for 60 min at 22OC and subsequently fixed in ethanol-acetic, Cunha et al., 1972; Stoltz et al., 1973). The purpose of this paper is to present 3: 1, 15 min. Each caecum was transferred new data obtained from electron micro- to a drop of acetic acid 45% on a slide and, scopical analysis of the virus infected after 5 min, squashed under siliconized nuclei as well as information on the rela- corerslip. The coverslips were removed in tionships between viral and chromosomal liquid nitrogen, and the preparations were DNA synthesis obtained from studies using dried, washed in 5% trichloroacetic acid at autoradiographic techniques. 4OC for 5 min, washed in three lo-mm baths of distilled water, and dried. MATERIAL AND METHODS The same procedure was used to study The strains of Rhynchosciara angelae the incorporation of uridine. The material were raised in the laboratory according to was incubated for 45 min in the same type the techniques described by Morgante et al. of culture medium with 10 ~1 of 3H-uridine (1970). The temperat,ure of 21°C was used (2.0 Ci/mmole, 1 mCi/l ml) from Schwarz in all the experiments and in the breeding BioResearch Inc., New York. The preparaand maint’enance of the larvae. The larval tions, after squashing and removal of the life of R. nngelne lasts 60 days at this tem- coverslips, were washed in two 30-min perature. Only infected cells of the intestibaths of ethylic alcohol 70° G.L. followed nal caeca have been studied. by 3 baths of 10 min in distilled water. The caeca for ultramicroscopical studies The dried preparations were covered with were removed and fixed in buffered glutarKodak AR10 film or with Kodak NTB2 aldehyde fixative prepared by mixing equal liquid emulsion. The caeca incubated with volumes of Sorensen buffer and 5% glutarthymidine were exposed during 8 days, and aldehyde. After rinsing in three lo-min those with uridine during 15 days, at 4°C changes of Sorensen buffer, the fixed mate- in a dark box and afterward developed, rial was postfixed in 1% 0~0, for 1 hr, fixed, and dried. The autoradiographic washed in distilled water, and left in 0.5% preparations were stained with methylene aqueous many1 acetate for 16-20 hr. After blue (Unna). Some preparations were washing and dehydration, the material was stained by Feulgen before being covered embedclcd in a mixture of 10% Epon 812 with the autoradiographic emulsion. The (Shell), 20% Araldite 6005, and 70% Feulgen staining was prepared according to dodecenyl succinic anhydride (DDSA) Gurr (1959) . Sections were cut with a diamond knife on Preparations were also stained with a Sorvall Porter-Blum ultramicrotome and Nicoletti’s (1959) lact,ic-acetic orcein (50 stained with lead citrate prepared accord- ml of lactic acid S570, 50 ml of glacial

FIG. 1. Section of a cell of the intestinal caecn from a 6-day-old normal larva of Rhyrlchosciara angelae showing a compact nucleolus. FIG. 2. Nucleus of a cell of the intestinal caeca from a 6-day-old infcctcd larva. Tht~ section shows the very much enlarged nucleolus with a spongy aspect. x9200. FIG. 3. Nucleus of a cell of the intestinal cwcu from a Gday-old infcctcsd larva. The nucleolus is alrcndy very much f&~l~grd and the spongy aspert is in development. X 13,800.

95

96

MORGANTE

ET

AL.

FIG. 4. Cytoplasm close to the nucleus in 3 ~,11 of the intwtinal 6-day-old larva. Compare ~vith Fig. 5. Xlg,SOO. FIG. 5. Cytoplasm close to the nucleus in a cell of the intestinal infected larva. The infected cell is not only very much enlarged concentration of rihosomes are much increased in relation to the normal

ww:i

from

a normal

caeca from a 6-day-old hut the quantity and cells. ~20,700.

