Archives of Insect Biochemistry and Physiology 10:215-228 (1 989) Properties, Synthesis, and Accumulation of Storage Proteins in Pieris rapae 1. Hak Ryul Kim, Sook J. Seo, and Richard T. Mayer Department of Biology, Korea University, Seoul, Korea (H.R.K., S.J.S.); Horticultural Research Laboratoy,Agricultural Research Service, U.S. Department of Agriculture, Orlando, F L (R.T.M.) Two kinds of storage proteins (SP-I, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited i n fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M, 77,000), while SP-2 has a molecular weight of 460,000 and i s composed of two types of subunits (M, 80,000 and 69,000). The pi values of SP-I and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from I-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken u p in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues. Key words: hemolymph, fat body, storage granules Acknowledgments: This work was supported by Basic Science Research Support Fund from the Ministry of Education, Republic of Korea. Received September 15,1988; accepted December 19,1988. Address reprint requests to Dr. R. T. Mayer, U.S. Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803. Sook J. Seo is now at Department of Biology, Cyeongsang National University, Jinju 660-701, Korea. Use of a company or product name by the U.S. Department of agriculture does not imply approval or recommendation of the product to the exclusion of others that may also be suitable. Some of these data were presented at a symposium of the 10th International Congress of the Korean Society of Science and Technology. 0 1989 Alan R. Liss, Inc. 216 Kim et al. INTRODUCTION The major proteins of larval hemolymph in holometabolous insects have been intensively studied since calliphorin was described in Calliphora erythrocephala [l].One of the more dramatic examples of developmental regulation is provided by an abundant set of hemolymph proteins collectively called storage proteins . Storage proteins are synthesized by larval fat body cells and secreted into hemolymph where they reach high concentrations during the last instar . Between the end of the feeding stage and pupation they are sequestered selectively by the fat body cells and accumulate in dense granules [4,5]. Proteins with these properties have been described in many dipteran and lepidopteran insects ; however, little information is available on storage proteins of Pieris r a p e (L.). We report here on the characterization, synthesis, and accumulation in fat body of storage proteins in the cabbage white butterfly, P. r a p e L. MATERIALS AND METHODS Insects Cabbage worms, P. r a p e L., were from a colony maintained within the Department of Biology, Korea University, and were reared on kale plants at 27 2 1°Cand 80 & 5%relative humidity with a photoperiod of 16hlight and 8 h dark. Preparation of Protein Extracts for Electrophoresis Hemolymph was collected into a small, chilled test tube after puncturing larvae and pupae in the abdominal region with a fine needle. A few crystals of phenylthiourea were added to the hemolymph to prevent melanization. Collected hemolymph was centrifuged at 5,000s for 20 min at 4°C to remove cellular debris and stored at - 70°C until used. Fat body was dissected from larvae and pupae in cold Ringer's solution (128 mM NaC1, 1.8 mM CaC12, 1.3 mM KCl), blotted on weighing paper, and the wet weights were recorded. Fat bodies (100 mg) were homogenized in Ringer's solution (0.5 ml) and centrifuged at 10,OOOg for 20 min at 4"C, and the supernatant was stored at - 70°C until used. Electrophoresis and Purification Slab gel electrophoresis (160 mm x 170 mm x 2 mm) was conducted on a 2.5% stacking gel and 5%polyacrylamide slab gel at 30 mA for 4 h at 4°C according to Davis . Disc gel electrophoresis was carried out on a 5% polyacrylamide separation gel and 2.5% stacking gel (tube size, 130 mm x 5.5 mm ID) at 2 mA per tube for 3 h at 4°C. Ten microliters of