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Storage proteins, vitellogenin and vitellin of wild silkworms, Antheraea yamamai, Antheraea pernyi and their hybrids
Cornp. Biochem. Physiol. Vol. 106B,No. 1, pp. 163-172, 1993 Printed in Great Britain
0305-0491/93 $6.00+ 0.00 © 1993PergamonPress Ltd
STORAGE PROTEINS, VITELLOGENIN A N D VITELLIN OF WILD SILKWORMS, ANTHERAEA YAMAMAI, ANTHERAEA PERNYI AND THEIR HYBRIDS MARCIA NORIKOYOKOYAMA,ZENTAKAJIURA,MASAONAKAGAKI,RYUZOTAKEI, MASARUKOBAYASHI*and KAZUYUKITANAKA* Laboratory of Silkworm Genetics and Pathology; and *Laboratory of Silkworm Physiology, Faculty of Textile Science and Technology, Shinshu University, Tokida, Ueda-Shi, Nagano-Ken, 386, Japan (Tel. 0268-22-1215 ext. 274; Fax 0268-22-4079) (Received 4 January 1993; accepted 12 February 1993) Abstraet--l. Hemolymph proteins, fat body proteins and ovary proteins of Antheraea yamamai, Antheraea pernyi and their hybrids were analyzed by SDS-PAGE. A 83 kDa peptide increased remarkably in larval hemolymph, but a 75 kDa peptide increased only in the hemolymph of female larvae. 2. Immunoblot analysis using antisera of Bombyx arylphorin and female-specific storage protein showed that the 83 kDa peptide was Antheraea arylphorin, the 75 kDa peptide was Antheraea femalespecific storage protein. 3. We found that a 36 kDa peptide was A. yamamai-specific serum protein, a 34 kDa peptide was A. pernyi-specific serum protein. These two peptides were accumulated in the hemolymph at constant level through larval instar and were detected in hybrids of A. yamamai and A. pernyi. 4. A 185 kDa peptide appeared in the hemolymph of female pharate pupae of A. yamamai and was accumulated in the ovaries of pupae. A 210 kDa peptide appeared in hemolymph of female pharate pupae of A. pernyi. A 170 kDa peptide was accumulated in the ovaries of A. pernyi pupae. These three peptides were related to vitellogenin and vitellin as shown by immunoblot analysis using anti-BmVg serum. It was also found that these three peptides were accumulated in the ovaries of Antheraea F~ hybrids. 5. A new female-specificprotein (24 kDa peptide) was discovered only in the female fat bodies.
INTRODUCTION
Major proteins in hemolymph have been analyzed in several lepidopteran species: Bombyx mori (Tojo et al., 1980), Calpodes ethlius (Palli and Locke, 1987), Galleria mellonella (Ray et al., 1987), Heliothis zea (Haunerland and Bowers, 1986), Hyalophora cecropia (Telfer et al., 1983), Papilio polyxenes (Ryan et al., 1986), Manduca sexta (Ryan et al., 1985), Spodoptera litura (Tojo et al., 1985), Trichoplusia ni (Jones et al., 1988). An increasing number of studies on the classification and characterization of insect hemolymph proteins have revealed important commonalities and differences. Because there is a wealth of information about lepidopteran hemolymph proteins, we can classify a protein that possesses unique chemical and immunological properties. Most insects have an arylphorin and a femalespecific storage protein at least, which are synthesized in larval fat body cells and are sequestrated in fat body cells as a protein granule during larval-pupal metamorphosis. The fat bodies of pupae degrade storage proteins to use them as source of amino acids. The only female fat bodies of pupae synthesize vitellogenin, a yolk protein precursor. The follicle Abbreviations--Ay × Ap = A. yamamai x A. pernyi; Ap x Ay = A. pernyi x A. yamamai; SP1 = storage protein 1; SP2 = storage protein 2; BmVg = Bombyx vitellogenin.
cells of ovaries uptake vitellogenin by endocytosis and storage it as a yolk protein granule. We have attempted to develop a new cross-breed of wild silkworms. In fact, it is difficult to obtain F 2 hybrids. F~ hybrids grow up normally, but have some abnormal features: Kobayashi et al. (1992) showed the abnormal spermatogenesis of reciprocal F~ hybrids, Shimada and Kobayashi (1992) reported that the Ap x Ay ovaries of pupae underwent oogenesis and mature eggs were produced, but most of the Ay x Ap ovaries degenerated during pupal-adult transformation. We have investigated hemolymph proteins and fat body proteins of two wild silkworms, A. yamamai and A. pernyi, and their hybrids. However, information on hemolymph proteins of the two wild silkworms is less than that of the other silkworms such as Bombyx mori and Hyalophora cecropia. There are some reliable reports: blue chromoprotein (Yamada and Kato, 1 9 9 1 ) and vitellogenin (Furusawa, 1991) of A. yamamai, and lectin of A. pernyi (Qu et al., 1987). We analyzed immunologically Antheraea arylphorin, Antheraea female-specific storage protein and Antheraea vitellogenin. Furthermore, we discovered two species-specific proteins in hemolymph (Ay 36kDa, Ap 34kDa), and a female-specific protein (24 kDa) in fat bodies. SDSpolyacrylamide gel electrophoretic analysis and
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MARCIANORIKOYOKOYAMAet al.
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immunoblot analysis have showed that A. yamamaispecific proteins and ,4. pernyi-specific proteins were coordinately accumulated in their hybrids. MATERIALS AND METHODS
Animals We used strains of Antheraea yamamai (Ay) and A. pernyi (Ap) which had been stored in our laboratory. Their hybrids (Ay x Ap, Ap × Ay) were provided by
a cross according to the method of Kobayashi et al. (1992). Larvae were reared on an artificial diet (Yakult Co., Tokyo, Japan) until the fourth instar. After the third ecdysis they were reared on the leaves of the Japanese oak, Quercus acutissima, at room temperature under a natural photoperiod. Antheraea pernyi diapauses in pupal stage. The diapausing pupae were stored at 5°C for 30 days to break diapause, and then incubated at 25°C for 15 days until adult emergence.
A V 0
3 6
PP P 9 12 M F M F
k Do 235 185 145 83 75
44 36
B
~ - 83 ~ - 75 --
58
--45
Fig. 1. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of Antheraea yamamai shown by SDS-polyacrylamide gel electrophoresis. Hemolymph and fat bodies were collected every two days from day 0 to day 12 of last larval instar, pharate pupae and day 0 of pupae. Each 40 #g of proteins was resolved on SDS-polyacrylamide gels. V shows the fifth instar, PP and P correspond to pharate pupae and pupation, respectively. M and F represent male and female preparations.
Analysis of major proteins in wild silkworms
165
A V 0
3
6
PP 9 12 M F
P M
F
kDcs ~-- 235 210 ""- 14,5 l# 83 ~---- 75
~--51
~--34
B
-83 "75 58 "45
-24 Fig. 2. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of Antheraea pernyi shown by SDS-polyacrylamidegel electrophoresis. Hemolymph and fat bodies were collected as described in Fig. 1. Each 40 #g of proteins was resolved on SDS-polyacrylamide gels. Symbols used are the same as those in Fig. 1.
