Archives of Insect Biochemistry and Physiology 10:115-130 (1 989) Storage Proteins of the Fall Webworm, Hyphantria cunea Drury Hak Ryul Kim, C.S Kang, and Richard T. Mayer Department of Biology, Korea University, Seoul, Korea (H.R.K., C.S. K.); Horticultural Research laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, Florida (R.T.M.) Two storage proteins, storage protein-I (SPI)and storage protein-2 (SP2),were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SPI is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr=76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr=74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6-8 days of the 7th instar. Key words: hemolymph, fat body, yolk protein, vitellogenin INTRODUCTION A common developmental process of holometabolous insects is the synthesis of storage proteins. Storage proteins are synthesized by fat bodies, released into the hemolymph during the last larval instar, and then selectively taken up by fat bodies during nonfeeding stages [l-51. Storage proteins have been Acknowledgments: This work was supported by Basic Science Research Support Fund from the Minister of Education, Republic of Korea. Received August 8,1988; accepted January 17,1989. Address reprint requests to Dr. R.T. Mayer, USDA, ARS, Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803. Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products o r vendors that may also be suitable. 0 1989 Alan R. Liss, Inc. Kimetal. 116 also purified and characterized and their titer determined during development [2,6,7]. However, information concerning their ultimate fate and functions was rather limited. The purpose of this work was to compare the quantities of storage proteins in hemolymph and fat body during last larval and pupal stages and describe their properties and synthesis and further determine the resemblance of storage protein to vitellogenin and ovary extract in the fall webworm, Hyphantria cunea. MATERIALS AND METHODS Insects Larvae of Hyphantria cunea Drury were reared on fresh willow leaves at 27°C 1°C and 75% 5% relative humidity with a photoperiod of 16 h light and 8 h dark. Sexes were segregated during larval and pupal stages. + + Chemicals All reagents for electrophoresis and immunological analysis as well as ampholytes (pH3-lo), including SDS,* acrylamide, TEMED, and molecular weight markers were purchased from Sigma (St. Louis, MO). Other chemicals were obtained from the following sources: Freund’s adjuvant was from Difco (Detroit, MI), Gracgs insect medium was from Gibco (Grand Island, NY), and [3H]leucine (specific activity, 5.0 Ci/mmol) was from DuPont Co. (Boston, MA). All chemicals were reagent grade. Preparation of Protein Extracts for Electrophoresis and Immunological Analysis Hemolymph was collected in a chilled test tube after puncturing the larvae and pupae with a needle. To prevent melanization, a few crystals of phenylthiourea were added to the hemolymph, which was then centrifuged at 10,OOOg for 10 min at 4°C to remove hemocytes and cellular debris. The supernatant was stored at - 70°C until used. Fat body was dissected from larvae and pupae in cold Ringer’s solution (128 mM NaCl, 1.8 mM CaC12, 1.3 mM KCl), and was washed with Ringer’s solution two or three times. Wet weights of fat bodies were measured after blotting on weighing paper. Fat bodies (100 mg) were homogenized in Ringer’s solution (0.5 ml) and centrifuged at 10,OOOg for 10 min at 4°C and the superatant was stored at - 70°C until used. Ovaries were dissected from adult females and rinsed in Ringer’s solution (pH 7.4), dried on filter paper, and homogenized in 0.2 ml, 50 mM Tris-HC1 buffer (pH 8.0, 0.5 M NaC1, 5 mM EDTA) per pair of ovaries and centrifuged at 10,OOOg for 10 min. The surface lipid layer was removed and the supernatant was used as the sample. Polyacrylamide Gel Electrophoresis PAGE was carried out using a 2.5% stacking gel and a 6% running gel (tube size, 150 x 6.5 mm ID) at 3 mA per gel . Hemolymph (30 mg proteiniml) *Abbreviations used: ANS = 8-anilino-I-naphthalene sulfonic acid; PAGE = polyacrylarnide gel electrophoresis; PAS = periodate Schiff reagent; SDS = sodium dodecyl sultate; SPI, SP2 = storage protein-I and -2; TCA = trichloroacetic acid; TEMED = N,N,N’,N’-tetrarnethylethylenediamine; Vg = vitellogenin; YPI, YP2, YP3 = major yolk proteins 1 , 2 , 3 . Storage Proteins of H. cunea 1 17 and supernatant from the fat body homogenate (15 mg proteidml) were each mixed with an equal volume of 0.1 M Tris-glycine buffer (pH 8.3) containing 20% sucrose and 0.006% bromphenol blue prior to applying them to the gel. Slab SDS-PAGE (160 x 170 x 3 mm) was also performed at room temperature using 3% stacking gels and 10% running gels, with 0.1 M Tris-glycine buffer (pH 8.3) containing 0.1 % SDS . Hemolymph (2.5 p1) and supernatant from the fat body homogenate (5 p1) were each mixed with 20 pl of sample buffer (4% SDS, 10% 2-mercaptoethanol, 0.006% bromphenol blue, 20% sucrose, 62.5 mM Tris-glycine, pH 6.8) in microcentrifuge tubes and boiled at 100°Cfor 3 min. Following electrophoresis, the gel was stained in 0.25% Coomassie brilliant blue and then destained overnight in 50% methanol containing 7% acetic acid, and then in 30% methanol containing 3.5% acetic acid. The gels were fixed in 7.5% acetic acid. Gel bands were scanned with a TG 2970 densitometer (Transidyne General Corp .,Ann Arbor, MI). Two-dimensional electrophoresis was performed in an effort to determine the subunit structure of storage protein. Hemolymph samples were first electrophoresed on 6% non-SDS polyacrylamide tube gels (tube size, 150 x 2.5 mm ID). Afterwards, the gels were incubated in sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-glycine, pH 6.8) for 2 h, and then electrophoresed on an 8-10% slab gel gradient SDS at 30 mA for 4 h. Composition of Protein After electrophoresis of the hemolymph from last instar larvae, the protein bands were stained in 0.25% Coomassie brilliant blue. Carbohydrates, lipids, and phosphates were proved with PAS stain [lo], Sudan black B [ll], and methyl green , respectively. Purification of Storage Protein, Vitellogenin, and Major Yolk Proteins Some proteins were present in large amounts in hemolymph during the last larval instar, but their titers were reduced after pupation. Conversely, these proteins were present in small amounts in fat body during the last larval instar but their concentrations were increased after pupation. These proteins were designated storage proteins. Some protein is present in hemolyph of female but not of male. This protein is called vitellogenin. Also, major protein bands on electrophoresis from ovary extract were designated major yolk proteins (YP1, YP2, YP3). Storage protein and Vg were purified using a gel-slicing method. Seven hundred fifty microliters of hemolymph collected from the last larval instar (for storage protein) and 1.O ml of hemolymph collected from 3-day-old female pupae (for Vg) were loaded onto 3-mm-thick slab gels (2.5%stacking gel and 6% running gel) and electrophoresed at 30 mA for 4 h each. Subsequently, the gels were incubated in 75% ammonium sulfate solution containing 0.0003% ANS for 10 min . Storage protein and Vg bands were observed under UV light and were excised from the gels, eluted, dialyzed against 50 mM Tris-glycine buffer (pH 8.3), and concentrated by freeze-drying. Ovary extracts (10 mg proteidml) were applied to 7.5% polyacrylamide slab gels to determine the major yolk proteins (YP1, YP2, YP3). After electropho- 118 Kimet al. resis, parts of the gel were stained with Coomassie brilliant blue and the bands corresponding to yolk proteins YP1, YP2, YP3 were excised and eluted from the gels by electrophoresis in Tris-glycine buffer, pH 8.3, at 100 V. Determination of Molecular Weights Molecular weights of the native storage proteins were determined as described by Hedrick and Smith . Standard marker proteins were a-lactalbumin (M, = 14,200), 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). In addition, the molecular weights of storage protein subunits were determined using the method of Lambin et al. with an SDS gradient gel (8-10%). Standard molecular weight markers were myosin (M, = 205,000), P-galactosidase (M, = 116,000), phosphorylase (M, = 97,400), bovine plasma albumin (M, = 66,000), egg albumin (M, = 45,000), and carbonic anhydrase (M, = 29,000). Isoelectric Focusing Isoelectric focusing of storage proteins was conducted on 6% polyacrylamide gels using 1%ampholytes (pH 3-10) as described by Wrigley . After polymerization, the empty space at one end of the