hemolymph and 15 pl of supernatant from the fat body homogenate (100 mgl0.5 ml Ringer's solution) were each mixed with an equal volume of 0.1 M Tris-HC1buffer, pH 6.8, containing 20% sucrose and 0.005% bromphenol blue. After electrophoresis, protein bands were stained in 0.1% Coomassie brilliant blue R-250. Slab SDS-PAGE* (160 mm x 170 mm x 2 mm) was performed at room temperature using a 3% *Abbreviationsused: DAB = 3,3-diaminobenzidine; PAGE = polyacrylamidegel electrophoresis; PBS = phosphate buffered saline; (140 mM NaCI, 2.7 mM KCI, 1.5 rnM KH2P04, 8.1 mM Na2HP04, pH 7.2; SDS = sodium dodecyl sulfate; SP-1 = storage protein-I; SP-2 = storage protein-2. Pieris rapae Storage Proteins 21 7 stacking gel and 10% separation gel containing 0.1% SDS . Purified SP-1 (10 pl) and SP-2 (10 p1) were mixed with 20 pl of sample buffer (4% SDS, 10% 2-mercaptoethanol, 0.006% bromphenol blue, 20% sucrose, 50 mM Tris-HC1, pH 8.3) in a microcentrifuge tube, heated for 2 min at lOO"C, and then applied to the gel. For the purification of storageprotein large slabgels (170mm x 180mm x 3 mm) were used. Electrophoresis was conducted using Tris-glycine buffer without SDS or 2-mercaptoethanol.After electrophoresis, the storage protein band was excised with a razor blade, eluted, dialysed against 50 mM Tris-glycine buffer (pH 8.3), and concentrated by freeze drying. Protein content was determined by the method of Lowry et al. . Determination of Molecular Weight The molecular weight of native storage protein was determined as described by Hedrick and Smith [lo]. Marker proteins (Sigma Chemical Co., St. Louis) were carbonic anhydrase (M, 29,000), chicken egg albumin (M, 45,000), bovine serum albumin (monomer, M, 66,000; dimer, M, 132,000), and urease (dimer, M, 240,000; tetramer, M, 480,000). Molecular weights of storage protein subunits were measured by the method of Laemmli  using a 10% SDS slab gel. Marker proteins (Sigma)were trypsin inhibitor (M, 20,100), carbonic anhydrase (M, 29,000), ovalbumin (M, 45,000), bovine serum albumin (M, 66,000), phosphorylase B (M, 97,400), P-galactosidase (M, 116,000), and myosin (M, 205,000). Isoelectric Focusing Isoelectric focusing was conducted on 5% polyacrylamide gels using 1% ampholytes (pH 3-10) as described by Wrigley [ll]. After electrophoresis, the gel was stained with a solution containing 27% isopropanol, 10% acetic acid, 0.05% Coomassiebrilliant blue, and 0.5% CuS04for 1h. Destaining was accomplished using a solution of 12%isopropanol, 7% acetic acid, and 0.5% CuS04. Preparation of SP-2 Antiserum Purified SP-2 (200 pg) in 0.5 ml saline solution was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into a rabbit. Three injections at 2-week intervals were made, and a booster injection was given 1week after the final injection. Blood was collected 1week after the booster injection, allowed to clot at 4°C overnight, and then centrifuged at 10,OOOg for 10 min. The supernatant was removed and stored at - 70°C until used. The specificity of antisera was determined by Ouchterlony  doublediffusion tests using 1%agarose (Sigma) prepared in 0.15 M potassium phosphate, pH 7.4, that contained 0.1% sodium azide. Incubation of Fat Body Fat body tissues (100 mg) were washed in Ringer's solution two or three times and incubated in 100 p1 of Grace's insect medium containing 20 pCi of L-[3,4,5-3H(N)]-leucinefor 5 h in a shaking incubator at 28°C. Following the incubation, fat body tissues were homogenized in Ringer's solution (0.3 ml) and centrifuged at 10,OOOg for 20 min at 4"C, and the supernatant was subsequently electrophoresed, stained, destained, and fluorographed. 