Archives of insect Biochemistry and Physiology 2:91-104 (1985) Effect of Metamorphosis on the Major Hemolymph Proteins of the Silkworm Su I1 Seong, Kwang E. Park, Masao Nagata, and Narumi Yoshitake Deparfmenf of Genetic Engineering, Suweon University, Suweon, South Korea (S. I. S.); Department of Sericulture, Agricultural College, Seoul National University, Suweon, South Korea (K.E.P.); and Laboratory of Sericultural Science, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, Japan (M.N., N.Y.) During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found t o be distributed i n the hernolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The M H P s in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of M H P s in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. M H P s in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the M H P s may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages. Key words: Bombyx mori, major hemolymph protein, metamorphosis INTRODUCTION Electrophoretic and immunological analyses have shown that some proteins are commonly found in the hemolymph and tissues during the metamorphosis of insects [l-41.Loughton and West  have demonstrated that some hemolymph proteins of Mulucosarna are transported into several tissues at the onset of larval-pupal transformation, but such transport is not observed during the early larval stage. Pate1 also examined the distribution, transport, and synthesis of hemolymph and tissue proteins of the cecropia Acknowledgments. We wish to express our thanks to Dr. Y. Tanada, Division of Entomology and Parasitology, University of California, for reviewing the manuscript. Received February 28,1984; accepted July 23, 1984. Address reprint requests to Dr. Su II Seong, Department of Genetic Engineering, Suweon University, P.O. Box 77, Suweon 170, South Korea. @ 1985 Alan R. Liss, inc. 92 Seongetal silkworm, Hyulophoru cecropia, during development. He argued that some proteins that are synthesized in one tissue are transported and accumulated in others. Such a protein transport from the hemolymph to several tissues is observed widely in many insects during metamorphosis [5-151. The detailed process of protein transport in insects has been well investigated with vitellogenin and storage proteins. Vitellogenin is a female-specific protein. It is synthesized by the fat body, and is secreted into the hemolymph and transferred to the egg as a yolk precursor during oogenesis [5-7,9,10]. Storage proteins in the hemolymph are also selectively taken into the fat body during the larval-pupal transformation and stored in protein granules as reserve materials for adult tissue formation [ll-151. We previously reported that three proteins appeared in the midgut at the onset of metamorphosis . These proteins were called the midgut-pupalspecific proteins. Our recent study showed that these proteins also occurred in the hemolymph and in other tissues of a metamorphosing silkworm. We named the proteins the major hemolymph proteins because of their abundant distribution in the hemolymph at metamorphosis. The aims of the present study are 1) to investigate the distribution of MHPs* in the hemolymph and various tissues during ontogenetic development with polyacrylamide gel electrophoresis and immunological assay, and 2) to establish the synthesizing sites of these proteins and their possible translocation during metamorphosis by means of radioisotope experiments in vivo. MATERIALS AND METHODS Insect The commercial hybrid race between Kinshu and Showa of the silkworm was used in the experiment. Larvae were reared on mulberry leaves at 25°C. Larvae in the final instar stopped feeding on day 8 after the last-larval molt and began to spin cocoons. After the completion of the cocoon, the mature larva ecdysed to a pupa. At 10 days after the larval-pupal ecdysis, the adult emerged and copulated and the female oviposited. The eggs entered into diapause at 25°C. The eggs, which had been activated by treatment with HC1 (sp. gr. 1.075, at 46°C for 5 min) 20 h after oviposition, soon began embryogenesis and ultimately hatched within 10 days at 25°C. Preparation of Protein Extracts for Electrophoresis and Immunologic Assay From day 3 of the fifth larval instar to adult emergence, the hemolymph from each sex was collected at 1- or 2-day intervals in a tube containing a small amount of phenylthiourea, and centrifuged at 5,OOOg for 10 min at 5°C. The supernatant was diluted five times with PBS (pH 7.4) just prior to acrylamide gel electrophoretic and immunologic assays. The fat body, midgut, ovary, and testis were separately removed and placed into iced PBS, and their wet weights were measured. The soluble *Abbreviations: major hernolymph protein = MHP; phosphate-buffered saline = PBS. Fate of Major Hernolymph Proteins 93 proteins were extracted by homogenizing the tissues in ten volumes of iced PBS, followed by centrifugation at about 20,OOOg for 10 min. The egg extracts were prepared by the same methods. Polyacrylamide G e l Electrophoresis Polyacrylamide gel (7.5%) was prepared on glass plates (0.2 x 15 