Studies on ribonucleic acid in the fat body of Philosamia cynthia ricini Donovan (Lepidoptera) during development

J. Insect

Phyriol.,1966, Vol. 12, pp. 789 to 801. Pergamon Press Ltd. Printed in Great Britain

STUDIES ON RIBONUCLEIC ACID IN THE FAT BODY OF PHILOSAMIA CYNTHIA RICINI DONOVAN (LEPIDOPTERA) DURING DEVELOPMENT SOHJI

TAKAHASHI

Department of Biology, College of Liberal Arts, Kobe University, Kobe, Japan (Receded 11 January 1966) A&txact-The fluctuation of RNA was determined using the fat body cells of l%Zosan& cynthia ricini during the course of development from the fifth larval instar to the early pupal stage. The ratio of total RNA to DNA was high in the early fifth instar, but it decreased afterwards to reach a minimum at pupation. This change was mainly accounted for by the reduction of RNA contained in the microsomal fraction. For the purpose of analysing further such changes in RNA, nucleic acids were isolated from the fat body cells by a slightly modified SDS-phenol method after injection of srP into the body cavity. The sRNA*, rRNA and DNA were fractionated by methylated serum albumin column chromatography. The ratio of sRNA to DNA was rather constant, while the ratio of rRNA to DNA decreased gradually during the progress of development. Incorporation of 8*P into sRNA and rRNA took place only in the early stage of the fifth instar. It is concluded that the RNA which exists in the fat body cells in the later stages of the fifth instar and pupal stage was synthesized during the first few days in the fifth larval instar.

INTRODUCTION

THE fat body of insects becomes laden with globules of fat, protein, and glycogen in the course of development (WIGGLESWORTH,1950; MUNSON, 1953). Recently, there have been found many kinds of enzymes related to the intermediary metabolism of these storage materials (KILBY, 1963). Morphologically, in the Philosaka silkworm, ISHIZAKI(1965) ob served the fat body cell by electron microscopy and reported profound changes in the subceIlular organization, especially in the ribosomes and endoplasmic reticulum during larval-pupal metamorphosis. From recent endocrinological knowledge, it may be recognized that intermediary metabolism and morphological change in the fat body cell are regulated by the moulting hormone, which activates the genes leading to production of RNA and protein (WIGGLESWORTH, 1957, 1963; PELLING, 1959; CLEVER and KARLSON, 1960; KARLSON, 1963). Study of the nucleic acids, therefore, will be indispensable for an understanding of the development of the fat body cell. This report deals with changes in RNA in the fat body of Philosamia cynthia ricini during the development from the fifth larval instar to the early pupal stage. * Abbreviations used: kieselguhr; rRNA-ribosomal

TCA-trichloroacetic RNA; sRNA-soluble 789

acid; MAK-methylated (transfer) RNA.

albumin-

790

SOHJITAKAHASHI MATERIALS

Animals Philosamia Cynthia ricini is a non-diapausing insect with five larval instars. All animals were reared on leaves of the castor-oil plant at 25°C in the laboratory. Females were exclusively used to avoid possible confusion due to sex difference, because sex-specific proteins and polypeptides are detected in some insects (TELFER and WILLIAMS, 1953 ; TELFER, 19.54; Fox et al., 1959). Four representative stages, the 3-day-old fifth instar, the mature fifth instar, the l-day-old pupa, and the 3-day-old pupa, and sometimes other appropriate stages were chosen, Preparation of fat body cells Fat body cells grow with the ageing of the last larval instar and fill up the body cavity in the prepupal and pupal stages. They form whitish leaf-like sheets or globulated masses. All procedures for the preparation of fat body cells were conducted at O”-4°C. The tergum was cut open and the fat body was taken out together with other organs, including Malpighian tubules, silk-glands, gut, and nerve cords. The fat body was cautiously freed from other tissues in O-9:(, NaCl solution under a dissecting microscope. The isolated fat body was washed three times in a large quantity of the NaCl solution and stored at -20°C until used. For the isotopic study, the cells were prepared in potassium phosphate buffer (O-05 M, plus 0.001 M MgCl,, pH 6.8) instead of the NaCl solution and were used at once without freezing. Unless otherwise stated, 40 individuals from the same group afforded an experimental sample. With the object of surveying homogeneity of the fat body cells, a part of the sample was shaken in the saline solution and suspended cells were collected by centrifuging. The cells were fixed, sectioned, and stained for light microscopy. The observations revealed that contaminating cells were less than 2 per cent of total cells in all stages studied. The contaminating cells were identified to be chiefly haemocytes and small fragments of tracheoles. CHANGES OF DRY WEIGHT AND AMOUNT OF RNA, PROTEIN, AND PHOSPHOLIPID PHOSPHORUS IN THE CELL HOMOGENATE