Tissue preparation Larvae were anesthetized on ice. Their forelegs were cut, and then hemolymph was collected in ice cold test tubes and mixed with nine volumes of phosphate-buffered saline (10 mM phosphate buffer at pH 7.0 with 0.15 M NaC1) containing a few crystals of phenylthiourea. The hemocytes in the mixture were removed by centrifugation at 10,000 rpm for 10 min. The supernatants were stored at -20°C. Fat bodies were dissected from the bled larvae in ice-cold saline. After being rinsed with distilled water, the fat bodies were weighed and immediately homogenized
with 10 volumes of phosphate-buffered saline. The homogenates were centrifuged at 10,000rpm for 10 rain. The supernatants were stored at - 2 0 ° C until the experiments were conducted (Kajiura and Yamashita, 1989). The ovaries of A. yamamai were collected at day 20 of pupal stage. The ovaries of A. pernyi were collected after diapause was broken as described above. Those of Ap x Ay were collected just before adult emergence. After being washed, the collected ovaries were homogenized with 10mM Tris-HCl buffer (6 M urea, 2% SDS, 0.35 M NaC1, I mM EDTA, pH 8.0). The homogenate was cen-
MARCIA NORIKO YOKOYAMAet al.
166
trifuged at 10,000 rpm for 10 min. The supernatant was stored at -20°C.
stacking gel and a 10% separating gel. Peptides were stained with 0.025% Coomassie Brilliant Blue R-250.
Determination of protein concentration
Purification of 83 kDa peptide The 83 kDa A. pernyi peptide was purified from 15 ml of hemolymph of the fifth instar larvae (day 12) by column chromatography: gel filtration with cellulofine GCL-1000 and cellulofine GCL-300 (Seikagaku Co., Tokyo, Japan), anion exchange chromatography with DE-52 (Whatman International Ltd., Maidstone, England) and adsorptive chromatography with hydroxyapatite (Seikagaku Co., Tokyo, Japan) (Yokoyama et al., unreported data).
Total proteins were collected as precipitates by 5% trichloroacetic acid. The precipitates were dissolved with 0.1 N NaOH containing 2% Na2CO3. The amount of protein was measured according to the method of Lowry et al. (1951) using bovine serum albumin as a standard.
SDS-polyacrylamide gel electrophoresis Diluted hemolymph were mixed with an equal volume of SDS-sample buffer (62.5mM Tris-HC1 buffer, pH 6.8, containing 1% SDS and 2% 2-mercaptoethanol), and homogenates of fat body were mixed with two volumes of SDS-sample buffer (Laemmli, 1970). The mixture was boiled for I min and then subjected to S D S - P A G E using a 6.0%
Immunoblot analysis Immunoblot analysis was carried out according to the method of Burnette (1981). Antisera of Bombyx arylphorin, Bombyx female-specific protein and
A V 0 M F
3 6 9 MFMFMFM
i~lllD
S PP P 12 F M F M FM F
kDo
"--210 ~"- 185 ""-145 + - 83 75 51 .~--44 ,, 36 +-34
B
Fig. 3. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of A. yamamai x A. pernyi (Ay x Ap) shown by SDS-polyacrylamide gel electrophoresis. Hemolymph and fat bodies were collected as described in Fig. 1. Each 40/~g of proteins was resolved on SDS-polyacrylamide gels. S represents spinning and the other symbols used are the same as those in Fig. 1.
167
Analysis of major proteins in wild silkworms
A
V 0 3 6 MFMFMFMFMF
S 9
12
PP
MFMFMF
P kD. 210 ~ . . 185 145 "~- 83 ~"75
~--
~-
51
..--4/, -~36 ~-- 34
B
--~-~~-
83 75
58 45
2t, Fig. 4. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of A. pernyi x A. yamamai (Ap x Ay) shown by SDS-polyacrylamide gel electrophoresis. Hemolymph and fat bodies were collected as described in Fig. I. Each 40 #g of proteins was resolved on SDS--polyacrylamidegels. Symbols used are the same as those in Fig. 3.
purified 83 kDa peptide were prepared according to the method of Kajiura and Yamashita (1989). Antiserum of Bombyx vitellogenin (BmVg) was kindly given by Dr Yamashita (Nagoya University, Japan). After electrophoresis on 10% SDSpolyacrylamide gels, peptides were electroblotted onto a nitrocellulose sheet in blotting buffer [100 mM Tris, 192mM glycine and 5% (v/v) methanol] for 60min at 2mA/cm 2 using Horizblot (Atto Co., Tokyo, Japan). The electroblotted nitrocellulose sheets were incubated for 15min in phosphatebuffered saline (PBS) and then incubated for 60 min with antisera diluted with 1:5000 in PBS containing 2% skim milk. After being washed with PBS/skim milk three times, the nitrocellulose sheets were incubated for 60 min with a 1:5000 dilution of peroxidase-conjugated goat anti-rabbit IgG in PBS/ skim milk. The bound antibodies were visualized with a Konika immunostaining HRP kit IS-50B (Konika, Tokyo, Japan) or a POD immunostain CBPB 106/I--L
set (Wako Pure Chemical Industries, Ltd, Osaka, Japan). RESULTS
Developmental changes in hemolymph peptides andfat body peptides Hemolymph peptides and fat body peptides of A.
yamamai were collected from day 0 of the last larval instar to the early pupal stage, and were analyzed by SDS-PAGE (Fig. 1). Five major peptides (235 kDa, 83 kDa, 75 kDa, 51 kDa and 36 kDa) were found in the hemolymph until the pharate pupal stage. The other three peptides (185 kDa, 145 kDa and 44 kDa) were detected at the pharate pupal stage (Fig. IA). The amounts of 235 kDa, 51 kDa and 36 kDa peptides in the hemolymph did not change very much through the larval instar, but those of 83 kDa peptide and 75 kDa peptide increased remarkably during the feeding stage and decreased at the early stage of
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MARCIANORIKOYOKOYAMAet al.
pupae. The 185 kDa and 44 kDa peptides appeared in the female hemolymph of pharate pupal stage (Fig. 1A). Fat body peptides consisted of four major peptides (83 kDa, 75 kDa, 58 kDa and 45 kDa) and many minor ones. There were a lot of 58 kDa peptide and 45 kDa peptide in larval fat bodies. After spinning, the 83 kDa and 75 kDa peptides were mainly accumulated in the fat bodies (Fig. 1B), and concurrently declined in the hemolymph (Fig. 1A). In A. pernyi, six major peptides (235 kDa, 145 kDa, 83 kDa, 75 kDa, 51 kDa and 34 kDa) were found in the hemolymph until the pharate pupal stage. The amount of 210 kDa peptide fluctuated through the fifth instar, and increased in the hemolymph of female pharate pupae (Fig. 2A). The peptides of 235 kDa, 145 kDa, 83 kDa and 75 kDa were detected in both A. yamamai and A. pernyi. However,
A
Hemolymph Bm Ay Ap M F M F M F ~
BmSP 1 =-
~C ¸
.
.
.
.
.
.
.
.
.
.
185 kDa, 44 kDa and 36 kDa peptides were specific to A. yamamai, while 210 kDa and 34 kDa peptides were specific to A. pernyi. The developmental changing patterns of 235 kDa, 83 kDa and 75 kDa peptides were the same between the two wild silkworms. Fat body peptides of A. pernyi consisted of five major peptides (83 kDa, 75 kDa, 58 kDa, 45 kDa and 24kDa) and many minor ones. 83 kDa, 75 kDa, 58 kDa and 45 kDa peptides were detected in the fat bodies of A. pernyi as well as in those of A. yamamai. Their developmental profiles matched those of the two wild silkworms. A 24 kDa peptide increased in the only female fat bodies from day 9 of the last larval instar. This peptide was a fat body female-specific peptide (Fig. 2B). Hemolymph and fat body peptides of Ay x Ap were analyzed by SDS-PAGE (Fig. 3). The 83 kDa
Fat Body Bm Ay Ap M F M F M F
kDa
.