218 Kimet al. In order to examine sequestration of storage protein into fat body tissues, fat body from 1-day-old fifth instar larvae was cultured, homogenized, and centrifuged as described above. Free amino acids in the supernatant (100 p1) were removed using a centrifuged desalting column [131. Sephadex G-50 was packed and equilibrated in 50 mm phosphate buffer (pH 7.5) in a 1-ml disposable syringe. After centrifugation at 1,600g for 4 min in a bench centrifuge, the effluent from the syringe was collected and used as the labeled protein source. The radioactivity recovered in the protein fractions was determined by direct scintillation counting of aqueous samples dispersed in 5.0 ml Aquascint (ICN, Irvine, CA) with a 40% counting efficiency. Fat bodies (50 mg) of various ages (3-day-old fifth instar larvae to 3-day-old pupae) were incubated in Grace's medium containing 3H-labeledproteins (1,000 dpd100 p1 medium) for 5 h in a shaking incubator at 28°C. Following the incubation, the fat bodies were washed in Ringer's solution three times and cut into 5-km-thick sections using a cryostat at - 40°C for autoradiography. Tissue Preparation for Light Microscopy The fat bodies were fixed in glutaraldehyde (5%with 50 mMcacodylate buffer, pH 7.4) at - 4°C for 18 h and washed. Five-micron-thick serial sections were cut using a cryostat at - 40°C and stored at - 20°C until used. Fat body sections were immunohistochemically stained by the method of Hsu et al. . Tissue sections were hydrated in distilled water and washed with PBS. After washing, the sections were treated with methanolic hydrogen peroxide followed by 5% normal rabbit serum to block the endogenous peroxidase activity and to reduce the nonspecific background staining. The primary antibody (anti SP-2, 100 11.) was applied over the section and allowed to incubate at 4°C for 24 h in a water-saturated atmosphere. Afterward, sections were washed in three changes of PBS (15 min each) and incubated for 1h at room temperature with the secondary antibody (biotinylated anti-rabbit IgG, Vectastain, Vector Laboratories, Burlingame, C A PK4001). Subsequently, tissue sections were rinsed for 15min in three changes of PBS and then incubated with a 1:lOO dilution of the avidin biotin peroxidase complex (Vectastain, PK4001) for 1 h. Sections were then placed in 200 ml of 0.075% fresh, filtered solution of DAB tetrahydrochloride in 50 mM Tris buffer, pH 7.6. The DAB treatment was accompanied by constant stirring. The sections were suspended in the DAB solution for 5 min, after which 0.003% hydrogen peroxide was added, and incubation was continued for an additional 15 min. Coverslips were placed on all slides in preparation for light microscopy. Antigen-antibody binding sites were visualized as the brown product of the reaction with DAB. Autoradiographic Procedure Dry-mount autoradiography was combined with immunohistochemistry, thereby allowing both storage protein accumulation and the storage protein uptake site to be confirmed in the same preparation. After the immunohistochemical staining procedure, the tissue sections were dehydrated in an ethanol series, air dried, and coated with Kodak nuclear emulsion NTB 2. After exposure for 30 days in light-proof desiccator slide boxes at 4"C, the autoradiograms were transferred to freshly prepared Kodak D-19 developer and developed at Pieris rapae Storage Proteins 21 g 15°C for 4 min. After briefly rinsing the slides in tap water (15"C), the autoradiograms were incubated for 5 minin Kodak fixer at 15°C. Rinsing in tap water at 18°Cwas followed by a 5 min wash in 10 mM PBS, pH 7.5, at room temperature. RESULTS Protein Profiles in Hemolymph and Fat Body During Development Aliquots of fat body extracts and hemolymph were electrophoresed to determine quantitative changes of storage proteins during the metamorphosis of the last larval instar to the pupal stage. Two storage proteins occur in hemolymph. Figure 1 shows the electrophoretic pattern of hemolymph proteins; the upper band on the gel (Fig. 1A) was designated SP-1, and the lower band was designated SP-2. Storage proteins in the hemolymph are present in high concentrationsuntil the early prepupal stage, at which time they decrease gradually and completely disappear at 5 days after pupation. Conversely, the