Purification and Cdna Cloning of Vitellogenin of the Wild

Journal of Insect Biotechnology and Sericology 77, 35-44 (2008)

Puriﬁcation and cDNA Cloning of Vitellogenin of the Wild Silkworm, Saturnia japonica (Lepidoptera: Saturniidae)

Yan Meng1,+, Chao Liang Liu2, Kunihiro Shiomi1, Masao Nakagaki1 and Zenta Kajiura1,＊

1 Laboratory of Silkworm Genetics and Pathology, Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1, Ueda, Nagano, 386-8567, Japan, and 2 Life Science School, Anhui Agricultural University, Changjiang West Road 130, Hefei, Anhui, 230036, China

(Received January 5, 2007; Accepted September 21, 2007)

We puriﬁed the major yolk protein, vitellin, from Saturnia japonica, by column chromatographies. SDS-PAGE and immunoblot analysis of the S. japonica vitellin (SjVn) showed that SjVn consisted of only a large subunit with a molecular size of approximately 200 kDa. We then cloned and sequenced cDNA of the S. japonica vitellogenin (SjVg), a SjVn precursor. The SjVg cDNA was 5731 nucleotides long and encoded 1776 amino acids for the entire subunit. The molecular weight of the predicted polypeptide was 200,000. Consensus motifs, such as GL/ICG (at the amino acid position 1592) and DGGR (located 17 residues upstream from the GL/ICG motif) were found in the deduced amino acid sequence. There is no RXRR motif, which is a cleavage site between the small and large subunits. Two polyserine regions were found in the deduced amino acid sequence. Key words: Saturnia japonica, vitellogenin, vitellin, RXRR motif, polyserine

Generally, biosynthesis of Vg in insects is transcription- INTRODUCTION ally regulated in tissue-, stage-, and sex-speciﬁc manners Vitellogenesis, through which yolk proteins are accu- (Dhadialla and Raikhel, 1990; Yano et al., 1994b; Liu et mulated in oocytes for subsequent utilization, is a crucial al., 2001). Vg synthesis is suppressed by a juvenile hor- part of embryogenesis for oviparous animals including in- mone in Lymantria dispar and Locusta migratoria (Wyatt, sects. Vitellogenin (Vg), the precursor of the major yolk 1988; Davis et al., 1990; Hiremanth and Jones, 1992). protein, is extraovarially synthesized by metabolic tissues, Characterization of a variety of insects’ Vg has shown such as the liver in vertebrate animals, the intestine in that in most species a primary Vg gene product with a nematodes, and the fat body in insects (Wahli et al., 1981; molecular mass of approximately 200 kDa is generated by Wang and Williams, 1982; Spieth et al., 1985; Wheeler proteolytic cleavage of large (140-190 kDa) and small and Kawooya, 1990). After being secreted into hemo- (40-60 kDa) polypeptides in the fat body and subsequent- lymph, Vg is transported to the developing oocytes by ly secreted into the hemolymph. However, we discovered selective receptor-mediated endocytosis and deposited as that the vitellogenin of the wild silkmoth, Antheraea per- vitellin (Vn). cDNA cloning and isolation of a number of nyi, with a molecular mass of 200 kDa, is secreted with- Vg genes from insects, vertebrates, and nematodes indi- out cleavage (Yokoyama et al., 1993; Liu et al., 2001). cated that they share several conserved features and are Vitellogenins secreted without cleavage have also been re- derived from a common ancestral gene (Nardelli et al., ported in higher hymenopteran insects of the suborder Ap- 1987; Trewitt et al., 1992; Yano et al., 1994a, b; Romans ocrita, the honeybee, Apis mellifera, the parasitoid wasps, et al., 1995; Chen et al., 1997). For most insect species, Pimpla nipponica, with a molecular mass of around 180 Vgs and Vns are large molecular phospholipoglycopro- kDa, and Encarsia formosa (Nose et al., 1997; Piulachs, teins, post-translationally processed by cleavage, lipida- et al., 2003; Donnell, 2004). tion, glycosylation, and phosphorylation, providing amino The Japanese wild silkworm family, Saturniidae, is di- acids, lipids, carbohydrates, phosphates, and other nutri- vided into two subfamilies, Saturniinae and Agliinae; and ents to the developing embryo (Kunkel and Nordin, 1985; eight genera, Actias, Antheraea, Attacus, Loepa, Rhodnia, Byrne et al., 1989; Kanost et al., 1990; Raikhel and Samia, Saturnia, and Aglia (Jinbo, 2005). We have stud- Dhadialla, 1992). ied variations of these species’ vitellogenins, and determined six full cDNA sequences of vitellogenins from