~- 75
B
BmSP2,-
~-
83
Fig. 5. Immunological identification of Antheraea arylphorin and Antheraea female specific storage protein. Hemolymph proteins and fat body proteins of Bombyx mori (Bin) were collected from day 7 of the fifth instar, those of Antheraea yamamai (Ay) and Antheraea pernyi (Ap) were collected from day 12 of the fifth instar. After SDS-polyacrylamide gel eleetrophoresis, each 30/zg of proteins was transferred to nitrocellulose filters. The nitrocellulose filters were reacted with antiserum against Bombyx femalespecific storage protein (A) and against Bombyx arylphorin (B).
169
Analysis of major proteins in wild silkworms
83k Ap Ay Bm
Ap83k
"
Fig. 6. Immunological relationship between Antheraea arylphorin and Bombyx arylphorin. Hemolymph proteins of Antheraea yamamai, Antheraea pernyi and Bombyx mori, and purified 83 kDa peptide of Antheraea pernyi were resolved on SDS--polyacrylamide gel electrophoresis, and subjected to immunoblot analysis with antiserum against purified 83 kDa peptide ofA. pernyi. Each 30/~g of proteins was applied on each lane. Lane 1: purified 83kDa peptide. Lane 2: hemolymph proteins of A. pernyi. Lane 3: hemolymph proteins of A. yamamai. Lane 4: hemolymph proteins of B. mori. peptide increased in the larval hemolymph of both females and males, while the 75 kDa peptide increased specifically in the female larval hemolymph from day 9 of the fifth instar. A 36 kDa A. yamamaispecific and a 34 kDa A. pernyi-specific peptide were detected in the hemolymph of the hybrid silkworm larvae. A 210 kDa A. pernyi female-specific peptide, a 185 kDa and a 44 kDa A. yamamai female-specific peptide increased in the hemolymph of female pharate pupae (Fig. 3A). The peptides of 83 kDa and 75 kDa in the fat bodies were slightly stained with Coomassie Brilliant Blue through the last larval instar, but strongly stained after spinning. A 24 kDa female-specific peptide was detected in the fat bodies after day 9 of the fifth instar. Developmental profiles of the other fat body peptides were the same as those of their parents (Fig. 3B). Figure 4 shows SDS-polyacrylamide gel electrophoretic analyses of hemolymph and fat bodies of Ap x Ay. The developmental profiles of the other hemolymph and fat body peptides of Ap x Ay were almost the same as those of Ay x Ap (Fig. 3). Storage proteins Bombyx mori has two storage proteins: storage protein 1 (SPI) is a hexamer composed of a methionine-rich 85 kDa subunit, and storage protein 2 (SP2) is a kind of arylphorin and also a hexamer composed of aromatic amino acid-rich two subunits (Kanost et al., 1990). The developmental profile of 83 kDa and of 75 kDa peptides in the hemolymph of wild silkworms corresponded to that of SP2 and of SP1, respectively. To determine whether the peptides of 83 kDa and 75 kDa are related to storage proteins or not, we analyzed them by immunoblot analysis with antisera of SP1 and SP2 (Fig. 5). On the one hand, anti-SP1 serum reacted to the 75 kDa peptide in the larval hemolymph and the fat bodies of the only female Antheraeas (Fig. 5A). On the other hand, anti-SP2 serum reacted unequivocally to the 83 kDa peptide in
the larval hemolymph and the pupal fat bodies of Antheraeas (Fig. 5B). Consequently, the 83 kDa peptide was immunologically related to arylphorin, while the 75 kDa peptide was immunologically related to a female-specific storage protein. Immunoblot analysis was also carried out with antiserum raised against 83 kDa Antheraea peptide (Fig. 6). The antiserum reacted obviously to 83 kDa peptide of A. yamamai and A. pernyi, but did not react to SP2. Vitellogenin and vitellin The 185 kDa A. yamamai peptide could be detected in the hemolymph and the ovaries by SDS-PAGE (Fig. 7). A 210 kDa A. pernyi peptide was major in the female hemolymph but minor in the ovaries. Instead of the 210 kDa peptide, a 170 kDa peptide was major in the ovaries of A. pernyi. The peptides of 185kDa A. yamamai, 170kDa and 210kDa A. pernyi were accumulated in the ovaries o f A p x Ay (Fig. 7A). To identify these peptides with vitellogenin or vitellin, we used an immunoblot analysis with antiBmVg serum (Fig. 7B). The 185kDa A. yamamai peptide in the hemolymph reacted to anti-BmVg serum, as did in the ovaries. The 210 kDa A. pernyi peptide in the hemolymph reacted to anti-BmVg serum. In the ovaries of A. pernyi, however, the 170 kDa peptide reacted to anti-BmVg serum. Furthermore, the three peptides were detected in the ovaries of Ap x Ay. Therefore, the peptides of 185 kDa A. yamamai and 210 kDa A. pernyi were immunologically related to vitellogenin, the 170 kDa A. pernyi peptide was related to vitellin. Furusawa (1991) reported A. yamamai vitellin composed of two subunits (180kDa and 40kDa). Figure 7 shows two peptides (45 kDa and 44 kDa) whose mobility was similar as of the 40 kDa subunit. However, neither the 45 kDa nor the 44 kDa peptide reacted with anti-BmVg serum, so that we could not identify the counterpart subunit of Antheraea vitellogenin and vitellin (Fig. 7B). DISCUSSION To investigate the physiological aspects of A. yamamai, A. pernyi and their hybrids, we analyzed hemolymph, fat body and ovary proteins by SDS-PAGE (Figs 1, 2, 3, 4 and 7). The peptides of 36 kDa A. yamamai and 34 kDa A. pernyi were found in the hemolymph of reciprocal Fl hybrids, and the peptides of 185kDa A. yamamai, 210kDa and 170 kDa A. pernyi were accumulated in the ovaries of these hybrids. These facts indicate that the F~ hybrids synthesize proteins of both parents without dominance. Arylphorins have been identified in all species that have been carefully examined, and in many cases immunological cross-reactivity has been observed over wide phylogenetic distances (Ryan et al., 1984).
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MARCIA NORIKO YOKOYAMAet al.
Telfer et al. (1983) showed also an immunological relationship between Hyalophora and Manduca arylphorin. The 83 kDa Antheraea peptide increased remarkably in the hemolymph of feeding larvae, declined after spinning, but increased in fat body concurrently. This peptide showed similar electrophoretic mobility to that of Bombyx SP2, and it was immunologically related to Bombyx arylphorin (Fig. 5). Antheraea peptide reacted to anti-SP2 serum, but Bombyx SP2 did not react to antibodies against
A Bm
Ay
the 83 kDa peptide (Fig. 6). These results indicate that Antheraea and Bombyx arylphorin not only have common antigenic sites but also species-specific antigenic sites, as did Samia and Bombyx arylphorin (Shimada et al., 1987). The 75 kDa Antheraea peptide moved faster than the 85 kDa Bombyx subunit of SP1, but was immunologically related to SP1 (Fig. 5). Interesting differences arise when we compared Antheraea with other Lepidopteran species. In Antheraeas the synthesis of arylphorins occurs in early
Ap
Ap,.Ay kDa -.,--210 "'-185
~'170
~-- 83 +- 75 ~.. 6 5
60 --,-- 45 " - - 44 4--34
B 4--- 210 170
Fig. 7. Identification of Antheraea vitellogenin and Antheraea vitellin. SDS-polyacrylamide gel electrophoretic analysis (A) and immunoblot analysis using anti-BmVg serum (B). Hemolymph proteins were collected from day 0 of female pupae, and ovaries proteins were collected from female pupae just before adult emergence. Each 30/zg of proteins was applied to each lane of polyacrylamide gels. H and O represent hemolymph and ovary, respectively.