same storage proteins in fat body were present in trace amounts until the early prepupal stages, after which they accumulated in large amounts (Fig. 1B). Purification of Storage Proteins Hemolymph from 3-day-old fifth instar larvae was subjected to nondenaturing PAGE, the storage protein bands were excised with sharp razor blades, and proteins were extracted and concentrated (SP1,20mg/ml; SP2,15 mg/ml).When the purified proteins were analyzed by disc gel electrophoresis, the same mobility of storage proteins was observed in the hemolymph and fat body (Fig. 2). Hemolymph and fat body of various ages were used to confirm the pure antibody for SP-2 antigen. This analysis was conducted in 1%agarose gel using the Ouchterlony double-diffusion test (Fig. 3). SP-2 antibody yielded single precipitation line with purified SP-2,3-day-oId fifth instar larval hemolymph, prepupal hemolymph, and 5-day-old pupal fat body but no line with 5-day-old pupal hemolymph, indicating pure antibody for only SP-2. Physiochemical Characteristics of SP-1 and SP-2 SP-1 was estimated to have a M, of 500,000 and composed of one subunit with a M, of 77,000 (Fig. 4). The M, of SP-2 was found to be 460,000 and composed of two kinds of subunits with M,s of 80,000 and 69,000, respectively (Fig. 4). Isoelectric focusing of the storage proteins was conducted on 5% polyacrylamide gel including ampholytes. SP-1 and SP-2 were determined to have PI values of 6.97 and 7.06, respectively. Biosynthesis of Storage Proteins Fat bodies from fourth and newly ecdysed fifth instar larvae were incubated and fluorographed as described in Materials and Methods; a negative reaction was observed (data not shown). Fat body was incubated at 1-day intervals from 1-day-old fifth instar larvae to 2-day-old pupae. High incorporation of radioactivity into storage proteins occurred in fat body from 1-day-old fifth instar larvae, with low incorporation in 2- and 3-day-old fifth instar larvae. No incorporation was found during the pupal stage (Fig. 5). 220 Kimetal. 5L1 5L3 PPE PPL PO PI p3 p5 A SPl SP2 + B I + Fig. 1. Electrophoretic patterns of hemolymph and fat body in f? rapae during the metamorphosis. A Hemolyrnph (10 PI).B: Fat body (15 PI). 5L1, I-day-old fifth instar larvae; 5L3,3-day-old fifth instar larvae; PPE, early prepupae; PPL, late prepupae; PO, newly ecdysed pupae; PI, 1day-old pupae; P3'3-day-oId pupae; P5,s-day-old pupae. Morphological Changes of Fat Bodies During Development Fat body tissue from different developmental stages was examined with a light microscope to determine the relationship between the morphological changes and physiological function. Nuclear and morphological changes in Pieris rapae Storage Proteins HL FB SP1 sP2 221 SPl+SP2 + Fig. 2. PAGE of hernolymph, fat body extract, and purified storage proteins. HL, male prepupal hemolyrnph (10 PI); FB, male prepupal fat body (15pl), SPI, 30 pl; SP2,30 pl; SP1 + SP2,20 pl + 10pl. Fig. 3. Ouchterlony double-diffusion analysis of the storage protein. The center well contained serum against storage protein-2 (20 PI). Wells 1 and 4 contained the purified storage protein-2 (20 PI). Wells 2 and 3 contained soluble proteins from 5-day-old pupal fat body (20 pI) and hernolyrnph (15 PI), respectively. Well 5 contained prepupal hernolyrnph (15 PI). Well 6 contained 3-day-old fifth instar larval hemolyrnph (15 pl). fat body tissues were observed after staining in hematoxylin only. Sections of fat body from 1-day-old fifth instar larvae showed normal cell morphology with distinct nuclei (Fig. 6: 5L1). Two to 3 days later, the nuclei became irregularly shaped, and small basophilic granules appeared around them (Fig. 6: 5L3). In pupae, the boundaries between the cytoplasm and the nucleus seemed to be 222 Kimetal. SP1 SP2 205 K 116K 97.4K 80 K 66K 69 K 77K 45K 29 K 20.1 K Fig. 4. Determination of molecular weight of storage protein subunits by electrophoresis in 10% polyacrylamide gel containingO.l% SDS by the