DEVELOPMENT

Olq

A NUCLEAR

POLI-HEDHOSIS

97

acetic acid, and 2 g of Gurr’s orccin). The to recently hatched larvae and to one hunoptical micrographs were made in a Zeiss dred 4th-stage larvae. No infection was obphotomicroscope. served among the old larvae, and in the recently hatched the results were similar to RESULTS those above. A simlar experiment was done with old larvae in molt, between the 3rd and 4th Infected caeca of old larvae were used stage. No infection was observed in one as the source of virus for the experimental hundred treated larvae. infection. The infected caeca were isolated A different experiment was made with and kept dry at 4%. The dry caeca were 4th-stage larvae. The larvae received injcctrituratcd and suspended in a solution of tions of 3 ,J of virus hydrolysis in the hemopH 10.7 composed of 0.07 M NaaCO, + coel. The number of injected larvae was 50, 0.05 M NaCl, when virus was necessary and none dcvclopcd polyheclrosis. The same for the infection. The times of hydrolysis hydrolysis used in the injections was tcstcd tested were 30, 60, 90, and 120 min. The given in the food to recentiy hat&d larbest results in experimental infections were vae, and the result was positive. The results of feeding or injecting virus obtained making the hydrolysis during 90 min, and this time was used in the in old larvae show that R. nngelae larvae experiments. develop a high resistance against the RPV The first experimental infections were as they age. made with larvae taken as soon as hatched Larvae of R. hollaenderi and of R. nzilfrom the eggs. A strain of R. angelae free leri were infected by the RPV of R. nngeof virus was used. A female lays about 1000 he. However, attempts to infect Sciarn eggs, half of which were used as control ocellaris with the RPV gave negative while the other half was used for the infec- results. tion. The larvae received food as soon as they hatched. The food for infection was Virus-Induced Ultrastructural Changes mixed with 0.5 ml of virus hydrolysis. SamAn analysis of the ultrastructural ples of 100 experimental and 100 control lar- changes in the infected cells was published vae were studied in every experiment. The by da Cunha et al. (1972). OnIy new asinfection is easily detected because, after pects, observed in cells of larvae infected 4 days of infection, the caeca show whitish just after hatching and fixed after 4 and spots, which correspond to the cells with 6 days, will be presented. RPV. The percent of infected larvae were RPV crystals were found in some nuclei 2, 8, 9, 13, 15, and 31 in five experiments. after 4 days of infection. Nuclei with RPV No infected larvae was found in the control crystals are common after 6 days of in five samples of 100 larvae each. infection. A similar experiment was made with old, A striking nuclear change is t.he very fast 4th stage larvae. Food with virus was given and enormous increase of the nucleoli. Sec-

6. Nucleoplnsm of a g-day-old infected cnecnl cell. The section shows the virogenic (VS), a segment of the nuc1eolu.s (Nu), the nuclear membrane (NM) and a large number of viral particles in organization. A large amount of paired open membranes are present among the viral particles. These membranes may he free or in connection with the viral outer membrane but have no relation with the nuclear mcmhrane. x 19,800. FIG. 7. Another region of t,he same nucleus of Fig. 6. The virogcnic stroma is more abundant in this l):trt of the srction. The paired open mcmbrnncs are present. The arrow indicates paired mcmhrancs, one being continuation of the outer viral membrane and the other independent. X 19,800. FIG. stroma

MORGANTE

ET

AL.

FIG. 8. Autoradiographic preparation of caecal cells of a normal l&day-old larva treated with tritiated uridine. Methylene blue-ITnna. FIG. 9. Autoradiographic preparation of caecal cells of an l&day-old infected larva. The caeca were treated with tritiated uridjne. Methylene blue-Unna.

