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Storage proteins of the fall webworm Hyphantria cunea drury.

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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 [8]. 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 [9]. 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 [12], 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 [13]. 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 [14]. 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. [15]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 [16]. 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 [17]. 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. [19]. 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 [22].
Collins and Downe [25], using a histochemical method, reported that the
hemolymph protein taken up by the fat body is glycolipoprotein. Kramer et
al. [6] 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 [28] to 92,000 in
Manduca sexta [6].
The PI value of purified SPl was determined to be 5.70. Storage proteins of
Diptera are known to be acidic [3]. 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 [26]. 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 [29] 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 [30]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. [33] 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.
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