method of Laemmli .Purified SP1 (10 1.1) and SP2 (10 1.1.) Protein markers used were myosin (205,000), p-galactosidase (116,000), phosphorylase (97,400), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), and trypsin inhibitor (20,100). obscured, possibly by granules, and the cytoplasm increasingly filled with storage granules (Fig. 6: PO). At the end of the pupal instar, the fat body was filled with many more granules (Fig. 6: P5). Analysis of SP-2 Granules Granules containing storage protein were analyzed in sections of fat body by immunohistochemical staining with SP-2 antiserum. Granules containing SP-2 were not detected in sections of fat body from 2-day-old fifth instar larvae (Fig. 7 5L2). Storage protein granules appeared soon after the prepupal stage (Fig. 7 PPI) and gradually increased in number toward the end of pupal life (Fig. 7: PO, P5). The number and size of the storage protein granules reached a maximum in 5-day-old pupae (Fig. 7: P5) but drastically decreased in the adult stage (Fig. 7: A3). SP-2 Uptake by Fat Body Incubation of fat body obtained from larvae and pupae of various ages with 3H- proteins was performed for 5 h. The highest uptake was attained during the late prepupal stage (Fig. 8). As shown in Figure 8, silver deposits (indicated as black specks) were dispersed throughout the fat body tissue. Some grains were overlapped by darker shading (arrows)which indicated the presence of the SP-2 antibody; other silver deposits had no shading (triangles) which Pieris rapae storage Proteins 5L1 5L2 5L3 PPI PO 223 p2 Fig. 5. Incorporation of [3H]-leucine into proteins by P rapae fat body at different stages. The fat bodies obtained from larvae and pupae of several ages were incubated in the presence of 20 pCi of [3H]-leucine for 5 h at 28°C and submitted to electrophoresis and fluorography as described in “Materials and Methods.” Stages as in Figure 1. indicated the absence of SP-2. Protein uptake by the fat body appears to be nonspecific as judged by these experiments. DISCUSSION The synthesis of storage proteins used in the formation of adult tissues occurs during the larval stages of the insect [1,3,6]. This is a distinct event in holometabolic insects, which do not feed during the pupal instar [l]. Storage proteins are synthesized by fat body cells, released into the hemolymph during the last larval instar, and taken up by the fat body of prepupae and pupae [15-191. In the present work, two storage proteins (SP-l,SP-2) have been identified in the hemolymph of last instar larvae of P. rupue. The concentrations of SP-1 and SP-2 in the hemolymph and the fat body of P. rupue change drastically during metamorphosis; these data coincide with the report of Tojo et al.  on the hemolymph storage protein in H. cecropia. The molecular weights of storage proteins have been reported to range from 235,000 for Galleria melonella  to 720,000 for Calpodes ethlius . Storage proteins were usually hexamers or trimers [2,18,22,23]. Most storage proteins have one or two types of subunits [4,24-261 with molecular weights from 72,000 for Rhynchosciuru umericunu  to 92,000 for Munducu sextu . Storage proteins of I? rapae appear to follow this pattern. Molecular weights of SP-1 and SP-2 of P. rupue were determined to be 500,000 and 460,000, respectively. SP-1 consists of one type of subunit with molecular weight of 77,000, whereas SP-2 224 Kimetal. Fig. 6. Light photomicrographs of fat body from /? rapae at different stages. 