Experimental methods Samples were homogenized in 3 volumes of 0.9% NaCl adjusted to pH 7.4. An aliquot of the homogenate was dried at 80°C for 10 hr and kept in a vacuum until the material reached constant weight. The dry weight was corrected for NaCI. The homogenate was mixed with 2.5 vol. of 10% TCA and centrifuged. The precipitate was resuspended in distilled water. The suspension was divided into two parts. One was used for the analysis of protein and the other for analyses of RNA, DNA, and phospholipid phosphorus by the method of VOLKINand COHN (1954). Protein was determined with the biuret reagent (GORNALL et al., 1949) and RNA with the orcinol reagent (BROWN, 1946). An interference in orcinol

RN’A IN FAT BODY

791

reaction by carbohydrate, which LINZEN and WYATT (1964) pointed out, could be avoided when the RNA fraction was isolated following faithfully the Schmidt, Tannhauser, and Schneider method. DNA was determined with the diphenylamine reagent (DISCHE and SCHWARZ, 1937). Phospholipid phosphorus was estimated by ALLEN’S method (1940). In the Philosamia fat body cells, mitotic figures are not observed from the beginning of the fifth instar to the fourth day after pupation and the DNA in the cell does not change significantly during this period (MINATO and ISHIZAKI,1963). Therefore, dry weight, RNA, protein, and phospholipid phosphorus could be meaningfully expressed as the ratio to DNA content of the cell homogenate.

FIG. 1. Changes of dry weight, RNA, protein, and phospholipid phosphorus. (a) Dry weight; (b) RNA; (c) protein; (d) phospholipidphosphorus. S-3-3-day-old Gf’thinstar; M-mature larva; P-pupal moulting; P-l-l-d&old pupa; P-33-day-old pupa. Samples were taken from six groups shown with various symbols.

792

SOHJI TAKAHASHI

Experimental results Dry we$qht. The ratio of dry weight to DNA tends to increase through the period from the early fifth instar to the mature larval stage and then decreases slightly at the prepupal stage. It increases again in later periods (Fig. la). RNA. The ratio of RNA to DNA shows the highest value in the early stages of the fifth instar among the stages examined (Fig. 1b). Then, it decreases to the lowest value at the time of pupation. It regains a slight rise in the 3-day-old pupa. The ratio ranges from 7 to 9 in the 3-day-old fifth instar and from 2 to 3 in the l-day-old pupa. Protein. The protein content in the homogenate shows a good correlation with the change of RNA, but not with that of the dry weight (Fig. lc). The ratio of protein to DNA keeps a higher level (N 50 to 70) for 4 days after the last larval moult, and then it begins to decrease to reach the minimum level (~30) in the period of larval-pupal moulting. The protein occupies approximately 70 per cent of the total dry weight of the cell in the early fifth instar, but 30 per cent in the l-day-old pupa and the 3-day-old pupa. Phospholipid phosphorus. The ratio of phospholipid phosphorus to DNA is 0.13 to 0.21 in the fifth larval instar and then decreases markedly to O-025 to 0.075 in the pupal stages (Fig. Id).