x 11cm) at pH 8.9 with a 3.125% stacking gel, pH 6.8. Electrophoresis was carried out 0.5 mh4 Tris-3.8 mM glycine buffer, pH 8.3, at a constant current of 1.5 mAl cm for 2 h. After electrophoresis, the proteins were stained overnight with 0.05% Coomassie Brilliant Blue R-250 in 7% acetic acid and then destained with 7% acetic acid. Densitometry was done at 600 nm with a chromatoscanner (SC-910, Shimadzu Co., Tokyo). After scanning, the areas under each peak of MHPs were quantitated by triangulation. Immunologic Assay Antiserum against components including MHPs was prepared by immunizing rabbits with a midgut homogenate of l-day-old pupae . Then a specific antiserum against MHPs was prepared by absorption of other antibodies; larval and adult homogenates were added to above antiserum until only arcs of MHPs were detected in immunoelectrophoresis. Immunoelectrophoresis was conducted at 5°C on a 12 x 9 cm plate using 1.2% agarose gel in vernal buffer, pH 8.6, p = 0.05 for 2 h at a constant current of 2 mAlcm. Upon the completion of electrophoresis the trough was filled with antiserum, and the plates were incubated overnight at 25°C. Double-diffusion tests were performed according to the method of Ouchterlony [17l in 1.5% agar suspended in NaCl-vernal buffer, pH 7.5. Diffusion was allowed to proceed overnight at 25°C. The gels, both for immunoelectrophoresis and double diffusion, were washed with PBS two or three times a day for more than a week, and then stained with a mixture of Thiazine Red R, Amido Black lOB, Light Green SF, acetic acid, and HgC12 [IS]. Autoradiography ['*C]Leucine (specific activity 342 mCilmmole) was used as a protein precursor. The first experiment examined the incorporation of labeled amino acid into hemolymph and fat-body proteins at each representative developmental stage of the silkworm; ie, a quantity of 2.5 pCi of [**C]leucine was injected into fifth-instar larva (3 days old), pupa (1day old), and adult (just after emergence). Hernolymph and fat body were moved at 3 h after treatment. The second experiment was designed to trace the transport of the labeled MHPs during metamorphosis. A number of fifth-instar larvae (3 days old) were injected each with 5 pCi of ['4C]leucine. The injected insects were fed and reared and their tissues were sampled at the desired interval. The collected hemolymph and tissues were submitted to slab gel electrophoresis as previously described. After staining, the gel was dried on a filter paper and was exposed about 2 months in the dark on Sakura x-ray film. 94 Seongetal Determination of Molecular Weights of MHPs Molecular weights of native MHPs were determined by electrophoresis on acrylamide gels of different concentrations following the method of Hedrick and Smith . Standard proteins (molecular weights; chymotrypsinogen, 2.5 x lo4; bovine serum albumin, 6.7 x lo4 (monomer), 13.4 x lo4 (dimer), 20.1 x lo4 (trimer); aldolase 14.7 X lo4) and MHPs were run on 5-10% polyacrylamide gels, and the mobility of each protein relative to the dye front (Bromophenol Blue) was plotted versus acrylamide gel concentrations. The molecular weights of MHPs were determined by the relation between molecular weights of the standard proteins and the slope of the lines of the above plot. RESULTS Distribution of MHPs in the Hemolymph and Tissues During Metamorphosis Three major proteins, MHP-a, -b, and -c, occurred in the hernolymph of the final-instar larva. The concentrations of these proteins increased from the middle period of the final larval instar, reached maximum levels at the pharate pupa, and decreased with the pupal-adult transformation (Fig. 1). MHPs, in the pharate pupa, were the major hernolymph proteins, accounting Fig. 1. Profiles of hernolymph proteins of the female silkworm during metamorphosis. V, fifth-instar larva; S, spinning larva (pharate pupa); P, pupa ( + pharate adult); A, adult. Number above each electrophorogram represents the day of the developmental stage which was sample a,b, c: MHP-a, -b, and -c. SPI, SP2: storage proteins. Vg: vitellogenin. SP1 (sex-limited) and SP2 (not sex-limited) diminish in the pupal hernolymph whereas Vg first appears in the hernolymph after pupation. Mobilities of SP2 and VC on electrophorogram are very close . One microliter of hernolymph was applied to each gel comb. Fate of Major Hernolymph Proteins 95 for 1170,17%, and 14% of the total hemolymph proteins for MHP-a, -b, and -c, respectively (Fig. 2). The electrophorogram also detected corresponding protein bands in the midgut, fat body, ovary, and testis metamorphosis, and even in diapausing eggs (30 days old after oviposition; Fig. 3A). Higher concentrations of these proteins were observed in the ovary and eggs than in other tissues. The results of acrylamide gel electrophoresis coincided well with those of immunoelectrophoresis. The antiserum prepared against pupal midgut homogenates formed three precipitin arcs corresponding to MHP-a, -b, and -c of the hemolymph and extracts of fat body, ovary, diapausing eggs, and testis as well as the midgut (Fig. 3B). These results suggested that MHPs are