*To whom correspondence should be addressed. Antheraea yamamai, A. pernyi, Samia cynthia ricini, S. Fax: +81-268-21-5331. Tel: +81-268-21-5337. cynthia pryeri, Bombyx mandarina (both Japan and Chi- Email: [email protected] na). We then reported on the purification, cDNA se- +Present address: Department of Agricultural and Environmental quencing, and mRNA expression of Antheraea pernyi Biology, Graduate School of Agricultural and Life Science, The vitellogenin (ApVg) (Liu et al., 2001), genetic variations University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, in Bombyx mandarina vitellogenin (BmaVg) of Japanese Japan. populations (Meng et al., 2006a), and gene organization 36 Meng et al. of Antheraea yamamai vitellogenin (AyVg) (Meng et al., ovaries, so we started the purification from approximate 2006b). Our results indicated that ApVg has only a large 350 mg vitellin in crude extraction, and obtained 56 mg subunit and no RXRR motif in the deduced amino acid purified vitellin. sequence, that stage- and sex-specific synthesis of ApVg is regulated at the level of RNA, that there are nine Bma- Gel electrophoresis Vgs, a major vitellogenin and eight variations, and that SDS-PAGE was performed according to the method of AyVg consists of seven exons and six introns. Laemmli (1970). Molecular sizes were estimated using Our preliminary experiment shows that two Vgs of Sa- high-molecular-weight calibration kits (Amersham Biosci- turnia japonica and Rhodinia fugax have only a large sub- ences). We used 6% stacking gel and 10% separating gel. unit, as well as ApVg. In this report we describe the purification and the primary structure of Saturnia japonica Immunoblot analysis vitellogenin (SjVg) in which no RXRR motif was found. We used antiserum raised against the vitellin of Anther- We discuss the relationship of vitellogenins from five Sa- aea pernyi (Liu et al., 2000). Immunoblotting was per- turniinae insects, S. japonica, A. yamamai, A. pernyi, S. formed according to the manual of ECL Plus Western cynthia ricini, and S. cynthia pryeri, and three Bombyci- Blotting Detection Reagents (Amersham Biosciences) dae insects, two B. mandarina and B. mori. with some modifications. After SDS-PAGE the gel was equilibrated in transfer buffer [100 mM Tris, 192 mM gly- cine, and 20% methanol (v/v)] for 15 min and electropho- MATERIALS AND METHODS retically transferred to polyvinilidenfluoride (PVDF) Experimental animals membrane (Immobilon™ -PSQ, Millipore) at 2 mA/cm2 for Wild silkworms of the species Saturnia japonica are 3 h. The blot was blocked with 5% non-fat dried milk in agricultural pests, because their larvae consume the leaves PBST [137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, of chestnut trees and walnut trees. Larvae of Saturnia ja- and 1.8 mM KH2PO4, pH 7.4 containing 0.1% Tween-20 ponica were collected at the Tokida campus of Shinshu (v/v)] for 1 h and then incubated for 1 h with the first an- University and reared on the leaves of the Japanese chest- tibody, a 1:5000 dilution of anti-ApVn serum in PBST. nut, Castanea crenata, or the Japanese walnut, Juglans The blot was rinsed 3 times briefly and then washed 1 × sieblodiana, at 24 ± 2°C in the rearing room. 15 min and 3 × 5 min with PBST. Then, a PVDF filter was reacted with the second antibody, peroxidase-conju- Preparation of tissues gated goat anti-mouse IgG 1:10000 diluted in PBST, for The mature ovaries of adult S. japonica were dissected another 1 h. The bound antibodies were exposed to Hyper and homogenized with 10 vol. of 10 mM phosphate-buffer film™ ECL (Amersham Biosciences). saline pH 7.0 containing 0.75% NaCl and 1 mM phenylm- etylsulfonylfluoride after weighing. The homogenates Total RNA preparation were centrifuged at 10,000 g for 10 min. The supernatants The fat bodies of female larvae of Saturnia japonica at were stored at −20°C until use (Kajiura and Yamashita, larval-pupal metamorphosis were dissected and rinsed 1989). with ice-cold 10 mM phosphate buffer