Analysis of major proteins in wild silkworms larval instars as well as in the ultimate instar (data not shown). It was also observed in Bombyx mori (Izumi et al., 1980), Manduca sexta (Riddiford and Hice, 1985) and Galleria mellonella (Ray et al., 1987). During larval-pupal transformation arylphorins of Bombyx and Galleria are mostly taken up by the fat bodies along with other hemolymph storage proteins. Antheraea arylphorin remains partly in the hemolymph, but the rest of arylphorin is sequestrated in the fat bodies during larval-pupal transformation (Figs 1, 2, 3 and 4). A large amount of arylphorin also remain in the hemolymph of Hyalophora and Manduca after wandering (Tojo et al., 1978; Telfer et al., 1983). The amount of 75 kDa Antheraea peptide increased only in the final instar (Figs 3 and 4) as did the Manduca methionine-rich storage protein (Riddiford and Hice, 1985), while in Bombyx mori it occurs in earlier instars (Izumi et al., 1988). Figure 7 shows that the 185kDa peptide was A. yamamai vitellogenin, and that the 210 kDa and 170 kDa peptides were A. pernyi vitellogenin and vitellin, respectively. Nevertheless, we could not identify the counterpart subunit of vitellogenin by immunoblot analysis. Antheraea pernyi vitellogenin and vitellin showed different gel electrophoretic mobility. The cause of the molecular heterogeneity of vitellogenin and vitellin remains to be determined. Differences exist between A. yamamai and A. pernyi with respect to a kind of peptides in the larval hemolymph. We found that a 34 kDa peptide was A. pernyi-specific in the hemolymph, whereas a 36 kDa peptide was A. yamamai-specific (Figs 1-4). These peptides were detected in the hemolymph from the first instar (data not shown). This is a feature of larval serum proteins. But they were not immunoreactable to antisera raised against 30 k proteins (Izumi et al., 1980) and against B. mori larval serum proteins (BmLSP) (Fujiwara and Yamashita, 1990) (data not shown). A 24kDa peptide is a fat body female-specific peptide, a new class of insect protein. There is little information about female fat body specific protein of other insects. We are now extending our studies to purification, characterization and molecular cloning of the 24 kDa peptide. Studies of this protein will develop our understanding about the development and differentiation of fat body. Acknowledgements--We wish to thank Dr O. Yamashita of Nagoya University for providing antiserum of BmVg, and Dr K. Kimura of Shinshu University for his technical assistance. The present work was supported in part by a Research grant from the Ministry of Education, Scienceand Culture, Japan. REFERENCES
Burnette W. N. (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. Biochem. 112, 195-203.
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Fujiwara Y. and Yamashita O. (1990) Purification, characterization and developmental changes in the titer of a new larval serum protein of the silkworm, Bombyx mori. Insect Biochem. 20, 751-759. Furusawa T. (1991) Carbohydrate metabolism and limited degradation of vitellin in the eggs of the Japanese oak silkworm, Antheraea yamamai, during diapause and embryonic development. In Wild Silkmoths '89 '90 (Edited by Akai H. and Kiuchi M.), pp. 81-88. Haunerland N. H. and Bowers W. S. (1986) Arylphorin from the corn earworm, Heliothis zea. Insect Biochem. 16, 617-625. Izumi S., Tojo S. and Tomino S. (1980) Translation of fat body mRNA from the silkworm, Bombyx mori. Insect Biochem. 10, 429-434. Izumi S., Sakurai H., Fujii T., Ikeda W. and Tomino S. (1988) Cloning of mRNA sequencecoding for sex-specific storage protein of Bombyx mori. Biochem. biophys. Acta 949, 181-188. Jones G., Hiremath S. T., Hellmann G. M. and Rhoads R. E. (1988) Juvenile hormone regulation of mRNA levels for a highly abundant hemolymph protein in larval Trichoplusia hi. J. biol. Chem. 263, 1089-1092. Kajiura Z. and Yamashita O. (1989) Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog. Arch. Insect Biochem. Physiol. 12, 99-109. Kanost M. R., Kawooya J. K., Ryan R. O., Hensden M. C. V. and Ziegler R. (1990) Insect haemolymph proteins. In Advances in Insect Physiology (Edited by Evans P. D. and Wigglesworth V. B.), Vol. 22, pp. 299-396. Kobayashi M., Yonekawa M., Yokoyama M. N., Nakagaki M. and Tanaka K. (1992) Ultramorphological characterization of spermatogenesis of interspecifichybrid (F l ) of Antheraea yamamai and Antheraea pernyi. Jap. J. appl. Entomol. Zool. 36, 231-237. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 337, 680-685. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-273. Palli S. R. and Locke M. (1987) Purification and characterization of three major hemolymph proteins of an insect Calpodes ethlius (Lepidoptera; Hesperiidae). Arch. Insect Biochem. Physiol. 5, 233-244. Qu X., Zhang C., Komano H. and Natori S. (1987) Purification of a lectin from the hemolymph of Chinese oak silkmoth (Antheraea pernyi) pupae. J. Biochem. 101, 545-551. Ray A., Memmel N. A. and Kumaran A. K. (1987) Developmental regulation of the larval hemolymph protein genes in Galleria mellonella. Roux's Arch. Dev. Biol. 196, 414-420. Riddiford L. M. and Hice R. H. (1985) Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta. Insect Biochem. 15, 489-502. Ryan R. O., Wang X. Y., Willot E. and Law J. H. (1986) Major hemolymph proteins from larvae of the black swallowtail butterfly, Papilio polyxenes. Archs Insect Biochem. PhysioL 3, 539-550. Ryan R. O., Keim P. S., Wells M. A. and Law J. H. (1985) Purification and properties of a predominantly female specific protein from the hemolymph of the larva of the tobacco hornworm, Manduca sexta. J. biol. Chem. 260, 782-786. Shimada T., Nagata M. and Yoshitake N. (1987) Characterization of arylphorin of the Eri-silkmoth, Samia cynthia ricini (Donovan) (Lepidoptera: Saturniidae). Appl. Ent. Zool. 22, 543-552.
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Shimada T. and Kobayashi M. (1992) Fertility of F~ hybrids between Antheraea yamamai (Guerin-Meneville) and Antheraea pernyi (G.-M.). In Wild Silkmoths '91 (Edited by Akai H., Kato Y., Kiuchi M. and Kobayashi J.), pp. 187-195. Telfer W. H., Keim P. S. and Law J. H. (1983) Arylphorin, a new protein from Hyalophora cecropia: comparisons with calliphorin and manducin. Insect Biochem. 13, 601-613. Tojo S., Betchaku T., Ziccardi V. J. and Wyatt G. R. (1978) Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia. J. cell. Biol. 78, 823-838.
Tojo S., Nagata M. and Kobayashi M. (1980) Storage proteins in the silkworm, Bombyx mori. lnsect Biochem. 10, 289-303. Tojo S., Morita M., Agui N. and Kimura K. (1985) Hormonal regulation of phase polymorphism and storage protein fluctuation in the common cutworm, Spodoptera litura. J. Insect Physiol. 31, 283-292. Yamada H. and Kato H. (1991) Characteristic properties of a blue chromoprotein in larval hemolymph of Antheraea yamamai. In Wild Silkmoths '89 '90 (Edited by Akai H. and Kiuchi M.), pp. 89--95.