common proteins that were found throughout the hemolymph and in various tissues during insect metamorphosis. The appearance and distribution of MHPs in the hemolymph and tissues during metamorphosis were examined with the antiserum specific to MHPs and the Ouchterloney's double-diffusion method (Fig. 4). The MHPs in the hemolymph and fat body began to appear from the middle period of the fifth instar, more or less earlier than in other tissues. The MHPs in the hemolymph and the above tissues gradually disappeared at the onset of pupaladult transformation, and they began to accumulate in the ovary. The MHPs were transported into the eggs and were retained in the oviposited dispausing eggs. Similar results were obtained with gel electrophoresis (data not shown). The MHPs that had accumulated in the eggs decreased gradually with embryogenesis and disappeared completely at the time of hatching (Fig. 5). The MHPs were found also in the male insect (Fig. 3). The protein profiles of the MHPs, except for the gonads, were not affected by the sex of the silkworm. Incorporation of ['4C]Leucine Into the Hernolymph and Tissue Proteins During Metamorphosis Figure 6 is an autoradiograph that illustrates the incorporations of [I4C]leucine into larval, pupal, and adult hemolymph proteins at 3 h after the L (+I (-1 Fig. 2. A representative scan of hemolymph proteins of the female silkworm (pharate pupa) (electrophoresed on 7.5% polyacrylamide gel, pH 9.4). a, b, c: MHP-a, -b, and -c. 96 Seongetal Fig. 3. Polyacrylamide gel (A) and immuno (B) electrophoretic profiles of hemolymph and tissue proteins at metamorphosis of the silkworm. A, rnidgut (stage of SI); B, female hemolymph (SI); C, male hemolymph (SI); D, female fat body (51); E, male fat body (51); F, ovary (PI);G, diapausing eggs (30 day old after oviposition); H,testis (SI). Antiserum was prepared against the homogenate of pupal midgut . injection of [14C]leucine. Most of the larval protein bands were actively labeled. The incorporation was also apparent in adult hemolymph proteins, but scarcely noticeable in the pupal hemolymph proteins. No incorporation of the labeled amino acid into the MHPs occurred during the larval-pupal transformation, although these proteins were abundant in the hemolymph at that time. To examine whether the fat body was involved in the biosynthesis of MHPs, the incorporation of labeled amino acid into the larval and pupal fat body proteins at 3 h after isotope injection was examined. Labeled amino acid was incorporated into the larval fat-body proteins, eg, MHP-b and -c, whereas the incorporation could hardly be seen in the pupal fat body (Fig. 7). The incorporation into the MHP-a, however, was not apparent in the larval fat body, since this protein had not yet appeared in the fat body at the time of isotope injection. Fate of Major Hernolymph Proteins 97 Fig. 4. Ouchterlony's double-diffusion tests with the female hemolymph (A), fat body (B), midgut (C),and ovary and diapausing eggs (D) during the ontogenetic development of the silkworm. In plate D, A stands for l-day-old and D for 30-day-old (diapausing) eggs after oviposition. Specific antiserum against the M H P s is located in the center wells. All other symbols are the same as in Figure 1. Next, we injected ['4C]leucine into 3-day-old larvae of the fifth instar, and traced the labeled hemolymph and tissue proteins throughout the metamorphic development. The radioactivities of hemolymph proteins were retained in considerable amounts up to the late period of the fifth larval instar, but thereafter they became weak during the pupal-adult transformation (Fig. 8). The labeled MHPs were detected throughout the pupal life and they vanished with adult emergence, The released MHPs, at the early period of the fifth larval instar, appeared in the fat body, in the midgut of 1-day-old pupa, and in mature eggs (Fig. 9.) Molecular Weights of MHPs Approximate molecular weights of native MHPs were calculated from the slope of the line in Figure 10. The molecular weights of MHP-a, -b, and -c were estimated at 27,000, 25,000, and 17,000, respectively. 98 Seongetal Fig. 5. Changes in the egg proteins during embryonic development. a, b, c: MHP-a, -b, -c; Vt: vitellin. Number above each electrophorogram represents the day of embryogenesis. H: hatched larva. Fig. 6. Autoradiograph (right) showing the incorporation of [14C]leucine into larval (V3), pupal (Po) and adult (A,) hernolymph proteins of female silkworm at 3 h after isotope treatment. a, b, c: MHP-a, -b, -c. Left: electrophorogram of hernolymph proteins. Fate of Major Hemolymph Proteins 99 Fig. 7. Autoradiograph (right) showing the incorporation of ['4C]leucine into larval (V,) and pupal (Po) fat body proteins of female silkworm at 3 h after isotope treatment. a, b, c: MHP-a, -b, -c. Left: electrophorogram of fat-body proteins. Fig. 8. Autoradiograph (right) showing [14C]-labeled proteins that were released in the hemolymphs of the late larval (V,), pupal (Po and P3), pharated adult (P,) and adult (&) of female silkworm from the third day of the fifth larval instar when isotope was treated. a, b, c: MHP-a, -b, c. Left: electrophorogram of hemolymph proteins. 