pH 7.0 containing 0.75% NaCl and then quickly frozen by liquid nitrogen. Purification of vitellin The tissues were stored at −80°C until use. Total RNA of To purify SjVn, we referred to the methods of Yokoya- fat bodies was extracted using ISOGEN (Wako) according ma et al. (1994), Kajiura et al. (1998), and Liu et al. (2001). to the protocol of the supplier. SjVn was purified from 4.5 gram of mature ovaries by column chromatography using anion-exchange chromatog- Reverse transcription/polymerase chain reaction raphy with DE52 (Whatman International Ltd. Madison, (RT-PCR) England) and hydrophobic column chromatography with First-strand cDNA was synthesized with Superscript™ I butyl-cellulofine (Seikagaku Kogyo, Tokyo, Japan). In the RNaseH− reverse transcriptase (Gibco BRL) and EcoRI- purification procedures, the presence of SjVn in the frac- NotI-oligo dT18 (ENdT) synthetic primer, using 1.5 μg of tions (5 ml each) obtained at each step was checked by the total RNA prepared from the fat bodies of the spin- SDS polyacrylamide gel electrophoresis (SDS-PAGE). ning larvae. Sense and antisense primers were designed The amount of protein was measured using bovine serum on the basis of the highly conserved vitellogenin cDNA albumin as a standard (Bradford, 1976). The amount of sequences among known wild silkworms. PCR was per- vitellin in the ovaries was calculated by the scanning den- formed under standard conditions with ExTaq DNA poly- sitometer CS9000 (SHIMADZU). The calculated percent- merase (TaKaRa) using the designed primers and the age of vitellin was about 70% of total proteins in the ENdT primer. Rapid amplification of cDNA ends (RACE) Vitellogenin of Saturnia japonica 37 was used to amplify SjVg cDNA ends (Frohman et al., access at http://www.cbs.dtu.dk/services/. 1988), which were purified by QIAEX Gel Extraction Kit (QIAEX) for direct sequencing. RESULTS AND DISCUSSION cDNA cloning and subcloning Vitellins Two overlapping cDNA fragments of 2.2 kb and 3.5 kb Yolk proteins of three wild silkworms, S. japonica, R. were obtained and inserted into the cloning vector, pGEM- fugax and S. cynthia pryeri, were analyzed by SDS-PAGE 3Zf (+) (Promega), and confirmed by SacI/ XbaI and (Fig. 1A). The electrophoretic analysis clearly shows that SacI/ SphI double enzyme digestion, respectively. Accord- their vitellins were approximately 200 kDa peptide of S. ing to the single enzyme restriction map of the recombi- japonica, approximately 200 kDa peptide of R. fugax, and nant plasmid DNA, two overlapping fragments of 1.0 kb peptides of approximately 160 kDa and 45 kDa of S. cyn- and 1.9 kb were subcloned into the vector again. thia pryeri. Immunoblot analysis showed the 200 kDa peptide was vitellin large subunit of S. japonica, and the DNA sequencing peptides of 160 kDa and 45 kDa were vitellin large sub- DNA sequencing was carried out by dideoxynucleotide unit and small subunit of S. cynthia pryeri, supporting the chain termination using the Big Dye™ Terminator v 1.1 result of SDS-PAGE (Fig. 1B). Vitellins of the two wild Cycle sequencing RP-100 kit from Applied Biosystems. silkworms, S. japonica and R. fugax, consisted of only a large subunit, while that of the wild silkworm, S. cynthia Amino acid sequences analysis pryeri, consisted of a small subunit and a large subunit. The primary structure of SjVg was deduced from cDNA sequences by GENETYX-SV/R 4.0. The signal SjVn purification and identification peptide prediction program of the for SjVg amino acid se- Anion-exchange column chromatography (2.0 cm × quences was SignalP 3.0. The prediction programs for 20 cm, DE-52) was utilized for separating the different post-translational modifications of glycosylation and phos- yolk proteins in the crude extracts from the 4.5 g ovaries phorylation were NetNGlyc 1.0 and NetPhos 2.0, respec- of S. japonica. SjVn was eluted by a linear NaCl gradient tively. The CBS web site offers all programs with free from 0.0 to 0.5 M in 25 mM Tris-HCl (pH 8.0) after the