0305-0491/93 $6.00+ 0.00 © 1993PergamonPress Ltd
STORAGE PROTEINS, VITELLOGENIN A N D VITELLIN OF WILD SILKWORMS, ANTHERAEA YAMAMAI, ANTHERAEA PERNYI AND THEIR HYBRIDS MARCIA NORIKOYOKOYAMA,ZENTAKAJIURA,MASAONAKAGAKI,RYUZOTAKEI, MASARUKOBAYASHI*and KAZUYUKITANAKA* Laboratory of Silkworm Genetics and Pathology; and *Laboratory of Silkworm Physiology, Faculty of Textile Science and Technology, Shinshu University, Tokida, Ueda-Shi, Nagano-Ken, 386, Japan (Tel. 0268-22-1215 ext. 274; Fax 0268-22-4079) (Received 4 January 1993; accepted 12 February 1993) Abstraet--l. Hemolymph proteins, fat body proteins and ovary proteins of Antheraea yamamai, Antheraea pernyi and their hybrids were analyzed by SDS-PAGE. A 83 kDa peptide increased remarkably in larval hemolymph, but a 75 kDa peptide increased only in the hemolymph of female larvae. 2. Immunoblot analysis using antisera of Bombyx arylphorin and female-specific storage protein showed that the 83 kDa peptide was Antheraea arylphorin, the 75 kDa peptide was Antheraea femalespecific storage protein. 3. We found that a 36 kDa peptide was A. yamamai-specific serum protein, a 34 kDa peptide was A. pernyi-specific serum protein. These two peptides were accumulated in the hemolymph at constant level through larval instar and were detected in hybrids of A. yamamai and A. pernyi. 4. A 185 kDa peptide appeared in the hemolymph of female pharate pupae of A. yamamai and was accumulated in the ovaries of pupae. A 210 kDa peptide appeared in hemolymph of female pharate pupae of A. pernyi. A 170 kDa peptide was accumulated in the ovaries of A. pernyi pupae. These three peptides were related to vitellogenin and vitellin as shown by immunoblot analysis using anti-BmVg serum. It was also found that these three peptides were accumulated in the ovaries of Antheraea F~ hybrids. 5. A new female-specificprotein (24 kDa peptide) was discovered only in the female fat bodies.
INTRODUCTION
Major proteins in hemolymph have been analyzed in several lepidopteran species: Bombyx mori (Tojo et al., 1980), Calpodes ethlius (Palli and Locke, 1987), Galleria mellonella (Ray et al., 1987), Heliothis zea (Haunerland and Bowers, 1986), Hyalophora cecropia (Telfer et al., 1983), Papilio polyxenes (Ryan et al., 1986), Manduca sexta (Ryan et al., 1985), Spodoptera litura (Tojo et al., 1985), Trichoplusia ni (Jones et al., 1988). An increasing number of studies on the classification and characterization of insect hemolymph proteins have revealed important commonalities and differences. Because there is a wealth of information about lepidopteran hemolymph proteins, we can classify a protein that possesses unique chemical and immunological properties. Most insects have an arylphorin and a femalespecific storage protein at least, which are synthesized in larval fat body cells and are sequestrated in fat body cells as a protein granule during larval-pupal metamorphosis. The fat bodies of pupae degrade storage proteins to use them as source of amino acids. The only female fat bodies of pupae synthesize vitellogenin, a yolk protein precursor. The follicle Abbreviations--Ay × Ap = A. yamamai x A. pernyi; Ap x Ay = A. pernyi x A. yamamai; SP1 = storage protein 1; SP2 = storage protein 2; BmVg = Bombyx vitellogenin.
cells of ovaries uptake vitellogenin by endocytosis and storage it as a yolk protein granule. We have attempted to develop a new cross-breed of wild silkworms. In fact, it is difficult to obtain F 2 hybrids. F~ hybrids grow up normally, but have some abnormal features: Kobayashi et al. (1992) showed the abnormal spermatogenesis of reciprocal F~ hybrids, Shimada and Kobayashi (1992) reported that the Ap x Ay ovaries of pupae underwent oogenesis and mature eggs were produced, but most of the Ay x Ap ovaries degenerated during pupal-adult transformation. We have investigated hemolymph proteins and fat body proteins of two wild silkworms, A. yamamai and A. pernyi, and their hybrids. However, information on hemolymph proteins of the two wild silkworms is less than that of the other silkworms such as Bombyx mori and Hyalophora cecropia. There are some reliable reports: blue chromoprotein (Yamada and Kato, 1 9 9 1 ) and vitellogenin (Furusawa, 1991) of A. yamamai, and lectin of A. pernyi (Qu et al., 1987). We analyzed immunologically Antheraea arylphorin, Antheraea female-specific storage protein and Antheraea vitellogenin. Furthermore, we discovered two species-specific proteins in hemolymph (Ay 36kDa, Ap 34kDa), and a female-specific protein (24 kDa) in fat bodies. SDSpolyacrylamide gel electrophoretic analysis and
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MARCIANORIKOYOKOYAMAet al.
164
immunoblot analysis have showed that A. yamamaispecific proteins and ,4. pernyi-specific proteins were coordinately accumulated in their hybrids. MATERIALS AND METHODS
Animals We used strains of Antheraea yamamai (Ay) and A. pernyi (Ap) which had been stored in our laboratory. Their hybrids (Ay x Ap, Ap × Ay) were provided by
a cross according to the method of Kobayashi et al. (1992). Larvae were reared on an artificial diet (Yakult Co., Tokyo, Japan) until the fourth instar. After the third ecdysis they were reared on the leaves of the Japanese oak, Quercus acutissima, at room temperature under a natural photoperiod. Antheraea pernyi diapauses in pupal stage. The diapausing pupae were stored at 5°C for 30 days to break diapause, and then incubated at 25°C for 15 days until adult emergence.
A V 0
3 6
PP P 9 12 M F M F
k Do 235 185 145 83 75
44 36
B
~ - 83 ~ - 75 --
58
--45
Fig. 1. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of Antheraea yamamai shown by SDS-polyacrylamide gel electrophoresis. Hemolymph and fat bodies were collected every two days from day 0 to day 12 of last larval instar, pharate pupae and day 0 of pupae. Each 40 #g of proteins was resolved on SDS-polyacrylamide gels. V shows the fifth instar, PP and P correspond to pharate pupae and pupation, respectively. M and F represent male and female preparations.
Analysis of major proteins in wild silkworms
165
A V 0
3
6
PP 9 12 M F
P M
F
kDcs ~-- 235 210 ""- 14,5 l# 83 ~---- 75
~--51
~--34
B
-83 "75 58 "45
-24 Fig. 2. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of Antheraea pernyi shown by SDS-polyacrylamidegel electrophoresis. Hemolymph and fat bodies were collected as described in Fig. 1. Each 40 #g of proteins was resolved on SDS-polyacrylamide gels. Symbols used are the same as those in Fig. 1.