DEVELOPMENT

OF

A

tions of nucleoli of a normal 6-day-old larva and of virus infected nuclei, 4 and 6 days after hatching are shown in Figs. 1, 3, and 2, respectively. Several sections of normal nuclei were studied. The section presented is that with the largest diameter found and probably an equatorial one. The normal nucleoli are compact and have a granular structure. The nucleoli of infected 4-day-old larvae are already much cnlarged and have compact regions surrounded by spongy expansions. The nucleoli of infected 6-day-old larvae are remarkable by their expansion and development occupying a large part of the nuclear volume. The aspect of these nucleoli is mostly spongy. The increase of the nucleoli in size and the expansion of the surface given by the spongy structure are related to the great increase in the number of ribosomes. Figures 4 and 5 show two cytoplasmic sections close to the nuclei. The first section, Fig. 4, is from a caecal cell of a g-day-old normal larva, while the second, Fig. 5, is from a 6-day-old infected larva. The increase in ribosomes is typical and found in every infected cell studied. The synthetic activity of the nucleoli is very high in the infected cells and will be shown in another section of this paper. The nuclei of 6-day-old RPV infected larvae show a very large development of the virogenic stroma and a great quantity of viral particles in organization (Figs. 6, 7). The aspects of the virogenic stroma and of the virus are similar to those described by da Cunha et al. (1972). However, a new aspect regarding the viral membrane was observed in 6-day-old larvae. Segments of unit membranes are present among the

NUCLEAR

99

POLYHEDROSIS

viral particles. These unit membranes may be free of viral material, paired, and with open ends. We did not find any connection of the viral membranes with the nuclear envelope (Figs. 6, 7). Membranes enveloping viral particles may have expansions going to large distances (Fig. 7). These observations support the conclusions of Stoltz et al. (1973). It seems t.hat the virus induces also a very intense synthesis of viral membrane material. When this material is accumulated it may self-assemble giving origin to membranes. The organization of the membranes may not depend on the presence of a viral particle as substrate and is not related topographically to the nuclear envelope. Nuclear

Activity

and RNA

Synthesis

The synthetic activity of the nucleoli was studied using tritiated uridine and autoradiography. The development of the nucleoli in normal and infected larvae was followed from the 6th to the 44th day. The nucleoli of normal cells are always considerably smaller than those of the infected cells of the same age. The normal nucleoli have regular shape. The nucleoli of the infected cells emit expansions that later may separate from the main nucleolar body forming smaller nucleoli that remain in contact wit.h the chromosomes or become loose in the nucleoplasm. Figures 8 and 9 show the incorporation of 3H-uridine in an B-day-old normal larva and in an infected larva of the same age. The difference between the nucleoli in the normal and in the infected nuclei increases with the progress of the infecticn. The incorporation of tritiated uridine is always very intense in the infected cells. Fig. 10

Fro. 10. Nucleus of a caecal ccl1 of a ZO-day-old infected larva. The caeca were treated with tritiakd uridine. The nucleolus with its expansions is clearly distinguishable. Methylene blue-Unna. FIG. 11. Autoradiographic preparation of a caecal cell of a 44-day-old infected larva. The caeca were treated with tritiated uridine. The much expanded nucleolus gave origin to several independent bodies. The chromosomes are losing their bandings and the chromosomal synthesis of RNA is low. Methylene blueUnna.

100

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_..-._FIGS. 12, 14, and 15. Aut,orndiographic prrparationn of c:wc:11 cells trcakl with ktinted thymidinr. The infected larva ~vas 40 days old. The nuclrus of Fig. 12 shows the “chromosomal I” pattern of thymidine incorporation. Only the chromosomes show silver grains. The pattern, silver grains only in the nuclei of Figs. 14 and 15 present the “nucleoplasmic”

DEVELOPMENT

OF

A

shows a nucleus of an infected cell of a 20day-old larva. Fig. 11 presents the pattern of uridine incorporation and the distribution of nucleolar material in a 44-day-old infected larva. The multibranched nucleolus and the detached segments are clearly visible. The synthesis of RXA in the chromosomes is also very intense from the beginning of the infection, decreasing only when the chromosomes start to lose their banding and dissociate (Fig. 11). Patterns Nuclei

of DNA

Synthesis

in Infected

Pavan et al. (1971) described the cytological aspects of the RPV infection as seen with a light microscope. It was shown that nuclei with equally enlarged chromosomes may have two different types of nucleoplasm, Fculgen positive or negative. The two types are related to the presence or absence of virogenie stroma. Autoradiographic analysis of DNA synthesis was made in order to see what are the cytological patterns of DNA synthesis and whether there is any sequential process. Treated infected larvae were killed daily from day 3 to day 9 of infection and processed for the autoradiography. Four main patterns of DNA synthesis were found. Some nuclei show thymidine incorporation mainly in the chromosomes. This pattern of incorporation is called ‘Lchromosomal I” (Fig. 12). These nuclei have Feulgen negative nuclcoplasm. Other nuclei show incorporation in the chromosomes and also in the nucleoplasm (Fig. 13). This is the “gencralized” pattern. The nuclei with this pnttern have Feulgen positive nucleoplasm. A third pattern is the “nucleoplasmic,” where