Fig. 1. SDS-polyacrylamide gel electrophoretic analysis of yolk proteins from Saturnia japonica (Sj), Rhodinia. fugax (Rf), and Samia cynthia pryeri (Scp). Each 10 µg of proteins in eggs was loaded on the lane. Markers of high-molecular-weight (HMW) and low-molecular-weight (LMW) are also loaded in both sides. B, Immunoblot analysis. Each 1 µg of egg proteins of the wild silkworms, S. japonica and S. cynthia pryeri, was subjected to SDS-gel electrophoretic analysis. After SDS-PAGE the peptides in the gel were transferred to a PVDF membrane and reacted with anti-ApVn serum. Vn, VnH, and VnL represent vitellin, vitellin heavy subunit (large subunit) and vitellin light subunit (small subunit), respectively. 38 Meng et al. nonabsorbed proteins flowed down from the column (frac- had only a large subunit with a molecular size of approxi- tion 9-10, Fig. 1A). The absorbance of each 5-ml fraction mately 200 kDa (Figs. 1 and 3). was read at 280 nm with Jasco Uvidec-510 (Japan Spec- troscopic Co., Ltd.). SjVn was detected in the highest Nucleotide sequence of Vg mRNA peak of fraction 20 (Fig. 2A). SjVn was salted out from The nucleotide sequence was determined by RT-PCR pooled fractions 18-25 by ammonium sulfate at 60% final and RACE. Two sense primers (5’UTR and 5Vgs sense 2) concentration. The precipitate was resolved in 2 ml of and two antisense primers (5Vgs anti 1 and ENdT) were 10 mM Na-phosphate buffer at pH 7.0 containing 1.0 M designed for RT-PCR to amplify two overlapping cDNA ammonium sulfate and subjected to hydrophobic chroma- fragments. A 2.2 kb product (primer pairs 5’UTR and tography (1.0 cm × 15 cm, butyl cellulofine). SjVn was 5Vgs anti 1) and a 3.5 kb product (primer pairs 5Vgs eluted by a linear gradient of ammonium sulfate from 2.0 sense 2 and ENdT) were obtained as expected. The two to 0.0 M (Fig. 2B). Fractions 21-33 were salted-out by the products were cloned into a pGEM cloning vector (Pro- same method as described above. The calculated percent- mega). The 5’end was directly sequenced by the 5’RACE age of SjVn by the gel scanning densitometer was about method. Figure 4 shows the entire nucleotide sequence 70% of total proteins in the ovaries. So we started the pu- and the deduced amino acid sequence. The length of SjVg rification from approximate 350 mg vitellin in the first cDNA is 5731 bp, which involves a long open reading crude extracts, and obtained 56 mg purified vitellin. frame encoding 1776 amino acids followed by a termina- Therefore, SjVn was effectively separated from other ova- tion codon (TAA). A consensus polyadenylation signal ry proteins through these steps with a final yield of ap- (AATAAA) lies 327 nucleotides downstream from the ter- proximately 16%. The SDS-PAGE results of the SjVn mination codon at nucleotide position 5711-5716 (indicated eluting processes from each column chromatography are by a wavy underline in Fig. 4). shown in Figure 3. It was evident that the purified SjVn Primary structure of SjVg deduced from cDNA Figure 4 shows the deduced amino acid sequences of SjVg from the cDNA nucleotide sequence. The first 18 residues of the 1776-residue-long primary structure are considered the signal peptide for trans-membrane secre- tion by the SignalP 3.0 program. Disregarding the signal sequence, the peptide consists of 1758 amino acid resi-