Tissue preparation Larvae were anesthetized on ice. Their forelegs were cut, and then hemolymph was collected in ice cold test tubes and mixed with nine volumes of phosphate-buffered saline (10 mM phosphate buffer at pH 7.0 with 0.15 M NaC1) containing a few crystals of phenylthiourea. The hemocytes in the mixture were removed by centrifugation at 10,000 rpm for 10 min. The supernatants were stored at -20°C. Fat bodies were dissected from the bled larvae in ice-cold saline. After being rinsed with distilled water, the fat bodies were weighed and immediately homogenized
with 10 volumes of phosphate-buffered saline. The homogenates were centrifuged at 10,000rpm for 10 rain. The supernatants were stored at - 2 0 ° C until the experiments were conducted (Kajiura and Yamashita, 1989). The ovaries of A. yamamai were collected at day 20 of pupal stage. The ovaries of A. pernyi were collected after diapause was broken as described above. Those of Ap x Ay were collected just before adult emergence. After being washed, the collected ovaries were homogenized with 10mM Tris-HCl buffer (6 M urea, 2% SDS, 0.35 M NaC1, I mM EDTA, pH 8.0). The homogenate was cen-
MARCIA NORIKO YOKOYAMAet al.
166
trifuged at 10,000 rpm for 10 min. The supernatant was stored at -20°C.
stacking gel and a 10% separating gel. Peptides were stained with 0.025% Coomassie Brilliant Blue R-250.
Determination of protein concentration
Purification of 83 kDa peptide The 83 kDa A. pernyi peptide was purified from 15 ml of hemolymph of the fifth instar larvae (day 12) by column chromatography: gel filtration with cellulofine GCL-1000 and cellulofine GCL-300 (Seikagaku Co., Tokyo, Japan), anion exchange chromatography with DE-52 (Whatman International Ltd., Maidstone, England) and adsorptive chromatography with hydroxyapatite (Seikagaku Co., Tokyo, Japan) (Yokoyama et al., unreported data).
Total proteins were collected as precipitates by 5% trichloroacetic acid. The precipitates were dissolved with 0.1 N NaOH containing 2% Na2CO3. The amount of protein was measured according to the method of Lowry et al. (1951) using bovine serum albumin as a standard.
SDS-polyacrylamide gel electrophoresis Diluted hemolymph were mixed with an equal volume of SDS-sample buffer (62.5mM Tris-HC1 buffer, pH 6.8, containing 1% SDS and 2% 2-mercaptoethanol), and homogenates of fat body were mixed with two volumes of SDS-sample buffer (Laemmli, 1970). The mixture was boiled for I min and then subjected to S D S - P A G E using a 6.0%
Immunoblot analysis Immunoblot analysis was carried out according to the method of Burnette (1981). Antisera of Bombyx arylphorin, Bombyx female-specific protein and
A V 0 M F
3 6 9 MFMFMFM
i~lllD
S PP P 12 F M F M FM F
kDo
"--210 ~"- 185 ""-145 + - 83 75 51 .~--44 ,, 36 +-34
B
Fig. 3. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of A. yamamai x A. pernyi (Ay x Ap) shown by SDS-polyacrylamide gel electrophoresis. Hemolymph and fat bodies were collected as described in Fig. 1. Each 40/~g of proteins was resolved on SDS-polyacrylamide gels. S represents spinning and the other symbols used are the same as those in Fig. 1.
167
Analysis of major proteins in wild silkworms
A
V 0 3 6 MFMFMFMFMF
S 9
12
PP
MFMFMF
P kD. 210 ~ . . 185 145 "~- 83 ~"75
~--
~-
51
..--4/, -~36 ~-- 34
B
--~-~~-
83 75
58 45
2t, Fig. 4. Developmental profiles of hemolymph proteins (A) and fat body proteins (B) of A. pernyi x A. yamamai (Ap x Ay) shown by SDS-polyacrylamide gel electrophoresis. Hemolymph and fat bodies were collected as described in Fig. I. Each 40 #g of proteins was resolved on SDS--polyacrylamidegels. Symbols used are the same as those in Fig. 3.
purified 83 kDa peptide were prepared according to the method of Kajiura and Yamashita (1989). Antiserum of Bombyx vitellogenin (BmVg) was kindly given by Dr Yamashita (Nagoya University, Japan). After electrophoresis on 10% SDSpolyacrylamide gels, peptides were electroblotted onto a nitrocellulose sheet in blotting buffer [100 mM Tris, 192mM glycine and 5% (v/v) methanol] for 60min at 2mA/cm 2 using Horizblot (Atto Co., Tokyo, Japan). The electroblotted nitrocellulose sheets were incubated for 15min in phosphatebuffered saline (PBS) and then incubated for 60 min with antisera diluted with 1:5000 in PBS containing 2% skim milk. After being washed with PBS/skim milk three times, the nitrocellulose sheets were incubated for 60 min with a 1:5000 dilution of peroxidase-conjugated goat anti-rabbit IgG in PBS/ skim milk. The bound antibodies were visualized with a Konika immunostaining HRP kit IS-50B (Konika, Tokyo, Japan) or a POD immunostain CBPB 106/I--L
set (Wako Pure Chemical Industries, Ltd, Osaka, Japan). RESULTS
Developmental changes in hemolymph peptides andfat body peptides Hemolymph peptides and fat body peptides of A.
yamamai were collected from day 0 of the last larval instar to the early pupal stage, and were analyzed by SDS-PAGE (Fig. 1). Five major peptides (235 kDa, 83 kDa, 75 kDa, 51 kDa and 36 kDa) were found in the hemolymph until the pharate pupal stage. The other three peptides (185 kDa, 145 kDa and 44 kDa) were detected at the pharate pupal stage (Fig. IA). The amounts of 235 kDa, 51 kDa and 36 kDa peptides in the hemolymph did not change very much through the larval instar, but those of 83 kDa peptide and 75 kDa peptide increased remarkably during the feeding stage and decreased at the early stage of
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MARCIANORIKOYOKOYAMAet al.
pupae. The 185 kDa and 44 kDa peptides appeared in the female hemolymph of pharate pupal stage (Fig. 1A). Fat body peptides consisted of four major peptides (83 kDa, 75 kDa, 58 kDa and 45 kDa) and many minor ones. There were a lot of 58 kDa peptide and 45 kDa peptide in larval fat bodies. After spinning, the 83 kDa and 75 kDa peptides were mainly accumulated in the fat bodies (Fig. 1B), and concurrently declined in the hemolymph (Fig. 1A). In A. pernyi, six major peptides (235 kDa, 145 kDa, 83 kDa, 75 kDa, 51 kDa and 34 kDa) were found in the hemolymph until the pharate pupal stage. The amount of 210 kDa peptide fluctuated through the fifth instar, and increased in the hemolymph of female pharate pupae (Fig. 2A). The peptides of 235 kDa, 145 kDa, 83 kDa and 75 kDa were detected in both A. yamamai and A. pernyi. However,
A
Hemolymph Bm Ay Ap M F M F M F ~
BmSP 1 =-
~C ¸
.
.
.
.
.
.
.
.
.
.
185 kDa, 44 kDa and 36 kDa peptides were specific to A. yamamai, while 210 kDa and 34 kDa peptides were specific to A. pernyi. The developmental changing patterns of 235 kDa, 83 kDa and 75 kDa peptides were the same between the two wild silkworms. Fat body peptides of A. pernyi consisted of five major peptides (83 kDa, 75 kDa, 58 kDa, 45 kDa and 24kDa) and many minor ones. 83 kDa, 75 kDa, 58 kDa and 45 kDa peptides were detected in the fat bodies of A. pernyi as well as in those of A. yamamai. Their developmental profiles matched those of the two wild silkworms. A 24 kDa peptide increased in the only female fat bodies from day 9 of the last larval instar. This peptide was a fat body female-specific peptide (Fig. 2B). Hemolymph and fat body peptides of Ay x Ap were analyzed by SDS-PAGE (Fig. 3). The 83 kDa
Fat Body Bm Ay Ap M F M F M F
kDa
.