100 Seong et al Fig. 9. Autoradiograph (right) showing [14C]-labeled proteins that had been transported into the fat body (FB) and midgut (MG) of a female pupa (Po) and mature eggs (EG), from the third day of the fifth larval instar when the labeled amino acid was injected. a, b, c: MHP-a, -b, -c. Left: electrophorogram of fat body (FB), midgut (MG), and mature eggs (EG) proteins. DISCUSSION Three proteins, MHP-a, -b, and -c, have similar characteristics: first, they are present as major hemolymph proteins, especially at the time of larvalpupal transformation; second, their appearance and distribution in the hemolymph and tissues are identical; third, they have relatively small molecular weights. They differ in their mobility in electrophorogram and in molecular weights from storage proteins and vitellogenin [15,20]. Moreover, they are entirely different from storage proteins, other hemolymph proteins 1151, and vitellogenin, a precursor protein of yolk in Bombyx mori [20,21]. Mobilities of MHPs on acrylamide gel electrophorogram are much faster than those of storage proteins and vitellogenin (Fig. 1).The low-molecular-weight proteins, reported in the hemolymph of Bombyx mori, are probably identical to MHPs. Izumi et a1  have reported ”30K proteins,” which are major plasma proteins in last-instar silkworm larvae and also in oocytes, in large quantities comparable to the amount of vitellin. They have further indicated that the biosynthesis of 30K proteins in the fat body is regulated during the development at the pretranslational level. Gamo  has studied three kinds of lipoproteins with low molecular weights in the silkworm hemolymph. The developmental profiles of these lipoproteins in the hemolymph are very similar to those of MHPs, showing stage specificity from the late larval to the middle period of the pharate adult. We have summarized in Figure 11 the appearance, distribution, and depletion of MHPs in the hemolymph and tissues during the ontogenetic development of the silkworm. The MHPs first Fate of Major Hemolymph Proteins 101 1€ 12 Y c w 8 i i E 10 20 30 MOLECULAR WEIGHT x ~ o - ' Fig. 10. The slope-molecular weight relation of standard proteins and MHPs. 1) Chymotrypsinogen (k = 4.3); 2) bovine serum albumin (BSA) (monomer; k = 6.5); 3) BSA (dimer; k = 10.4); 4) BSA (trimer; k = 14.6); 5) aldolase (k = 10.6). a) MHP-a (k = 4.3); b) MHP-b (k = 4.4); c) MHP-c (k = 3.8). Molecular weights of standard proteins are presented in Materials and Met hods. HL FB MG TS ov EG ---. -- -. - --. -. --- 102 Seong et al appeared in the hemolymph and the fat body during the middle period of the final larval instar and accumulated in the hemolymph. At the onset of metamorphosis, the MKPs were also detected in other tissues such as the midgut and gonad. After larval-pupal ecdysis, the MHPs in the hemolymph decreased and their concentrations in the ovary increased. The MHPs in the tissues, except for the ovary, vanished at adult emergence, but those in the eggs were retained. Finally, the MHPs in the eggs disappeared with embryogenesis. Autoradiographic studies showed that at least two MHPs might be synthesized in the fat body and secreted into the hemolymph at the early period of the fifth larval instar as in the case with other hemolymph proteins. The larval fat body is believed to be the main synthesizing site for hemolymph proteins [24-261. But further establishment whether or not synthesis of MHPs in the fat body could be firmly confirmed by in vitro experiment. Translocation of the MHPs during metamorphosis might be suggested by the fact that the radioactivities of the protein bands, which were labeled at the early period of the fifth larval instar, were detected in the fat body, midgut, and ovary during pupal and adult developments. Thus, after being released into the hemolymph from their synthesizing sites at the early period of the fifth larval instar, the MHPs could be entered intact into each tissue at the onset of larval-pupal transformation. The MHPs in the individual tissue might be utilized for adult tissue formation just as storage proteins [ll-151, since these proteins almost disappeared from the hemolymph and most tissues at the onset of pupal-adult transformation. Pate1  also demonstrated the similar phenomena of the synthesis, distribution, transport, and breakdown of specific proteins in specific tissues of the cecropia silkworm, Hyalophoru cecropiu, during development and differentiation. It is noteworthy that the MHPs entered the ovary in greater abundance than in other tissues, and were utilized during embryogenesis together with other yolk proteins, such as vitellogenin. The preferential transport of hemolymph MHPs into the ovary during metamorphosis suggested that these proteins acted as one of the yolk proteins in egg formation. Thus, the MHPs in coincidence with other yolk protein, eg, vitellogenic female-specific protein, were absorbed by the follicle cell and accumulated in the egg yolk . 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