Fig. 2. Elution profiles of vitellin of Saturnia japonica. We purified SjVn from 4.5 gram of mature ovaries. A, anion-exchange chromatography, DE52. After the nonabsorbed proteins flowed down from the column, SjVn was eluted by a linear NaCl gradient from 0.0 to 0.5 M in 25 mM Tris-HCl (pH 8.0). B, hydrophobic column chromatography, butyl cellulofine. The proteins of fractions 18-25 collected in step A Fig. 3. SjVn in each purification step and identification of were salted out by ammonium sulfate at a final concentration SjVn subunit composition by SDS-PAGE. Lane 1, 20 µg crude of 60%, resolved in 2 ml of 10 mM Na-phosphate buffer (pH yolk proteins. Lane 2, 20 µl of fraction No. 22 from DE52 col- 7.0, containing 1.0 M ammonium sulfate), and subjected to umn chromatography, Lane 3, 20 µl of fraction No. 30 from bu- butyl cellulofine. SjVn was eluted by a linear gradient of am- tyl cellulofine column chromatography. M, molecular weight monium sulfate from 2.0 to 0.0 M. marker with sizes written on the left side. Vitellogenin of Saturnia japonica 39 dues and has an average molecular weight of 201.4 kDa, site of Antheraea pernyi has evolved from RXRR to which is consistent with the molecular size of purified vi- RSLH, that of SjVg has evolved from RXRR to RLRH tellin (Fig. 3). Eleven cysteines were found at conserved (thick wave-underlined in Fig. 3), resulting in SjVg not locations, with two near the N-terminal residues at posi- being recognized by Vg convertase any longer and being tions 179 and 217, and nine around the C-terminal resi- sequestered by the developing oocytes as a single long dues (positions 1306, 1320, 1442, 1594, 1709, 1718, polypeptide. Indeed, no predicted β-turn adjacent to 1722, 1736, and 1774). A highly conserved short motif, RSRH of SjVg was found by the Chou-Fasman method in GL/ICG (Nose et al., 1997), is also present at amino acid GENETYX-SV/R. Thus, the evolutionary loss of the SjVg positions 1592-1595. Additionally, the DGXR motif (Tufail cleavage site is due to local modifications in the amino et al., 2000), which occurs in almost all insect vitello- acid. genins examined thus far, is located 17 residues upstream of the GL/ICG motif. However, there is no R(K)-X- Phylogenetic tree R(K)-R motif, which is the putative cleavage site in the We have determined Vg cDNA sequences from seven deduced amino acid sequences of the previtellogenin of wild silkworms (reference and accession numbers in Bombyx mori (Yano et al., 1994b), Aedes aegypti (Chen parentheses): Saturnia japonica (this report; AB190809), et al., 1994), Anthonomous grandis (Trewitt et al., 1992), Antheraea pernyi (Liu et al., 2001; AB049631), An- and Lymantria dispar (Hiremath and Lethoma, 1997). theraea yamamai (Meng et al., 2006b; AB055843), Samia SjVg has five possible cleavage sites with an RXXR, al- cynthia ricini (AB055844), Samia cynthia pryeri though pro-Vg is not apparently cleaved there. (AB190810), Japanese Bombyx mandarina (Meng et al., Following the conserved domain search we concluded 2006a; AB239763), and Chinese Bombyx mandarina that the N-terminus of SjVg showed a highly conserved (Meng et al., 2006a; AB055845). We clarified seven Vg property at the lipoprotein N-terminal domain except for primary structures in six species: one in Bombycidae the polyserine areas, and that the C-terminus of SjVg in (Bombyx mandarina) and the other five included in the the von Willebrand factor (vWF) type D domain was con- Saturniidae (Antheraea, Samia and Saturnia). Additional- served very well. ly, the Vg cDNA sequences of Bombyx mori and Lymant- ria dispar have been reported by Yano et al. (1994b; RXRR motif in vitellogenins of silkworms D13160) and Hiremath and Lehtoma (1997; U60186), re- The cloning and characterization of Vg cDNAs from spectively. Thus, the Vg cDNA sequences of nine lepi- several species of insects have been reported (Trewitt et dopteran insects have been determined. al., 1992; Kageyama et al., 1994; Yano et al., 1994b; Ro- A homology search showed that SjVg had 80.7-82.2% mans et al., 1995; Hiremath and Lehtoma, 1997; Hirai et identity to the other four Vgs of Saturniidae, 58.0-58.4% al., 1998; Lee et al., 2000a, b). Results of these studies identity to the Vgs of Bombycidae, and 44.8% identity to revealed that the subunits of Vgs are derived from the pri- the Vg of Lymantriidae, while the Vgs of Antheraea had mary translation product encoded by a single contiguous 81.2-82.3% identity to the Vgs of Samia. Because each mRNA, and that the pro-Vg cleavage site conserves the identity of Vg in Saturniidae is the almost same, if the paired basic consensus sequence R/KXR/KR or RXXR, evolutionary rate of Vg amino acid sequence is constant specifically recognized by convertases, the subtilisin-like in Saturniidae, the common ancient Vg of Antheraea, protein endoproteases (Barr, 1991; Chen and Raikel, Samia, and Saturnia would have diverged at almost the 1996). Additionally, the cleavage motif is immediately same time. One after another, three phylogenetic trees of preceded by or followed by a predicted β-turn, a require- insect vitellogenins have been reported by Comas et al. ment for optimal recognition by this family of endoprote- (2000), Sappington et al. (2002), and Donnell (2004). ases (Brakch et al., 1993; Sappington and Raikhel, 1998). Their results showed that lepidopteran Vg’s appear to be Table 1 shows a comparative investigation of eight Vgs the the most similar to one another. They cluster together, from seven lepidopteran insects in terms of cleavage site with the Vg line represented by L. dispar (Noctuoidea) motifs, their nucleotide sequences, and subunit molecular being ancestral to A. pernyi and B. mori (Bombycoidea) weights. We propose that the RXRR motif is the con- Vg’s. We constructed a phylogenetic tree according to the served Vg convertase recognition site of lepidopteran silk- entire Vg amino acid sequences less the signal peptides to worms. There were four codons, AGG, AGA, CGG, and have a look at whether the identity is proportional to their CGA, encoding the first arginine; however, the second evolutionary classification (Fig. 5). It is evident that Sa- and third arginines were rigorously mono-encoded by turnia japonica clustered with four other Saturniidae wild CGT. The four codons of the first arginine were clearly silkworms and three insects of Bombycidae, then formed divided into AGR (Saturniidae) and CGR (Bombycidae). a larger group that included the gypsy moth, Lymantria Hence, similar to how the presumed ancestral cleavage dispar (Lymantriidae). The topologies are consistent with 40 Meng et al. Vitellogenin of Saturnia japonica 41

