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Effect of metamorphosis on the major hemolymph proteins of the silkworm.

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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
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 [2] 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 [4]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.
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 [16]. 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.
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
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 [16]. 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].
['*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
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.
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 [19]. 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
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
[20]. One microliter of hernolymph was applied to each gel comb.
Fate of Major Hernolymph Proteins
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
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
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
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.
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 [16].
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
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.
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
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.
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.
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 [22] 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 [23] 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
i i E
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.
-- -.
- --.
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
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 [4] 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 [27].
From the origin and utilization of the MHPs, we believe that these proteins
can be classified as reserve proteins that are synthesized in the larval stage
and utilized in later developmental stages like the other storage proteins and
vitellogenin. But further investigation is needed to determine and clarify the
functions of MHPs in detail in the individual tissue formation of the adult
insect. Seong [16] has suggested that the MHPs are involved in midgut
differentiation during metamorphosis. Another question is why the MHPs
are not affected by the sex of the insect. Are the excessive hemolymph MHPs
in the male silkworm excreted by catabolism into uric acid at the time of adult
emergence [28,29]?
Fate of Major Hernolyrnph Proteins
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