CHANGES

OF RNA AND PROTEIN

IN THE SUBCELLULAR

FRACTIONS

Experimental methods Fractionation of cell homogenate by ultracentrsfugation. The cells suspended in 0.9% NaCl solution were homogenized with a teflon homogenizer at 1000 rev/min for 5 min. The cell homogenate was mixed with a buffered sucrose solution (O-25 M sucrose, 0.001 M MgCl,, O-025 M KCl, 0.05 M Tris adjusted to pH 76 with HCl). It was separated into four subcellular fractions with a method slightly modified from LITTLEFIELDand KELLER (1957). Fraction 1 is a sediment of cell homogenate centrifuged at 600 xg for 10 min. This fraction was composed of nuclei and cell debris as well as a large quantity of fat droplets and glycogen granules. Fraction 2 the supernatant of the 600 x g fraction was centrifuged at 5000 xg for 10 min, then the sediment was resuspended in the sucrose solution. It was centrifuged again at 13,000 x g for 10 min. The sediment is referred to as Fraction 2. Fraction 3 is a microsomal fraction which was sedimented by centrifuging the mixture of the 5000 xg supematant and the 13,000 xg supernatant at 105,000 xg for 2 hr. Before this centrifugation, the concentration of MgCl, was raised from 0.001 M to 0.05 M, since the author’s unpublished data show that this concentration favours sedimentation of ribosomes prepared from fat body microsomes by the method of TAKANAMI(1960). Fraction 4 is the post-microsomal supematant, a yellowish clear fluid obtained after centrifugation at 105,000 xg.

793

RN’A IN FAT BODY

Experimentalresults RNA. The ratio of RNA in each fraction to total DNA was calculated.

In Fraction 1, the ratio is approximately 1 and does not fluctuate significantly from stage to stage (Fig, 2a). In Fraction 2, the ratio also remains between 1-O and l-2

FIG. 2. Changes of RNA in subcellular fractions. (a) Fraction 1; (b) Fraction 2; (c) Fraction 3; (d) Fraction 4. (b)

Cd)

a % ' 20 .c : e p IO

a

30 A 0 20

A

q

0 ~

Ow-+--FA . . FIG. 3. Changes of protein in subcellular fractions. (a) Fraction 1; (b) Fraction 2; (c) Fraction 3 ; (d) Fraction 4.

794

SOHJI TAKAHASHI

(Fig. Zb). The fluctuation of total RNA described above is mainly due to the change of RNA in Fraction 3 (Fig. 2~). In this microsomal fraction, the ratio is the highest (RNA/DNA = 5) in the 3-day-old fifth instar, while the ratio in the l-day-old pupa is only one-tenth as great (RNA/DNA = 0.5). A slight rise of the total RNA in the 3-day-old pupa may also be ascribed to the increase of the microsomal RNA. The ratio of RNA to DNA in Fraction 4 declines slightly during development (Fig. Zd). Protein. Fluctuations of protein in the subcellular fractions are shown in Fig. 3. In every fraction, the ratio of protein to DNA is the highest in the 3-day-old fifth instar and the lowest in the l-day-old pupa. In the 3-day-old pupa, a slight increase is found in Fractions 1 and 4, but not in Fractions 2 and 3. In addition, it is shown that more than 50 per cent of the total protein in the cell is found in Fraction 4, the post-microsomal supematant, throughout the four stages. CHROMATOGRAPHIC