NUCLEAR

POLYHEDROSIS

101

incorporation is obscrvcd in the Feulgen positive n~~clcoplasn~ but not in the chromosomes (Figs. 14, 15). It becomes easier to set that there is no chromosomal DNA synthesis or very little, when the chromosomes are d&ached from the nucleoplasmic mass (Fig. 15). A last pattern is the ‘Lcllromosomal II,” where thymidine incorporation occurs mainly in the chromosomes and is weak or negative in the nucleoplasm (Fig. 16). The “~l~ron~080mal I and II” patterns differ because I occurs at initial and II at advanced stages of the infection. The chromosomes in ‘Lchronlosonlal II” pattern do not show banding, arc in dissociation, and the nuclei already have RPV crystals in organization (Fig. 171. The EM analysis shows that a large quantity of viral material is present within and around the chromosomes (da Cunha et al., 1972) at t,liis stage of the infection. The same nuclei studied successively with lactic-orccin and phase, Fculgen, and autoradiography show that the nucleoplasmic synthesis of DNA is related to the virogenic stroma (Figs. 18-23). The extracliromosomal incorporation of thymidine is observed only when Feulgen positive st’romn is present (Fig. 12). The occurrence of the four patterns of DNA synthesis lead to the idea of a possible succession of them in time. Samples of nuclei were studied in the infected glands of different ages. The nuclei were classificcl and recorded according to the pattern of thymidine incorporation they showed. Table 1 shows the frequencies of the patterns of DNA synthesis in intestinal eaecn cells. Preparations of at least 10 larvae of each age from day 3 to day 9 of infection (see Fig. 24) were used for the scoring. The

nucleoplasm. While the unstained nuclei of Fig. 14 give negative images of the chromosomes (arrows), the stained nucleus of Fig. 15 shows well the chromosomes without thymidine incorporation (arrows). The preparations of Figs. 12 and 15 were stained with methylcnc blue-Unna, and that of Fig. 14 was left unstained. FIG. 13. Autoradiographic preparations of caeca cells of a 12-day-old infected larva. The nucleus shows the “gcnc~mlizcd” patt,ern of thymidine incaorporntion, chromosomes, and nucleoplasm marked with silver grams. Unstained.

102

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23

Fm. 16. Autorndiographic preparations of caccit cell of an infected caeca were treated with tritinted thymidine. The nucleus presents pnttrm of thymidine incorporation , ,Slvor grains nixinly on chromosom(~s.

44-day-old

larva,

the “&romosomal Unstained

The

11”

DEVELOPMENT

FREQUENCIES

OF

OF

A

NUCLEAR

103

POLTHEUROSIS

TABLE 1 FOUR PATTERNS OF TRITIATED THYMIDINE INCORPORATION CI”LLS OF Rhynchosciara Angelae INFECTED BY RPV VIRUS

THE

OF

Patterns

IX NUCLEI

of incorporation

Days of infection

Chromosomal I

Generalized

Nucleoplasmic

Chromosomal II

3 4 .5 6 7 8 9

0.753 0.621 0.179 0.040 0.174 0.040 0.071

0 024 0.287 0 535 0.464 0.258 0.371 0.387

0.063 0.089 0.05 0.086 0,033

0.092 0.223 0.338 0.512 0.503 0.509

results indicate the following sequence: “chromosomal I” + “generalized” and “nucleoplasmic” -+ “chromosomal II”. The sequence of patterns suggests that entering the cell the RPV induces first an increase of chromosomal DNA synthesis, i.e., the “chromosomal I” pattern. Some cells have nuclei with exceptionally large and well banded chromosomes but do not show any sign of viral DNA synthesis. It seems that these cells stay much longer at “chromosomal I” stage. These nuclei were already described by Pavan et al. (1971). Synthesis of viral DNA becomes apparent only after a large amount of chromosomal DNA and RNA are produced, i.e., the “generalized” pattern. The “nueleoplasmic” pattern seems to be the phase of the “generalized” pattern when the viral DNA is being synthesized in the nucleoplasm, but the chromosomes are in the interval between two cycles of DNA synthesis. The final pattern is the “chromosomal II,” which occurs when the cell is being disrupted. There is, at this stage, little synthe-