gel was filled with protective solution (1.5%ampholytes and 10%glycerol) and then connected to an electrophoretic chamber. The upper chamber contained 10 mM H3P04and the lower chamber 20 mM NaOH. Gels were prerun for 30 min at 200 V and then purified SP1 and SP2 in 0.1 ml of 1.5%ampholytes and 20% sucrose solution was gently placed at the end of the gel and was run at a constant 1 mA per gel up to 450 V. After electrophoresis, one gel was stained and the other gel used for pH determination. For staining, gels were fixed in a 4% sulfosalicylic acid/12.5% TCA solution which is exchanged eight times at 1h intervals and then finally stained with a solution containing 27% isopropanol, 10% acetic acid, 0.04% Coomassie brilliant blue, and 0.5% CuS04for 1h. Destaining was accomplished using a solution of 12% isopropanol, 7% acetic acid, 0.5% CuS04. For pH determination, one gel was sliced at 0.3 cm intervals, added to tubes containing 1.0 ml of distilled water, incubated for 24 h, and then the pH measured. Preparation of the Antiserum and Immunological Analysis Purified SP1 (600 pg/ml), Vg (400 pg/ml) were mixed with an equal volume of Freund’s complete adjuvant (0.5 ml) and injected subcutaneously into rabbits three times every other day with a fourth injection given 1 week later. Booster injections (0.5 ml protein and 0.5 ml Freund’s incomplete adjuvant) were given 2 weeks after the fourth injection. Blood was collected 1week after the fifth injection, allowed to coagulate at 40°C overnight, and centrifuged at 10,OOOg for 10 min. The supernatant was used in the immunological tests. Immunodiffusion tests were conducted on 1%agarose gel in 10 mM veronal buffer (pH 8.6) containing 0.1% sodium azide for 3 days at room temperature as described by Ouchterlony . Gels were stained in 1%amido black 10B and destained in 2% acetic acid. Rocket immunoelectrophoresis was carried out according to Laurel1 [181. One percent agarose in 10 mM veronal buffer (pH 8.6) containing 0.1% sodium Storage Proteins of H. cunea 119 azide was mixed with an appropriate amount of anti-SP1 serum to yield 5% anti-SP1 serum. This mixture was coated on a glass plate (6.5 X 6.5 cm). Electrophoresis was conducted in 10 mM veronal buffer (pH 8.6) at 50 V for 18 h. After electrophoresis, the gel was washed in 0.15 M NaCl for 48 h and stained in amido black 10B. Tandem-crossed immunoelectrophoresis was carried out as described by Axelson et al. . One percent agarose was coated on glass plates and each sample was applied to well and electrophoresed at 55 V for 2 h. The remaining sample-free gel was removed and replaced with antibody containing gel and electrophoresis was conducted in the second dimension at 50 V for 18 h. In Vitro Synthesis of Proteins Fat body tissues were washed in Ringer’s solution two or three times and preincubated in Grace’s insect medium for 10 min. Fat body (approximately 100 mg) was incuabated in 100 pl of Grace’s insect medium containing 20 pCi of L-[3,4,53H(N)]leucinefor 5 h in a shaking incubator at 30°C. Following incubation, labeled proteins were electrophoresed, stained, destained, and soaked in autoradiographic fluid (sodium salicylate) for 10 min for fluorography and dried. The dried gel was exposed to Kodak X-Omat X-ray film at - 70°C for 1 week. RESULTS Quantitative Changes of Storage Proteins in Hemolymph and Fat Body During Development Hemolymph of H . cuneu was analyzed by electrophoresis to show changes in storage proteins during the last larval and pupal instars. As shown in Figures 1 and 2, both sexes have two storage proteins in the hernolymph. The protein in the upper band on the gel was called SP1, and protein in the lower band designated SP2. In general, SP1 is present in greater amounts than SP2 (Figs. lB, 2B). The hemolymph storage proteins begin to appear on the 4th day of the 7th larval instar and reach their peak on the 8th day. The hemolymph storage proteins begin to decrease during the prepupal stage, are drastically reduced immediately after pupation, and completely disappear by the 3rd day of the pupal instar. Conversely, storage proteins in fat body do not appear until late in the 7th instar, and accumulate in significant amounts from the prepupal period to newly ecdysed pupae and then remain in high concentration (Figs. 1, 2). Also SP1 in both males and females, as determined by rocket immunoelectrophesis, peaks on the 8th day of the 7th larval instar and drastically decreases after the prepupal stage (Fig. 3). These data corroborate those obtained from the electrophoretic experiments above (Figs. 1,2). Properties of Storage Protein SP1 and SP2 were subjected twice to electrophoresis on 6% polyacrylamide gels. The electrophoretic pattern shown in Figure 4 indicates that SP1 and SP2 are highly purified. Two-dimensional electrophoresis was carried out to determine the number Kimet a\. 