~- 75
B
BmSP2,-
~-
83
Fig. 5. Immunological identification of Antheraea arylphorin and Antheraea female specific storage protein. Hemolymph proteins and fat body proteins of Bombyx mori (Bin) were collected from day 7 of the fifth instar, those of Antheraea yamamai (Ay) and Antheraea pernyi (Ap) were collected from day 12 of the fifth instar. After SDS-polyacrylamide gel eleetrophoresis, each 30/zg of proteins was transferred to nitrocellulose filters. The nitrocellulose filters were reacted with antiserum against Bombyx femalespecific storage protein (A) and against Bombyx arylphorin (B).
169
Analysis of major proteins in wild silkworms
83k Ap Ay Bm
Ap83k
"
Fig. 6. Immunological relationship between Antheraea arylphorin and Bombyx arylphorin. Hemolymph proteins of Antheraea yamamai, Antheraea pernyi and Bombyx mori, and purified 83 kDa peptide of Antheraea pernyi were resolved on SDS--polyacrylamide gel electrophoresis, and subjected to immunoblot analysis with antiserum against purified 83 kDa peptide ofA. pernyi. Each 30/~g of proteins was applied on each lane. Lane 1: purified 83kDa peptide. Lane 2: hemolymph proteins of A. pernyi. Lane 3: hemolymph proteins of A. yamamai. Lane 4: hemolymph proteins of B. mori. peptide increased in the larval hemolymph of both females and males, while the 75 kDa peptide increased specifically in the female larval hemolymph from day 9 of the fifth instar. A 36 kDa A. yamamaispecific and a 34 kDa A. pernyi-specific peptide were detected in the hemolymph of the hybrid silkworm larvae. A 210 kDa A. pernyi female-specific peptide, a 185 kDa and a 44 kDa A. yamamai female-specific peptide increased in the hemolymph of female pharate pupae (Fig. 3A). The peptides of 83 kDa and 75 kDa in the fat bodies were slightly stained with Coomassie Brilliant Blue through the last larval instar, but strongly stained after spinning. A 24 kDa female-specific peptide was detected in the fat bodies after day 9 of the fifth instar. Developmental profiles of the other fat body peptides were the same as those of their parents (Fig. 3B). Figure 4 shows SDS-polyacrylamide gel electrophoretic analyses of hemolymph and fat bodies of Ap x Ay. The developmental profiles of the other hemolymph and fat body peptides of Ap x Ay were almost the same as those of Ay x Ap (Fig. 3). Storage proteins Bombyx mori has two storage proteins: storage protein 1 (SPI) is a hexamer composed of a methionine-rich 85 kDa subunit, and storage protein 2 (SP2) is a kind of arylphorin and also a hexamer composed of aromatic amino acid-rich two subunits (Kanost et al., 1990). The developmental profile of 83 kDa and of 75 kDa peptides in the hemolymph of wild silkworms corresponded to that of SP2 and of SP1, respectively. To determine whether the peptides of 83 kDa and 75 kDa are related to storage proteins or not, we analyzed them by immunoblot analysis with antisera of SP1 and SP2 (Fig. 5). On the one hand, anti-SP1 serum reacted to the 75 kDa peptide in the larval hemolymph and the fat bodies of the only female Antheraeas (Fig. 5A). On the other hand, anti-SP2 serum reacted unequivocally to the 83 kDa peptide in
the larval hemolymph and the pupal fat bodies of Antheraeas (Fig. 5B). Consequently, the 83 kDa peptide was immunologically related to arylphorin, while the 75 kDa peptide was immunologically related to a female-specific storage protein. Immunoblot analysis was also carried out with antiserum raised against 83 kDa Antheraea peptide (Fig. 6). The antiserum reacted obviously to 83 kDa peptide of A. yamamai and A. pernyi, but did not react to SP2. Vitellogenin and vitellin The 185 kDa A. yamamai peptide could be detected in the hemolymph and the ovaries by SDS-PAGE (Fig. 7). A 210 kDa A. pernyi peptide was major in the female hemolymph but minor in the ovaries. Instead of the 210 kDa peptide, a 170 kDa peptide was major in the ovaries of A. pernyi. The peptides of 185kDa A. yamamai, 170kDa and 210kDa A. pernyi were accumulated in the ovaries o f A p x Ay (Fig. 7A). To identify these peptides with vitellogenin or vitellin, we used an immunoblot analysis with antiBmVg serum (Fig. 7B). The 185kDa A. yamamai peptide in the hemolymph reacted to anti-BmVg serum, as did in the ovaries. The 210 kDa A. pernyi peptide in the hemolymph reacted to anti-BmVg serum. In the ovaries of A. pernyi, however, the 170 kDa peptide reacted to anti-BmVg serum. Furthermore, the three peptides were detected in the ovaries of Ap x Ay. Therefore, the peptides of 185 kDa A. yamamai and 210 kDa A. pernyi were immunologically related to vitellogenin, the 170 kDa A. pernyi peptide was related to vitellin. Furusawa (1991) reported A. yamamai vitellin composed of two subunits (180kDa and 40kDa). Figure 7 shows two peptides (45 kDa and 44 kDa) whose mobility was similar as of the 40 kDa subunit. However, neither the 45 kDa nor the 44 kDa peptide reacted with anti-BmVg serum, so that we could not identify the counterpart subunit of Antheraea vitellogenin and vitellin (Fig. 7B). DISCUSSION To investigate the physiological aspects of A. yamamai, A. pernyi and their hybrids, we analyzed hemolymph, fat body and ovary proteins by SDS-PAGE (Figs 1, 2, 3, 4 and 7). The peptides of 36 kDa A. yamamai and 34 kDa A. pernyi were found in the hemolymph of reciprocal Fl hybrids, and the peptides of 185kDa A. yamamai, 210kDa and 170 kDa A. pernyi were accumulated in the ovaries of these hybrids. These facts indicate that the F~ hybrids synthesize proteins of both parents without dominance. Arylphorins have been identified in all species that have been carefully examined, and in many cases immunological cross-reactivity has been observed over wide phylogenetic distances (Ryan et al., 1984).
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MARCIA NORIKO YOKOYAMAet al.
Telfer et al. (1983) showed also an immunological relationship between Hyalophora and Manduca arylphorin. The 83 kDa Antheraea peptide increased remarkably in the hemolymph of feeding larvae, declined after spinning, but increased in fat body concurrently. This peptide showed similar electrophoretic mobility to that of Bombyx SP2, and it was immunologically related to Bombyx arylphorin (Fig. 5). Antheraea peptide reacted to anti-SP2 serum, but Bombyx SP2 did not react to antibodies against
A Bm
Ay
the 83 kDa peptide (Fig. 6). These results indicate that Antheraea and Bombyx arylphorin not only have common antigenic sites but also species-specific antigenic sites, as did Samia and Bombyx arylphorin (Shimada et al., 1987). The 75 kDa Antheraea peptide moved faster than the 85 kDa Bombyx subunit of SP1, but was immunologically related to SP1 (Fig. 5). Interesting differences arise when we compared Antheraea with other Lepidopteran species. In Antheraeas the synthesis of arylphorins occurs in early
Ap
Ap,.Ay kDa -.,--210 "'-185
~'170
~-- 83 +- 75 ~.. 6 5
60 --,-- 45 " - - 44 4--34
B 4--- 210 170
Fig. 7. Identification of Antheraea vitellogenin and Antheraea vitellin. SDS-polyacrylamide gel electrophoretic analysis (A) and immunoblot analysis using anti-BmVg serum (B). Hemolymph proteins were collected from day 0 of female pupae, and ovaries proteins were collected from female pupae just before adult emergence. Each 30/zg of proteins was applied to each lane of polyacrylamide gels. H and O represent hemolymph and ovary, respectively.