5L1, I-day-old fifth instar larvae; 5L3, 3-day-old fifth instar larvae; PO, newly ecdysed pupae; P5, 5-day-old pupae; 5LI-P0, nuclei (arrows); P5, storage granules (arrows). Hernatoxylin staining. X 400. consists of two types of subunits with molecular weights of 80,000 and 69,000. This suggests that the storage proteins of P. r a p e are hexamers. The storage proteins of P. r a p e have PIS that are almost neutral. The pH of P. r a p e hemolymph is 6.9, which is a little higher than those reported for other insect species [27-291. In general, insect storage proteins are acidic and dissociate into subunits at more alkaline pHs . Munn et al.  indicated that the pH of the hemolymph of C. erythrocephulu is about 6 during the larval instar but increases to ca. 7 during the pupal instar. Calliphorin, the storage protein of C. erythrocephulu, is used during the pupal instar for the synthesis of adult tissues, suggesting some physiological significance for the dissociation of calliphorin at about pH 6.5. However, other storage proteins, e.g., protein-6 of R. urnericunu  and manducin of M.sextu , do not dissociate at pHs as high as 9.0, indicating the diversity of storage proteins in the various species of insects. Biosynthesis of storage proteins by fat body has been reported previously [2,21,25,26]. Bianchi et al. reported that fat body from Muscu domesticu synthesized storage protein during the last larval instar (feeding stage), ceased the synthesis at the prepupal stage, and accumulated storage proteins after the prepupal stages. The synthesis of P. r a p e storage proteins by the fat body was also confirmed in last instar larvae. Pieris rapae Storage Proteins 225 Fig. 7. lrnrnunohistochernical staining of F! r a p e fat body at different stages with SP-2 antiserum. Cont, control not treated with SP-2 antiserum, 5-day-old pupae; 5L2, 2-day-old fifth instar larvae; PPI, late prepupae; PO, newly ecdysed pupae; P5, 5-day-old pupae; A3, 3-dayold adult; arrows, storage protein granules. x 400. Tojo et al. reported that during the larval-pupal transformation rough endoplasmic reticula and mitochondria decreased in number in fat body cells, while the cytoplasm increasingly filled with lipid and protein granules. A comparable morphological difference was observed in the fat body cells after hematoxylin staining during the metamorphosis of I? rape. At the beginning of the pupal instar, nuclear shape change and storage granule accumulation suggest 226 Kirnetal. Fig. 8. lmmunohistochemically stained autoradiograms of late prepupal fat body indicating uptake of 13H] SP-2 (arrows) by the fat body. Other [3H-]proteins were also taken up (triangles). A: x 400; B: X 1,000. that fat body cell function might be switched from a biosynthetic role in larvae to a storage role after pupation. As shown in Figure 7, storage granules seem to contain many different proteins or others in addition to storage protein-2. Some authors reported that storage granules contain protein, lipid, and urate [4,31,32]. It has been suggested that the fat body cells in Calliphoru larvae synthesize and export proteins into the hemolymph and also take up protein from the hemolymph 1331. We have used an autoradiographic-immunohistochemical experiment to confirm the uptake of storage proteins into the fat body in P. rupue. Fat body cells from late prepupae of P. r a p e had the highest storage Pieris rapae Storage Proteins 227 protein uptake. Our observations, together with those of others on Lepidoptera, indicate that the synthesis and deposition in the fat body of a class of storage protein is a general process in insect metamorphosis. LITERATURE CITED 1. Munn, EA, Greville G D The soluble protein of developing Culliphoru erythrocephlu, particularly, calliphorin, and similar proteins on other insects. J Insect Physiol25,1935(1969). 2. Levenbook L: Insect storage proteins. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, Vol10, pp 307-346 (1985). 3. Chrysanthis G, Marmaras VJ, Christodolou C: Major hemolymph proteins in Cerutitis capitafa: Biosynthesis and secretion during development. Whilhelm Rouxs Arch 190,33 (1981). 4. Tojo S, Betchaku T, Ziccardi VJ, Wyatt GR: Fat body protein granules and storage proteins in the silk worm, Hyulophoru cecropia. J Cell Biol78,823 (1978). 5. Ueno K, Natori S: Activation of fat body by 20-hydroxyecdysone for the selective incorporation of storage protein in Surcophugu peregrinu larvae. Insect Biochem 12,185 (1982).. 