ANALYSES

AND *“P-INCORPORATION

STUDIES

Experimental methods Isotope and injection. szP (specific activity 465 mc/pmole P) was obtained as ortho-phosphoric acid from the Radio Chemical Centre, England. It was diluted with 0.01 M HCl, then neutralized with NaHCO,, and injected into the body cavity at a dose of 5 PC/g body weight. Suitable period of 3aP-treatment. Fat body was taken from 3-day-old fifth-instar larvae and l-day-old pupae at the intervals of 1, 2, 3, 5, 10, 15, and 25 hr after injection. The cells were washed with phosphate buffer, homogenized, and precipitated with an equal volume of 10% TCA. The RNA was fractionated by the Schmidt, Tannhauser, and Schneider method, and the amount of RNA and its radioactivity were measured. In the 3-day-old fifth instar, the specific radioactivity increased linearly for the first 2 hr after injection and showed the highest activity (1200 counts/min./l00 pg RNA) 3 hr after. However, in l-day-old pupae activity was hardly detectable in the RNA fraction at any time interval. It was decided to take the fat body cells 3 hr after injection. Preparation of fat body and isolation of the nucleic acids. The fat body was taken 3 hr after s2P-injection, and washed with the buffer solution. The tissue from 10 individuals afforded an experimental sample, and samples were taken every day from the l-day-old fifth instar to the 3-day-old pupa. After homogenization of the cells in 0.9% NaCl buffered at pH 6.8, the cell homogenate was combined with sodium deoxycholate and sodium dodecyl sulphate to achieve a final concentration of 1 per cent in each. It was then mixed with an equal volume of 90% phenol solution (9 volumes of phenol+ 1 volume of 0.05 M phosphate buffer containing 0.001 M MgCl,), stirred for 2 hr, and centrifuged. The aqueous phase was drawn off, mixed again with an equal volume of the phenol solution, and shaken for a few minutes. The mixture was centrifuged and the upper water layer was obtained. After addition of potassium acetate to achieve a concentration of 2 per cent, the aqueous layer was mixed with 2.5 volumes of alcohol to precipitate the RNA, DNA, and glycogen together.

RNA IN FAT BODY

795

Methylated serum albumin column chromatography. MAK columns were made by the procedure of MANDEL and HER~HJXY (1960). Bovine serum albumin powder ‘Fraction V’ purchased from Armour Pharmaceutical Co., U.S.A., was methylated. The alcohol precipitate containing the nucleic acids and glycogen was dissolved in 50 ml of O-2 M NaCl buffered’solution (0.05 M Na,HPO,-KHQO,, 0.001 M MgCl,, adjusted to pH 6.8) and adsorbed onto the column (2 x 14 cm). Glycogen was first eluted with 80 ml of 0.2 M NaCl buffered solution at 30°C and then RNA and DNA were eluted with a linear gradient of 0.2 M to 1-O M NaCl buffered solution. Detmmination of sedimentation coejkient. RNA was precipitated from the chromatogram fractions with alcohol. The sedimentation coefficient of RNA was determined by an analytical centrifuge (Hitachi UCA-2, Tokyo) in an 0.1% NaCl solution containing 0.001 M MgCl, and adjusted to pH 7.0 with NaHCO,. Analysis of nucleotide composition. sRNA and rRNA preparations were digested with 1 N HCl at 100°C for 1 hr. The hydrolysate was subjected to one-dimensional paper chromatography using Toyo No. 51A filter paper and the solvent of tertbutanol-HCl (SMITH and MARKHAM, 1950). The nucleotide composition was determined according to MARKHAMand SMITH (1951). Experimental results Fractionation of nucleic acids with MAK column chromatography. The nucleic acids were resolved by column chromatography into three components in every stage. These are referred to as ‘Fraction l’, ‘Fraction 2’, and ‘Fraction 3’ in the chromatogram (Fig. 4). Fraction 1. This was eluted from the column with O-3 M to 0.45 M NaCl. It was composed of two subpeaks through the fifth instar and the early pupal stage, but these were treated together. On the basis of the tests listed in Table 1, this fraction was assumed to be sRNA. Its nucleotide composition was of the GC-type. Table 1 shows an example of the analyses of this sRNA in the 3-day-old fifth instar. Fraction 2. This was eluted with 0.5 M NaCl. As Table 1 indicates, this fraction was regarded as DNA. Fraction 3. This fraction was eluted with 0.6 M to O-8 M NaCl. The fraction appeared as one peak in the chromatograms (Fig. 4), but the analytical centrifuge showed that it was composed of three components, i.e. 27 S, 18 $3, and 10 S in 0.1% NaCl solution containing 0.001 M MgCI, (Table 1). From the evidence, this fraction is regarded as the rRNA. The nucleotide composition was also of the GC-type and the ratio of the nucleotides was unchanged in all stages. Changes in the chromatographic pattern of the RNA on metamorphosis The ratio of sRNA to DNA was estimated to be constant (sRNA/DNA = 2) from the chromatograms excepting that of the last stage, in which the ratio falls to about 1, or half of that found in other stages (Fig. 4). The rRNA content changes significantly on metamorphosis. It is highest in the first 4 days of the fifth instar and decreases with the progress of development. 49