120 A B Fig. 1 . A: Polyacrylarnide gel electrophoresis of female hernolymph (7 PI each) and fat body (15 PI each) of H. cunea at different stages. A: Late 6th instar larvae. B: Day 2,7th instar larvae. C: Day 4,7th instar larvae. D: Day 6,7th instar larvae. E: Day 8,7th instar larvae. F: Prepupae. C: I-day-old pupae. H: 3-day-old pupae. I : 5-day-old pupae. J:7-day-old pupae. K: 9-day-old pupae. B: Densitornetric scanning of A. Storage Proteins of H. cunea 121 A Fig. 2. A: Polyacylarnide gel electrophoresis of male hernolymph (7 pI each) and fat body (15 pI each) of H . cunea at different stages. Stages are the same as those given in Figure 1 . B: Densitornetric scanning of A. of subunits of SP1 and SP2. The first electrophoretic dimension using a nonSDS condition shows SP1 and SP2. The second electrophoretic dimension perfomed in the presence of SDS, reveals a single band for SPl and two bands for SP2 (Fig. 5). Molecular weights of native SP1 and SP2 were estimated to be 460,000 and 450,000, respectively (Fig. 6). SP1 was found to be composed of a single subunit type with a molecular weight of 76,000 when analyzed under denaturing PAGE conditions. SP2 consists of two kinds of subunits which have molecular weights 122 Kimet al. Fig. 3. Rocket irnrnunolectrophoresis of male and female hemolymph at different times with anti-SPI. Samples (2 pl each) were loaded. Stages are as shown in Figure 1 . of 74,100 and 72,400 (Fig. 7). Based on the molecular weights of the native storage proteins and their subunits and comparing the intensity of subunit bands as determined densitometerically, both SP1 and SP2 appear to be hexamers, with SP2 consisting of three each of the two types of subunits (Fig. 5B, C). SP1 and SP2 gave positive reactions with PAS, Sudan black B, and methyl green staining, indicating that they contain carbohydrates, lipids, and phos- B A Fig. 4. A: Polyacylamide gel electrophoresis of SP1 and SP2 purified from hernolymph. HL: Last larval hemolymph, 7 pl. FB: Pupal fat body, 15 pI. SP1 : storage protein-1,50 pl. SP2: Storage protein-2,80 pl. B: Densitornetric scanning of A. Storage Proteins of A 0 NON-SDS H. cunea 123 HL M B C SPl - 1+2 2 1 - SP2a SP2b - Fig. 5. A: Determination of the number of subunits in storage proteins 1 and 2 by twodimensional gel electrophoresis of larval hernolymph. The hernolymph sample (2.5 pl) was electrophoresed on 6% non-SDS disc polyacrylarnide gel first and then electrophoresed on 8-10% SDS gradient slab gel at total 30 rnAfor4 h. Arrows indicate the subunits of SP1 and SP2. B: Densitornetricscanning of A. C:SDS PAGE of H. cunea SPI and SP2 (1 21, SP2 (2) and SPI (1). + 124 Kimet al. I , 5 10 ( 20 I I ' 0 0 my.x l u a Fig. 6. Determination of native molecular weights for storage proteins 1 and 2 according to the method of Hedrick and Smith 1141. The molecular weight standards are as follows: C ) carbonic anhydrase, 29,000; E) chicken egg albumin, 45,000; B1) bovine serum albumin, monomer, 66,000; B2) bovine serum albumin, dimer, 132,000; U2) urease, dimer, 240,000; U3) urease, te t ramer, 480,000. phate. The P1 values of SP1 and SP2 were estimated to be 5.70 and 5.50, respectively. Similarities Between SPl and Vg and Ovary Extract Non-SDS PAGE, immunodiffusion, and tandem-crossed immunoelectrophoresis were perfomed, using antibodies against SP1, Vg, to determine if there was any similarity between SP1 and Vg and ovary extract. Female hemolymph from 4-day-old pupae have a band that corresponds to the SP1 band, but 0.82 0.94 0.90 0.98 LOGY1 Fig. 7. Determination of subunit molecular weights for SP1 and SP2 (a,a,b) by electrophoresis in linear gradient of polyacrylamide gel in the presence of SDS. The protein standards used were myosine (205,000), P-galactosidase (116,000), phosphorylase (97,000), bovine plasma albumin (66,000), egg albumin (45,000), and carbonic anhydrase (29,000). Storage Proteins of H. cunea 125 hemolymph from males of the same age pupae does not (Fig. 8). SP1 might be similar to Vg electrophoretically. Also, anti-SP1 serum shows one precipitin line with SP1 and a continuous line with the hemolymph of last instar larvae and fat body of newly ecdysed pupae, and adult ovary extract but not with the wing of adult (Fig. 9). The similarity between SP1 and ovarian extract was also confirmed by the continuity between arcs from SP1 and from ovarian extract (Fig. 10). Figure 11 shows that the ovarian protein extract makes a clear continuous precipitin line with anti-SP1 and anti-Vg, indicating that SP1 and Vg and ovary extract are immunologically similar. Biosynthesis of Storage Protein Fat body from 7th instar larvae was incubated in Grace’s medium containing [3H]leucine, homogenized, and then the homogenote was subjected to electrophoresis and fluorography to determine when storage proteins were synthesized. Fat body was incubated at 1 day intervals to determine the exact time of synthesis. Male fat body exhibits storage protein synthesis activity during the fourth day, whereas female fat body exhibits continuous synthetic activity from day 4 to day 8, but no biosynthesis activity during the prepupal and pupal stages (Figs. 12, 13). In addition, fat body from male and female 7th instar larvae was incubated with [3H]leucine and fat body tissue and medium were separated, subjected to electrophoresis and fluorography. As shown in Figure 14, storage protein was present in medium as well as in fat body tissue, indicating that storage proteins are released into hemolymph after synthesis. DISCUSSION In general, storage proteins are synthesized in the fat body during the last larval stages, released into the hemolymph, and then selectively reabsorbed into the fat body after pupation. Tissue localization of storage protein during development was reported in Lepidoptera [6,20-221 and in Diptera [23,24]. The present work with Hyphuntriu cuneu shows two major storage proteins in hemolymph during the last larval instar and in fat body during the pupal instar. The developmental patterns of storage proteins in hemolymph and fat body reported here are similar with those of Hyulophovu cecropia, where storage proteins in the hemolymph peak during the last larval instar and spinning stages, decrease during the pupal stage, and accumulate in the form of granules in fat body after pupation . Collins and Downe , using a histochemical method, reported that the hemolymph protein taken up by the fat body is glycolipoprotein. Kramer et al.  indicated that the storage protein of Munducu sextu is a conjugated protein containing 2% lipid, of which a major portion is phospholipid, and 3.5% carbohydrate. Both SP1 and SP2 of Hyphuntriu cuneu were found to be phosphatecontaining glycoliproproteins. SP1 has a native molecular weight of 460,000 and consists of one subunit (M, = 76,700), whereas SP2 has a molecular weight of 450,000 and is composed of different subunits (My=74,100 and 72,400). Both SP1 and SP2 are hexamers. Storage proteins in most insects are macromole- 126 Kimetal. A B C D - -YP1 -YP2 -YP3 + 9 8 10 11 Fig. 8. Nondenaturing polyacrylamidegel electrophoretic analysis of hemolymph and ovary. A: Last instar larval hernolymph, 7 PI. B: Female hemolymph of 4-day-old pupae, 7 PI. C: Male hemolymph of 4-day-old pupae, 7 PI. D: Ovary extract, 20 PI. YPI, major yolk protein-I; YP2, major yolk protein-2; YP3, major yolk protein-3; Vg, vitellogenin. Fig. 9. Double diffusion precipitation pattern of anti-SP1 (AB, 15 PI)against purified SP1 (SPI, 20 PI),larval hemolymph (LH, 2.5 PI), pupal fat body extract (PF, 15 PI), adult ovary extract (OV, 15 PI),and adult wing extract (W, 20 PI). Fig. 10. Tandem-crossed immunoelectrophoretic pattern of purified SP1 (15 PI) and adult ovary extract (15 PI)with anti-SPI. Fig. 11. Double diffusion precipitation pattern of adult ovary extract (OV, 15 pl) against anti-SP1 (aspl, 15 PI), and anti-Vg (avg, 15 PI). Storage Proteinsof H. cunea A B C D E F 127 G Fig. 12. Autoradiogram of [3H]leucine-labeledfat body proteins from male larvae and pupae separated