Nuclei studied 81 261 1277 586 615 272 212

sis of viral DNA and a very irregular pattern of tritiated thymidine incorporation in the chromosomes. DISCUSSION

The infection of R. angelae cells by DNA polyhedrosis virus, RPV, causes intense cellular growth with great enlargement of the polytene chromosomes. The exceptionally large chromosomes permit a detailed analysis of the chromosomal changes caused by the virus and of the relationships between the viral multiplication and the genetic material of the infected cell. Experimental infection of the larvae with RPV was obtained only at the initial stages of the larval life. Fourth-instar larvae and larvae at the molt between third and fourth instars were not infected when receiving virus in the food or injected in the hemocoel. However, larvae infected when young and examined at these late stages show cells at the beginning of the infective process. This shows that at late larval stages the

FIG. 17. Nucleus from an infected caeca cell in a l&day-old larva. The nucleus and the II” pattern of thymidine inchromosomes present the structure that gives “chromosomal corporation, chromosomes swollen, not banded and segmented. Phase and lactic-acetic orcein. FIG. 18-23. Two nuclei of caecal cells from infected 20-day-old larvae. The same two nuclei, left and right figures, are presented as seen: (a) under phase and stained with lacticacetic orcein, Figs. 1%21; (b) stained by Feulgcn, Figs. 19 and 22; (c) autoradiographical preparation with tritiated thymidine and Feulgcn. Figs. 20 and 23. Both nuclei are at the “nucteoplnsmic” stage of DNA synthtxis. The microgmi~hs show the corrrlation between thymidinc incorporation and Prulgen-posif ivc virogrnic stromn. TIIP crystals present 110 not belong to the nuclei shown, bul to neighbor ct~lls.

104

MOHGANTE

ET

AL. -CHROMOSOMAL - - - GENERALIZED NUCLEOPLASMIC --CHROMOSOMAL

70

I2 k! a

I II

-

60

-

50

-

40-

30-

2 20

-

IO -

3

4

5

DAL

7

8

9

FE. 24. Graphic rqxesentation to show the frequencies of the four Ixtttrrns thymidine incorporation in the period from 3 to 9 dnys of infection. The infcctcd hq’ RPV Then hatching from the eggs.

infection may progress from ccl1 to cell, despite the fact that normal late larvae arc resistant to virus received in the food or injected into the hcmocoel. RPV of R. angeIae is infective to larvae of the closely related species R. hollaenderi and R. millets but not to larvae of Sciara ocellmis, which belongs to a different genus but the same family, Sciaridae. The viral infection causesa very remarkable stimulation of RNA and DNA synthesis. The increase of RNA synthesis starts soon after the infection, and it is observed along the chromosomes and mainly in the nucleoli, which grow to enormous size. Cells at the beginning of the infection show a higher concentration of ribosomes than the normal cells. One of the first effects of oncogcnic viruses in cells of mammals is to cause an increase in the rate of synthesis of the DNA of the infected cells (see YoshikawaFukada and Ebert, 1971; Eckhart, 1971; Howatson, 1971, for reviews). The results here reported indicate that the processesof the RPV infection in R. nngelne have some similarity to those found in mammals, in cells infcctcd with oncogcnic viruses. Three