6. Roberts DE, Brock HW: The major serum proteins of dipteran larvae. Experientia 37, 103 (1981). 7. Davis BJ: Disc electrophoresis-11. Method and application to human serum proteins. Ann NY Acad Sci 122,404 (1964). 8. Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 (1970). 9. Lowry OH, Rosebrough NJ, Farr AL, Randall RT: Protein measurement with the Folin phenol reagent. J Biol Chem 293,265 (1951) 10. Hedrick JL, Smith AJ: Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 126,155 (1968). 11. wrigley C W Gel electrofocusing: A technique for analyzing multiple protein samples by isoelectricfocusing. Sci Tools 25,17 (1968). 12. Ouchterlony 0: Handbook of Immunodiffusion and Immunoelectrophoresis. Ann Arbor Science Publisher, Ann Arbor (1968). 13. Maniatis T, Fritsch EF, Sambrook J: Molecular cloning, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY pp 464-467 (1982). 14. Hsu S, Raine L, Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase technique. J Histochem Cytochem 29,1577 (1981). 15. Chippendale GM, Kilby BA: Relationship between the protein of the haemolymph and fat body development of Pieris brussicue.J Insect Physioll5,905 (1969). 16. Chippendale GM: Selective protein storage by the fat body of the Augoumois grain moth, Storogu cerurellu. Insect Biochem 1,122 (1971). 17. Kinnear JF, Thomson JA: Nature, origin and fate of the major haemolymph proteins in Culliphoru. Insect Biochem 5,531 (1975). 18. Roberts DB, Wolfe J, Akam ME: The developmental profiles of two major haemolymph proteins from Drosophilu melunoguster. J Insect Physiol23,871(1977). 19. Marinotti 0, Bianchi AG: Uptake of storage protein by Muscu domesticu fat body. J Insect Physiol32,819 (1986). 20. Miller SG, Silhacek DL: Identification and purification of storage proteins in tissues of the greater wax moth Galleria mellonellu (L.) Insect Biochem 22,277 (1982). 21. Locke J, McDermid H, Brac T, Atkinson BG: Developmental changes in the synthesis of haemolymph polypeptides and sequestration by the prepupal fat body in Culpodes eth2ius Stoll (Lepidoptera: Hesperidae). Insect Biochem 12,431 (1982). 22. Munn EA, Feinstein A, Greville GD: The isolation and properties of the protein calliphorin. Biochem J 124,368 (1971). 23. Thomson JA, Radok KR, Shaw DC, Whitten MJ, Foster GG, Birt LM: Genetics of lucillin, a storage protein from the sheep blowfly, Luciliu cuprinu. Biochem Genet 14,145 (1976). 24. Kramer SJ, Mundall EC, Law JH: Purification and properties of manducin, an amino acid storage protein of the haemolymph of larval and pupal Munducu sextu. Insect Biochem 10, 279 (1980). 228 Kimetal. 25. Tojo S, Nagata M, Kobayashi M Storageproteins in the silkworm Bombyx rnori. Insect Biochem 10,289 (1980). 26. de Bianchi AG, Marinotti 0:A storage protein in Rhynchosciara americam (Diptera:Sciaridae). Insect Biochem 14,453 (1984). 27. Loughton BG: An investigation of haemolymph proteins in Lepidoptera. J Insect Physiol 11,1651 (1965). 28. Greville GD, Munn EA, Feinstein A: A major soluble protein (calliphorin) of developing Culliphoru erythrocephala (Diptera). Abstr 7th Int Congr Biochem Tokyo 589 (1967). 29. Wyatt GR, Chinzei, Chino H Purification and properties of vitellogenin and vitellin from Locustu migratoriu. Insect Biochem 11,1(1981). 30. de Bianchi AG, Marinotti 0, Epinoza-Fuentes FP, Pereira SO: Purification and characterization of Muscu domesticu storage protein and its developmental profile. Comp Biochem Physiol [B] 76,861 (1983). 31. Nair KS, George JC: A histological and histochemicalstudy of the larval fat body of Anthrenus vorax Waterhouse (Dermestidae, Coleoptera). J Insect Physiol10,509 (1964). 32. Locke M, Collins JV: Protein uptake into multivesicular bodies and storage granules in the fat body of an insect. J Cell Biol36,453 (1968). 33. Price GM, Bossman T: The electrophoretic separation of proteins isolated from the larva of the blowfly, Culliphoru erythrocephala. J Insect Physiol12,741(1966).