o n 10% polyacrylamide gel in the presence of SDS. Tissues from male larvae and pupae at different stages were incubated for5 h in the presence of 20 pCi of [3Hlleucine (specific activity, 5 Ci/mmol). Each sample was prepared for electrophoresis as described in Materials and Methods. The dried gel was exposed to X-ray film for 1week. Arrow indicates storage proteins. Stages are as shown in Figure 1. A B C D E F G Fig. 13. Autoradiogram of [3H]leucine-labeledfat body proteins from female larvae and pupae separated on 10% polyacylamide gel in the presence of SDS. Tissues from female larvae and pupae at different stages were cultured for 5 h in the presence of 20 PCi of [3Hlleucine (specific activity, 5 Ciimmol). Each sample was prepared for electrophoresis as described in Materials and Methods. The dried gel was exposed to X-ray film for 1 week. Arrow indicates storage proteins. Stages are as shown in Figure 1. 128 Kimetal. uu FEMALE MALE Fig. 14. Autoradiogram of [3H]leucine-labeled fat body proteins from seventh instar larvae separated on 10% polyacrylamide gel in the presence of SDS. Tissues from day 6 of seventh instar larvae were cultured for 5 h in the presence of 20 FCi of [3H]leucine(specific activity, 5 Ci/mmol). Labeled proteins were recovered from both the medium (M) and fat body cell (C) fractions. cules having molecular weights of approximately 500,000 and consisting of six subunits [24,26,27]. Generally, these proteins are composed of either a single type of subunit [2,22], or two types of subunits [6,27].The molecular weights of the subunits range from 72,000 in Periplantea americana  to 92,000 in Manduca sexta . The PI value of purified SPl was determined to be 5.70. Storage proteins of Diptera are known to be acidic . Larval hemolymph, in general, has an acid pH which increases when metamorphosis and adult organ formation begin. During metamorphosis, storage proteins are dissociated into subunits which become involved in organ formation or metabolism. For example, the pH of the hemolymph in Calliphora erythrocephala is pH 6.0 during the larval stages, but increases to pH 7.0 during the pupal stages when calliphorin begins to be dissociated and utilized . The hemolymph of Hyphantria cunea is acidic during the larval stages and increases to over pH 7.0 after pupation (unpublished data). Kawaguchi and Doira  reported that in Bombyx mori the female specific larval protein disappears in hemolymph during pupation and a new female Storage Proteins of H. cunea 129 specific protein appears in hemolymph after pupation; they suggested that the female specific larval protein is storage protein-1 and that the female specific pupal protein is vitellogenin. Ogawa and Tojo also reported that storage protein of Bombyx mori is involved in the formation of yolk protein. The present results indicated that SP1 is immunologically similar to Vg and the ovarian proteins, and its electrophoretic mobility corresponds to Vg. Therefore, SP1 of Hyphantria cunea is considered to act as Vg during the pupal instar and to be involved in ovarian protein formation. Biosynthesis of storage protein by the fat body has been reported previously [7,24,31,32]. Bianchi et al.  reported that fat body from Musca domestica actively synthesizes storage protein during the last larval instar (feeding stage), ceases synthesis at the prepupal stage, and actively accumulates storage proteins after the prepupal stage. The synthesis of Hyphantria cunea storage proteins by the fat body was also confirmed in last instar larvae. Males show strong synthetic activity only on the 4th day of the 7th larval instar, and females synthesize storage proteins more slowly over a longer period, i.e., from the 4th to 8th day of the 7th larval instar. Further investigations are required to determine the physiological significance of this difference. LITERATURE CITED 1. Thomson JA: Major patterns of gene activity during development in holometabolous insects. Adv Insect Physiol 11,321 (1975). 2. Tojo S, Nagata M, Kobayashi M: Storage protein in the silkworm, Bombyx mori. Insect Biochem 20,289 (1980). 3. Robert DB, Brock HW: The major serum proteins of Dipteran larvae. Experientia 37, 103 (1981). 4. Levenbook L, Bauer AC: The fate of the larval storage protein calliphorin during adult development of Calliphora vicina. Insect Biochem 24,77 (1984). 