SOHJI T-HI

796

O.D. b2

h II II

0.1 1260 rnp) 0.3

(a) 500

0.2

0. I

0i 0.2 (b) 500

0.2

0.1

0t

5

500

0.3

0.2

0.0

0.1

0.4

i,

0.6

0.2 -1

0

0

I.0 500

06

0.2

0.6 0.4

0.1

0.2 b1

0 I.0

500

0.0

0.2

0.6 0.4

0. I

0.2 0 I:

0 Tube

number

(5 ml/tube)

FIG. 4. Changes in chromatographic patterns of RNA during metamorphosis. (a) l-day-old fXth instar; (b) 3-day-old fifth instar; (c) mature larva; (d) l-day-old pupa; (e) 3-day-old pupa. l -a-: O.D. at 260 q; O- - 0- -: *T-radioactivity (counts/min./ml fraction); l - a-: NaCl concentration.

797

RNA IN FAT BODY

TABLE ~-CHARACTERIZATION OF THE FRACTIONS ISOLATBDBY MBTEWLATEDSBRUM ALBUMIN COLUMN CHROMATOGWI’Hy

Fraction

1

2

3

Orcinol Diphenylamine RNase sensitivity DNase sensitivity NaCl (M) S (10-s M Mg++)

+ -

+

+ -

Nuctc

INTO sRN’A, THE COURSE

Fifth&star

sRNA rRNA DNA l

0.6-0+3 27 S, 18 S, 10 s (%) 23.0 30.9 24.5 21.6

(%) 21.1 29.2 24.3 24.4

GU CY UR

Day

0:

0*3-0*45

composition

.TABLE 2-**P-INCORPORATION

+

+

rRN’A,

AND DNA

IN THE FAT BODY

DURING

OF DEVELOPMENT

larva

Pupa

1

2

3

4

5

6 (mature)

1

2

3

1065* 5800 120

850 6700 15

20 6640 10

8 3400 10

12 25 10

15 30 8

10 15 8

10 10 10

10 20 8

Radioactivity (counts/min.)

calculated from the chromatographic results.

200

200

00

.

L

d vi

(b)

(a)

300

;O

0

.8

100

Non-labeled

a

35

10

"

Non-labeled

P or D.W. 20 40 60 60 Intervol.

100 hr

after

35__ 10 " 3zP-injection

P or D.W. 20 40.60

60 100

FIG. 5. Dilution of radioactivity in RNA with non&belled phosphorus. (a) l-dayold t?fth inatar; (b) 4-day-old fifth instar. a-non-labelkd phosphorus injected; S.A.-specific activity (cotmts/min/lO~g RNA); each point O-controls. represents radioactivity in fat body RNA prepared from one individual.