of tritintrd larvae were

phasesof DNA synthesis were found in the process of infection. The first phase is characterized by a very intense synthesis of chromosomal DNA as shown by autoradiography. The second phase is marked by intense synthesis of viral DNA, while the cycles of chromosomal DNA synthesis continue. The last phase, “chromosomal II” occurs when the cells are already very damaged. The chromosomesshow irregular synthesis of DNA and the synthesis of viral DNA is very much decreased. The findings reported are in agreement with the idea that the first effect of the virus is to change the control of the synthcsis of DNA and of RNA in the infected cells. The chromosomal DNA multiplies at high rate, RNA is produced very actively all along the chromosomes and the synthetic activity of the nucleoli are raised to high level. The bulk of the synthesis of viral DNA is evident only after the host cell DNA and RNA machinery is amplified. The large sizes of the cells, nuclei, and chromosomes in the tissues of Rhynchoscictrtr nngelrre l)crniit a visualization of the synthetic ~~roccsscsand of the cytological clinngcs involvctl in the viral infection.

DEVELOPMENT

OF A NUCLEAR

Some of these processesmay be similar to those which occur in cells of higher organisms infected by oncogenic viruses. ACKNOWLEDGMENTS The authors are very thankful to Misses Jair& Marques, Thelma Picard, and Ivonetc Romeo as well as to Mr. JoLo B. Campos and Mr. Miguel A. J. da Silveira for their technical aid. Thanks are also due to Misses Therezinha M. Ungaretti and Ilze Lamber Jorge for their help. The authors are grateful also to Miss Patricia F. Sanders for her aid with the manuscript.

REFERENCES A. B., PA~.~N, C., BIESELE, J. J., RIESS, R. W., AND SIM~ES, L. C. G. 1972. An ultrastructural study of the development of a nuclear polyhedrosis with effects on giant polytene chromosomes. ,Studies in Genetics VIII. Univ. Texas P&l., 7213, 117-143. DIAZ, M., AND PAVAN, C. 1965. Changes in the chromosomes induced by microorganism infection. Proc. Nnt. Acad. Sci. U.S., 54, 1521-1527. ECKHART, W. 1971. Genetic modification of cells by viruses. BioScience, 21, 171-173. GURR, E. 1959. “Methods of Analytical Histology and Histochemistry.” Williams & Wilkins, Baltimore, Maryiand. HOWATSON, A. F. 1971. Oncogenic viruses: a survey of their properties. In “Comparative Virology” (K. Maramorosch and E. Kurstak, DA

CUNHA,

cds.),

POLYHEDRORIS

105

pp. 509-537. Ac:&mic~ Prc~ss, Sew York J. S., i%tRQURS, J., 1)A CTTNHA, A. B., AND ROMEO, I. 1970. Sobre a cria@io de nlguns Sciaridae (Diptera). Rev. Brctsil. Entomol., 14, 33-40. NICOLETTI, 8. 1959. An efficient method for salivar? gland preparation for Drosophila Droso/~hilo Znjom. S~JW., 33, 181. Pav,!~, C., AND BASILX, R. 1966. Invertrbratc pathology, c>-tology and development. J. Znvertebr. Pathol., 8, 131-132. PECAN, C., AND DA CUNHA, A. B. 1968. Chromosome activities in normal and in infecird cells of Sciaridae. ‘(Seminar on Chromosomes,” Xltcleus (Suppl.), 183-196. PAVAN, C., DA CUNWA, A. B. AND MORSOLETTO, C. 1971. Virus-chromosome relationships in cells of Rhynchoscinra angelae. Caryologia 24, 371389. REYKOLDS, E. S. 1963. The USC of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. STOLTZ, D. B., PAVAN, C., AND DA CUNHA, A. B. 1973. Nuclear polghcdrosis virus: a possible example of de nova intranuclear membrane morphogenesis. ;J. Gen. Viral., 19, 145-150. TERRA, W. R. 1972. Aspectos bioquimicos da hemolinfa e do casulo coletivo de Rhynchosciarn americana. Ph.D. thesis, Departnmcnto de Bioquimica, Instituto de Quimica, Universidade de Srio Paulo. YOSHIKAW,4-FUK.4D.4, M., AND EBERT, J. D. 1971. Interactions of oncogenic viruses and animal cells. BioScience, 21, 357-366. h’hW:.4NTli,