5. Marinotti 0, Bianchi AG: Uptake of storage protein by Musca domestica fat body. J Insect Physiol32,819 (1986). 6. Kramer SJ, Mundall EC, Law JH: Purification and properties of manducin, an amino acid storage protein of the haemolymph of larval and pupal Manduca sexta. Insect Biochem 10, 279, (1980). 7. Miller S, Silhacek D: Identification and purification of storage proteins in tissues of the greater wax moth, Galleria1 mellonella (L.). Insect Biochem 22, 177 (1982). 8. Davis BJ: Disc electrophoresis-11. Method and application to human serum proteins. Ann NY Acad Sci 121,404 (1964). 9. Laemli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 (1970). 10. Caldwell RC, Pigman W: Disc electrophoresis of human saliva in polyacrylamide gel. Arch Biochem Biophys 210,91 (1965). 11. Chippendale CM, Beck SD: Hemolymph proteins of Ostrinia nubilalis during diapause and prepupal differentiation. J Insect Physiol12,1692 (1966). 12. Cutting JA, Roth RF: Staining of phosphoproteins on acrylamide gel electrophoresis. Anal Biochem 54,386 (1973). 13. Hartman BK, Udenfriend S: A method for immediate visualization of proteins in acylamide gels and its use for preparation of antibodies to enzymes. Anal Biochem 30,391 (1969). 14. Hedrick JL, Smith AJ: Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 226,155 (1968). 15. Lambin P, Rochu D, Fine JM: A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulphate (SDS)-polyacrylamidegradient gel. Anal Biochem 74,567 (1976). 130 Kirnetal. 16. Wrigly CW: Gel electrofocusing: A technique for analyzing multiple protein sample by isoelectric focusing. Sci Tools 25,17 (1968). 17. Ouchterlony 0: Antigen-antibody reactions in gels. Acta Pathol Microbiol Immunol Scand [A]26, 507 (1949). 18. Laurel1 CB: Quantitative estimaton of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 25,45 (1966). 19. Axelson NH, Kroll J, Weeks B: A Manual of Quantitative Immunoelectrophoresis: Methods and Applications. Universitetsforlaget,Oslo. pp 15-59 (1973). 20. Chippendale GM, Kilby BA: Relationship between the proteins of the hemolymph and fat body during development of Pieris brussicue. J Insect Physiol25,905 (1969). 21. Chippendale GM: Metamorphic changes in hemolymph and midgut proteins of the southwestern corn borer, Diutrueu grundiosellu. J Insect Physiol26, 1909 (1970). 22. Tojo S, Butchaku T, Ziccardi VJ, Wyatt GR Fat body protein granules and storage proteins in the silk moth, Hyulophoru cecropia. J Cell Biol78,823 (1978). 23. Kinnear JF, Martin MD, Thomson JA: Developmental changes in the late larva of Calliphoru stygiu. 111. The occurrence and synthesis of specific tissue proteins. Aust J Biol Sci 24, 275 (1971). 24. Roberts DB, Wolf J, Akam ME: The developmental profiles of two major haemolymph proteins from Drosophilu melunoguster. J Insect Physiol23, 871 (1977). 25. Collins JV, Downe AER Selective accumulation of haemolymph proteins by fat body of Galleria mellonellu. J Insect Physiol26, 1697 (1970). 26. Munn EA, Feinstein A, Greville GD: The isolation and properties of the protein calliphorin. Biochem J 224,367 (1971). 27. Thomson JA, Radok KR, Shaw DC, Whitten MJ, Foster GG, Birt LM: Genetics of lucilin, a storage protein from the sheep blowfly, Luciliu cuprinu. Biochem Genet 24,145 (1976). 28. Bianchi AG, Marinotti 0: A storage protein in Rhynchosciuru umericunu (Diptera Sciaridae). Insect Biochem 24,453 (1984). 29. Kawaguchi Y, Doira H: Gene-controlled incorporation of hemolymph protein into the ovaries of Bombyx mori. J Insect Physiol19,2083 (1973). 30. Ogawa K, Tojo S: Quantitative changes of storage proteins and vitellogenin during the pupal adult development in the silkworm, Bombyx mori (Lepidoptera: Bombicidae). Appl Ent Zoo1 26,288 (1981). 31. Munn EA, Greville GD, Price GM: The synthesis in vitro of the protein calliphorin by fat body from the larva of the blowfly, Calliphoru erythrocephulu. J Insect Physiol25, 1601(1969). 32. Kinnear JF, Thomson JA: Nature, origin and fat of the major haemolymph proteins in Culliphoru. Insect Biochem 5,531 (1975). 33. Bianchi AG, Marinotti 0, Espinoza-Fuentes FP, Pereira SD: Purification and characterization of Muscu domesticu storage protein and its developmental profile. Comp Biochem Physiol [B], 76,861 (1983).