798

SOHJI

T-HI

3aP-incorporation into RNA. As shown in Fig. 4, the sRNA was labelled only within the first 2 days of the fifth instar, whereas the rRNA was labelled up to the fourth day of the instar. The incorporation into the three fractions, i.e. sRNA, rRNA, and DNA, was examined in every stage. The results are summarized in Table 2. Chase experiment. 32P was injected into the l-day-old fifth instar larvae at a dose of 10 PC/g body weight. Three hr later, some of the larvae were injected with 0.05 ml of 1 M phosphate buffer solution (pH 7.0) and the others with 0.05 ml distilled water as control. The same procedure was repeated with 4-day-old fifthinstar larvae. After the second injection, fat body was removed from groups of insects at several intervals, and the RNA fraction was prepared by the method of Schmidt, Tannhauser, and Schneider. Figure 5(a) shows that the radioactivity in the RNA was diluted with the injection of non-labelled phosphorus in the l-dayold fifth instar, but in the 4-day-old fifth instar no effect is evident (Fig. 5b). This indicates that RNA synthesized in the fat body cells is more stable in the 4-day-old fifth instar than in the l-day-old fifth instar. DISCUSSION

The growth of the cell may roughly be estimated by the rise of dry weight, and a gain of dry weight may be attributed to the storage of nutritional materials such as the fat droplets and glycogen granules (BUTTERWORTHet al., 1965 ; ISHIZAKI, 1965). The present study shows that this ratio decreases temporarily at the time of cocoon formation. This decrease may be ascribed partly to the consumption of glycogen (author’s unpublished data; BUTTERWORTHet al., 1965) and partly to protein decrease in the present experiments. Some destinations of protein from the fat body are the ovary in Rhodnius (VANDERBERG, 1963), and the blood globulins in Bombyx (FUKUDAet al., 1959). It has been reported that protein and some amino acids are used for synthesis of glycogen and fatty acids (WIGGLESWORTH,1942; CLEMENTS, 1959). Recently, KILBY (1963) has reviewed the synthesis and breakdown of many enzymes in the fat body. These findings may also hold in the fat body of Philosamia. Judging from the RNA fluctuations, it seems likely that protein synthesis decreases in the later stage of the fifth instar. It may be said that the fat body begins to accumulate fat and glycogen with the loss of protein in the late stage of the fifth instar. Studies of cell fractions from the ultracentrifuge proved that the change in total RNA content is due mainly to microsomal RNA. The marked fluctuation of the latter at the time of pupation suggests the degradation of ribosomes. This suggestion is in accord with the electron microscopic observations of ISHIZAKI(1965). Decline in phospholipid phosphorus content in the pupal stage may be concerned with changes in the membranous structures in the cell, i.e. endoplasmic reticulum, mitochondria, and nuclear membrane, during the larval-pupal metamorphosis. In histochemical studies of enzymes in the Philosamia fat body cell, SAITO and ICHIKAWA(1964) have reported that acid-phosphatase, which is generally found in degenerating cells, is active at the time of pupation. From these considerations,