Fig. 4. Nucleotide sequence of SjVg cDNA and the deduced amino acid residues. Nucleotides are numbered on the right. The termination codon (TAA) is in boldface with an asterisk. The polyadenylation signal (AATAAA) is underlined. Amino acids are numbered from the translation initiation methionine on the left. The ﬁrst 18 residues with a thick underline constitute the predicted signal peptide. Two polyserine regions are underlined, while RXXR motifs are shown by wavy lines. DGQR and GICG motifs are shown with a dotted line. The 11 conserved cysteines are indicated with bold and underlined letters. Boxed amino acids are asparagine-linked glycosylation sites. The phosphorylation sites, serines, threonines, and tyrosines predicted by the NetPhos 2.0 program are marked with bold letters. 42 Meng et al.

Table 1. Comparison of RXRR motifs and their nucleotide sequences among eight insects of Bombycoidae Amino acid Subunit molecular Insects Nucleotide sequence motif weight (kDa) Saturnia japonica RLRH AGATTGCGTCAT 203.2 Antheraea pernyi RSLH AGATCGCTTCAT 201.6 Antheraea yamamai RSRR AGATCACGTCGT 161.1, 40.4 Samia cynthia ricini RIRR AGGATACGTCGT 161.1, 40.5 Samia cynthia preyri RTRR AGGACACGTCGT 161.0, 40.6 Bombyx mandarina (JPN) RSRR CGGTCGCGTCGT 161.5, 41.3 Bombyx mandarina (CHN) RSRR CGGTCGCGTCGT 161.6, 41.3 Bombyx mori RSRR CGATCGCGTCGT 161.9, 41.2

Fig. 5. Phylogenetic tree based on known Vg amino acid sequences of lepidopteran insects using the neighbor-join- ing method from the Tree View package. The tree was constructed from the alignment of full-length amino acid sequences with the signal peptides using the ClustalW computer program (Thompson et al., 1994). Vitellogenin (accession number D89547) of the chicken, Gallus gallus, is deﬁned as an outgroup. The numbers to the left or above a node in- dicate 1000 bootstrap iterations supporting the branch. GenBank accession numbers of the other vitellogenins were described in the text. their morphological differentiation to a high degree. We evolution of vitellogenin genes among the wild silk- believe that Vg could be considered a reliable molecular worms. evolutionary base for classifying insects within an order. A DDBJ BLAST search of amino acids indicated that ACKNOWLEDGMENTS SjVg had 18-19% identity to the vitellogenin of nematodes, 18-24% identity to that of vertebrates, and 20-80% The present work was supported in part by the National to that of insects. Furthermore, SjVg shares 18% similari- Bio-resources Project (RR2002) of the Ministry of Educa- ty to the hemolymph clottable protein precursor, a vitello- tion, Science, Sports and Culture of Japan. This work was genin-related protein responsible for clot formation in supported by Grant-in-Aid for Global COE Program by crustaceans (Hall et al., 1999). the Ministry of Education,Culture,Sports,Science,and We have reported the vitellogenin gene structure of An- Technology. theraea yamamai (Meng et al., 2006b). Cloning and sequencing of the vitellogenin genes of S. japonica and A. REFERENCES pernyi, which are single subunit type, are in progress. Such the gene analyses will bring us a new insight about Barr, P.J. (1991) Mammalian subtilisins: The long-sought diba- Vitellogenin of Saturnia japonica 43

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