Analysis of major proteins in wild silkworms larval instars as well as in the ultimate instar (data not shown). It was also observed in Bombyx mori (Izumi et al., 1980), Manduca sexta (Riddiford and Hice, 1985) and Galleria mellonella (Ray et al., 1987). During larval-pupal transformation arylphorins of Bombyx and Galleria are mostly taken up by the fat bodies along with other hemolymph storage proteins. Antheraea arylphorin remains partly in the hemolymph, but the rest of arylphorin is sequestrated in the fat bodies during larval-pupal transformation (Figs 1, 2, 3 and 4). A large amount of arylphorin also remain in the hemolymph of Hyalophora and Manduca after wandering (Tojo et al., 1978; Telfer et al., 1983). The amount of 75 kDa Antheraea peptide increased only in the final instar (Figs 3 and 4) as did the Manduca methionine-rich storage protein (Riddiford and Hice, 1985), while in Bombyx mori it occurs in earlier instars (Izumi et al., 1988). Figure 7 shows that the 185kDa peptide was A. yamamai vitellogenin, and that the 210 kDa and 170 kDa peptides were A. pernyi vitellogenin and vitellin, respectively. Nevertheless, we could not identify the counterpart subunit of vitellogenin by immunoblot analysis. Antheraea pernyi vitellogenin and vitellin showed different gel electrophoretic mobility. The cause of the molecular heterogeneity of vitellogenin and vitellin remains to be determined. Differences exist between A. yamamai and A. pernyi with respect to a kind of peptides in the larval hemolymph. We found that a 34 kDa peptide was A. pernyi-specific in the hemolymph, whereas a 36 kDa peptide was A. yamamai-specific (Figs 1-4). These peptides were detected in the hemolymph from the first instar (data not shown). This is a feature of larval serum proteins. But they were not immunoreactable to antisera raised against 30 k proteins (Izumi et al., 1980) and against B. mori larval serum proteins (BmLSP) (Fujiwara and Yamashita, 1990) (data not shown). A 24kDa peptide is a fat body female-specific peptide, a new class of insect protein. There is little information about female fat body specific protein of other insects. We are now extending our studies to purification, characterization and molecular cloning of the 24 kDa peptide. Studies of this protein will develop our understanding about the development and differentiation of fat body. Acknowledgements--We wish to thank Dr O. Yamashita of Nagoya University for providing antiserum of BmVg, and Dr K. Kimura of Shinshu University for his technical assistance. The present work was supported in part by a Research grant from the Ministry of Education, Scienceand Culture, Japan. REFERENCES
Burnette W. N. (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. Biochem. 112, 195-203.
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Fujiwara Y. and Yamashita O. (1990) Purification, characterization and developmental changes in the titer of a new larval serum protein of the silkworm, Bombyx mori. Insect Biochem. 20, 751-759. Furusawa T. (1991) Carbohydrate metabolism and limited degradation of vitellin in the eggs of the Japanese oak silkworm, Antheraea yamamai, during diapause and embryonic development. In Wild Silkmoths '89 '90 (Edited by Akai H. and Kiuchi M.), pp. 81-88. Haunerland N. H. and Bowers W. S. (1986) Arylphorin from the corn earworm, Heliothis zea. Insect Biochem. 16, 617-625. Izumi S., Tojo S. and Tomino S. (1980) Translation of fat body mRNA from the silkworm, Bombyx mori. Insect Biochem. 10, 429-434. Izumi S., Sakurai H., Fujii T., Ikeda W. and Tomino S. (1988) Cloning of mRNA sequencecoding for sex-specific storage protein of Bombyx mori. Biochem. biophys. Acta 949, 181-188. Jones G., Hiremath S. T., Hellmann G. M. and Rhoads R. E. (1988) Juvenile hormone regulation of mRNA levels for a highly abundant hemolymph protein in larval Trichoplusia hi. J. biol. Chem. 263, 1089-1092. Kajiura Z. and Yamashita O. (1989) Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog. Arch. Insect Biochem. Physiol. 12, 99-109. Kanost M. R., Kawooya J. K., Ryan R. O., Hensden M. C. V. and Ziegler R. (1990) Insect haemolymph proteins. In Advances in Insect Physiology (Edited by Evans P. D. and Wigglesworth V. B.), Vol. 22, pp. 299-396. Kobayashi M., Yonekawa M., Yokoyama M. N., Nakagaki M. and Tanaka K. (1992) Ultramorphological characterization of spermatogenesis of interspecifichybrid (F l ) of Antheraea yamamai and Antheraea pernyi. Jap. J. appl. Entomol. Zool. 36, 231-237. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 337, 680-685. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-273. Palli S. R. and Locke M. (1987) Purification and characterization of three major hemolymph proteins of an insect Calpodes ethlius (Lepidoptera; Hesperiidae). Arch. Insect Biochem. Physiol. 5, 233-244. Qu X., Zhang C., Komano H. and Natori S. (1987) Purification of a lectin from the hemolymph of Chinese oak silkmoth (Antheraea pernyi) pupae. J. Biochem. 101, 545-551. Ray A., Memmel N. A. and Kumaran A. K. (1987) Developmental regulation of the larval hemolymph protein genes in Galleria mellonella. Roux's Arch. Dev. Biol. 196, 414-420. Riddiford L. M. and Hice R. H. (1985) Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta. Insect Biochem. 15, 489-502. Ryan R. O., Wang X. Y., Willot E. and Law J. H. (1986) Major hemolymph proteins from larvae of the black swallowtail butterfly, Papilio polyxenes. Archs Insect Biochem. PhysioL 3, 539-550. Ryan R. O., Keim P. S., Wells M. A. and Law J. H. (1985) Purification and properties of a predominantly female specific protein from the hemolymph of the larva of the tobacco hornworm, Manduca sexta. J. biol. Chem. 260, 782-786. Shimada T., Nagata M. and Yoshitake N. (1987) Characterization of arylphorin of the Eri-silkmoth, Samia cynthia ricini (Donovan) (Lepidoptera: Saturniidae). Appl. Ent. Zool. 22, 543-552.
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Shimada T. and Kobayashi M. (1992) Fertility of F~ hybrids between Antheraea yamamai (Guerin-Meneville) and Antheraea pernyi (G.-M.). In Wild Silkmoths '91 (Edited by Akai H., Kato Y., Kiuchi M. and Kobayashi J.), pp. 187-195. Telfer W. H., Keim P. S. and Law J. H. (1983) Arylphorin, a new protein from Hyalophora cecropia: comparisons with calliphorin and manducin. Insect Biochem. 13, 601-613. Tojo S., Betchaku T., Ziccardi V. J. and Wyatt G. R. (1978) Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia. J. cell. Biol. 78, 823-838.
Tojo S., Nagata M. and Kobayashi M. (1980) Storage proteins in the silkworm, Bombyx mori. lnsect Biochem. 10, 289-303. Tojo S., Morita M., Agui N. and Kimura K. (1985) Hormonal regulation of phase polymorphism and storage protein fluctuation in the common cutworm, Spodoptera litura. J. Insect Physiol. 31, 283-292. Yamada H. and Kato H. (1991) Characteristic properties of a blue chromoprotein in larval hemolymph of Antheraea yamamai. In Wild Silkmoths '89 '90 (Edited by Akai H. and Kiuchi M.), pp. 89--95.