RNA IN FAT BODY

it

799

is suggested that the decrease of the RNA content in the cell is caused, not only by reduced rate of RNA synthesis, but also by structural degradation of the site of protein synthesis. SHIGEMATW(1964) has analysed the ssP-RNA in the posterior silk gland of Bombyx by MAK column chromatography. His chromatographic patterns are substantially the same as those from Philosamia fat body RNA. The sRNA which was eluted from the MAK column with O-3 M to 0.45 M NaCl seems to have two components, judging from the chromatogram. Resolution of these by other methods, however, has not yet succeeded. It is noteworthy that the ratio of sRNA to DNA is nearly constant during the progress of development. The constancy of this ratio might be a general phenomenon in a growing cell, as KJELDGAARD and KURLAND(1963) reported it in a bacterium, Escherichia coli. The constancy, however, was not retained in the 3-day-old pupa, in which the ratio becomes smaller. The reason for this may be that the fat body cells were already confronted with some physiological degradation or dissociation for adult development in this stage. The fluctuation pattern of rRNA revealed by MAK column chromatography is quite similar to that of the microsomal RNA prepared by the ultracentrifuge. The optical density profiles show only one peak of rRNA, but it was shown by an analytical ultracentrifuge that the rRNA prepared by chromatography contained three components, 27 S, 18 S, and 10 S. Recently. BARTH et al. (1964) have reported that RNA from the fat body of diapausing Antheraea pupae is composed of 28 S and 16 S rRNA and 4-8 S ‘transfer’ RNA. Presumably, the insect 27-28 S and 16-18 S components correspond respectively to the 28 S and 18 S components from mammalian sources (KIRBY, 1964). The 10 S component may be an artefact produced by the degradation of 27 S, 18 S, or both. This point requires further study. SWJAKIAN(1959) has studied the nucleotide composition of total RNA in Bombyx mod, and reported that the ratio GU + CY/AD + UR tended to decrease during the course of metamorphosis. The present paper suggests that this may be a result of decrease in the rRNA/sRNA ratio during metamorphosis. This interpretation assumes, of course, that Bombyx and Philosamia are similar to each other in the nucleotide composition of sRNA as well as of rRNA. This assumption is not unreasonable according to the table concerning bulk RNA from different Lepidoptera given by BENSAMet al. (1963). The 3aP-incorporation into the fat body cell is worth noting. Radioactivity is detected in sRNA only within the first 2 days of the fifth instar and in rRNA within the first 4 days of the fifth instar and hardly detected in later stages. Moreover, it appears that the turnover of the newly synthesized RNA is very slow after the middle stage of the fifth instar. A slight rise of the ratios of RNA/DNA and protein/DNA in the 3-day-old pupa is consistent with the electron microscopic observation of ISHIZAKI (1965) that ribosomes and mitochondria appear again in the cytoplasm just before the dissociation of the fat body cells. This point requires further study however, because

800

significant sap-incorporation experiments.

SOHJI TAKAHASHI

in that

stage was not detected

in the present

Acknowledgements-The author wishes to thank Prof. M. ICHIKAWA,Laboratory of Developmental Biology, University of Kyoto, under whose supervision this work was carried out. He is also indebted to Dr. H. ISHIZAKIof the same laboratory for his valuable suggestions and stimulating criticism. Grateful acknowledgement is made to Prof. G. R. WYATT, Department of Biology, Yale University, for reading the manuscript. This work was supported by a grant for Scientific Research from the Ministry of Education in Japan.

REFERENCES ALLEN R. J. L. (1940) The estimation of phosphorus. Biochem. J. 34,858-865. BARTHR. H., BUNYARDP. P., and I&MILTON T. H. (1964) RNA metabolism in pupae of the oak silkworm, Antherueu pernyi: The effects of diapause, development, and injury. Proc. nutn. Acad. Sci. U.S.A. 52, 1572-1580. BEN~AMA., I(ITAzuMEY., and YEAS M. (1963) Rib onucleic acid metabolism in the silk gland. Exp. Cell Res. 31, 329-339. BROWNA. H. (1946) Determination of pentose in the presence of large quantities of glucose. Arch. Biochem. 11, 269-278. BWITBRWORTH F. M., BODENSTEIN D., and KING R. C. (1965) Adipose tissue of Drosophila melanogaster--I. An experimental study of larval fat body. J. exp. 2001. 158, 141-154. CLEMENTSA. N. (1959) Studies on the metabolism of locust fat body. J. exp. Biol. 36, 665-675. CLEVER U. and KARLSON P. (1960) Induktion von Puff-Vertiderungen in den Speicheldriisenchromosomen von Chironomus tentans durch Ecdyson. Exp. Cell Res. 20,623-626. DISCHE2. and SCHWAFUK. (1937) Mikromethode zur Bestimmung verschiedener Pentosen nebeneinander bei Gegenwart von Hexosen. Mihrochimica Actu 2, 13-19. Fox A. S., MEAD C. G., and MUNYONI. L. (1959) Sex-peptide of Drosophila melanoguster. Science, N.Y. 129, 1489-1490. FUKUDAT., SUDO M., MATSUDAM., HAYASHIT., KUROSET., and FL~RKIN M. (1959) Formation of the silkproteins during the growth of the silkworm larva (Bombyx mori). Proc. 4th int. Congr. Biochem. 12, 90-99. GORNALLA. G., BARDAWILLC. J., and DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751-766. Is~~uu
RNA IN FAT BODY

801

MANIXL J. D. and HERSHEYA. D. (1960) A fractionating column for analysis of nucleic acids. Analyt. Biochem. 1,66-77. Mm R. and SMITH J. D. (1951) Chromatographic studies of nucleic acids4. The nucleic acid of the turnip yellow mosaic virus, including a note on the nucleic acid of the tomato bushy stunt virus. Biochem. J. 49,401-#06. MINATO K. and ISHI~AKIH. (1963) Changes in nuclear DNA in the fat-body cell during metamorphosis. (In Japanese.) Dobutsuguku Zusshi 72, 330-331. MUNSONS. C. (1953) The hemocytes, pericardial cells and fat body. In Insect Physiology (Ed. by ROEDERK. D.), pp. 226-231. John Wiley, New York. PBLLING G. (1959) Chromosomal synthesis of ribonucleic acid as shown by incorporation of uridine labelled with tritium. Nature, Land. 184,655-656. SAITOT. and ICHIKAWAM. (1964) Histochemical studies of the enzymatic activities during insect metamorphosis. (In Japanese.) &fi.J. exp. Mmh. 18, 4049. SHIGWlATsUH. (1960) Protein metabolisms in the fat body of the silkworm, Bon&m mori L. Bull. seric. Exp. Sta., Tokyo 16, 141-170. SHIGEMA~UH. (1964) Recent advance in studies on the fibroin synthesis by the posterior silkgland of silkworm. SABCOJ. 1,21-29. SISSAKIAN N. M. (1959) Discussion. Proc. 4th int. Congr. Biochem 12, 180-183. SMITH J. D. and MARKHAMR. (1950) Chromatographic studies on nucleic acids-2. The quantitative analysis of ribonucleic acids. Biochem. J. 46, 509-513. TAKANM~IM. (1960) A stable ribonucleoprotein for amino acid incorporation. Biochim.

biophys. Acta 39, 318-326. TELFER W. H. (1954) Immunological studies of insect metamorphosis-II. The role of a sex-limited blood protein in egg formation by the cecropia silkworm. g. gen. Physiol. 37,

539-558. TELFW W. H. and WILLIAMS C. M. (1953) I mmunological studies of insect metamorphosis-1. Qualitative and quantitative description of the blood antigens of the cecropia silkworm. 3. gen. Physiol. 36,389-413. VANDERBBRG J. (1963) Synthesis and transfer of DNA, RNA and protein during vitellogenesis in Rhod&us prolixus (Hemiptera). Biol. Bull., Wooa!sHole 125,556-575. VOLKINE. and COHN W. E. (1954) Estimation of nucleic acids. In Methods of Biochemical Analysis 1,287-303. Interscience, New York. WIGGLE~WORTH V. B. (1942) The storage of protein, fat, glycogen and uric acid in the fat body and other tissues of mosquito larvae. J. exp. Bbl. 19, 56-77. WIGGL~SWORTHV. B. (1949) The utilization of reserve substances in Drosophila during Aight. J. exp. Biol. 26, 150-163. WIGGLFSWORTHV. B. (1950) The Principles of Insect Physiology (4th Ed.), pp. 289-293. Methuen, London. WIGGLIZ~~ORTH V. B. (1957) The action of growth hormones in insects. In Symp. Sot. exp. Biol. 11,204-227. Cambridge University Press. WIGGLESWORTH V. B. (1963) The action of moulting hormone and juvenile hormone at the cellular level in Rhodnius prolixus. J. exp. Biol. 40, 231-245.