Homologues of fibroin L-chain and P25 of Bombyx mori are present in Dendrolimus spectabilis and Papilio xuthus but not detectable in Antheraea yamamai

Low molecular mass protein components of fibroin, whose electrophoretic patterns before and after the reductive cleavage of disulfide bonds were similar to those of L-chain and P25 of Bombyx mori, were identified in fibroin samples of Bombyx mandarina, Dendrolimus spectabilis and Papilio xuthus but not of Antheraea yamamai. Fibroin of A. yamamai is suggested to form a dimer of H-chain. Full length cDNA sequences were cloned for the homologues of L-chain and P25 from B. mandarina, D. spectabilis and P. xuthus. The deduced sequences of L-chain and P25 of B. mandarina are almost identical to those of B. mori, each containing a single amino acid change. Homologues of L-chain and P25 of D. spectabilis and P. xuthus show about 50% overall identity, respectively, with those of B. mori, but essential structural features; i.e. the three Cys residues in an L-chain and the eight Cys residues and one of the potential N-glycosylation sites in P25, are conserved in both species. These results, together with the published results for Galleria mellonella, suggest that the three-components (H-chain, L-chain and P25) complex of fibroin is rather common among Lepidopteran silk-producing insects, in contrast to the H-H dimer type found in the saturnid silkworm.     

cDNA and deduced amino acid sequences of apolipophorin-IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.     

Distinctive presence of peritracheal athrocytes in Bombyx mori L. and Bombyx mandarina M. as compared to their absence in several other lepidopteran species

Pericardial cells are present in a wide variety of insects and are thought to constitute the majority of 'athrocytes (nephrocytes)'. In contrast, peritracheal athrocytes have only been observed in Bombyx mori L. Although peritracheal athrocytes have a distinct morphology, it is unknown whether these cells are common to all lepidopterans. We anatomically compared eight lepidopteran species: Bombyx mori L. and Bombyx mandarina M. (Bombycidae); Samia cynthia ricini D. (Saturniidae); Agrius convolvuli L. (Sphingidae); Spodoptera litura F. and Mythimna separata W. (Noctuidae); Pieris rapae L. (Pieridae); and Glyphodes pyloalis W. (Crambidae). Of these species, only Bombyx mori L. and Bombyx mandarina M. possess peritracheal athrocytes.     

野桑蚕、蓖麻蚕及家蚕基因组的RFLP分析

以家蚕Bombyx mori丝素重链基因、丝胶基因1和胰凝乳蛋白酶抑制因子13基因为探针,对野桑蚕B.mandarina、蓖麻蚕Philosamia cynthia ricini和家蚕B.mori基因组DNA进行限制性片段长度多态性分析。结果发现,在野桑蚕、蓖麻蚕基因组中存在着家蚕丝素重链基因、丝胶基因1的同源序列,而在中日野桑蚕以及蓖麻蚕品种间存在着限制性酶切位点差异;丝胶基因1在中国野桑蚕基因组的EcoRⅠ酶切图谱较日本野桑蚕与家蚕更为一致,表明家蚕与中国野桑蚕亲缘关系更近。此外,在野桑蚕基因组中发现了家蚕胰凝乳蛋白酶抑制因子13基因的同源序列,并且在家蚕品种间以及中日野桑蚕之间也存在着多态性。这些结果表明不同绢丝昆虫在适应生存环境的进化过程中,基因组发生了结构改变。     

Evidence of selection at melanin synthesis pathway loci during silkworm domestication

The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm (Bombyx mandarina) more than 5,000 years ago. During domestication, body color between B. mandarina and B. mori changed dramatically. However, the molecular mechanism of the silkworm body color transition is not known. In the present study, we examined within- and between-species nucleotide diversity for eight silkworm melanin synthesis pathway genes, which play a key role in cuticular pigmentation of insects. Our results showed that the genetic diversity of B. mori was significantly lower than that of B. mandarina and 40.7% of the genetic diversity of wild silkworm was lost in domesticated silkworm. We also examined whether position effect exists among melanin synthesis pathway genes in B. mandarina and B. mori. We found that the upstream genes have significantly lower levels of genetic diversity than the downstream genes, supporting a functional constraint hypothesis (FCH) of metabolic pathway, that is, upstream enzymes are under greater selective constraint than downstream enzymes because upstream enzymes participate in biosynthesis of a number of metabolites. We also investigated whether some of the melanin synthesis pathway genes experienced selection during domestication. Neutrality test, coalescent simulation, as well as network and phylogenetic analyses showed that tyrosine hydroxylase (TH) gene was a domestication locus. Sequence analysis further suggested that a putative expression enhancer (Abd-B-binding site) in the intron of TH gene might be disrupted during domestication. TH is the rate-limiting enzyme of melanin synthesis pathway in insects. Real-time polymerase chain reaction assay did show that the relative expression levels of TH gene in B. mori were significantly lower than that in B. mandarina at three different developmental stages, which is consistent with light body color of domesticated silkworm relative to wild silkworm. Therefore, we speculated that expression change of TH gene may contribute to the body color transition from B. mandarina to B. mori. Our results emphasize the exceptional role of gene expression regulation in morphological transition of domesticated animals.     

Nucleotide sequence variation in mitochondrial COI gene among 147 silkworm (Bombyx mori) strains from Japanese, Chinese, European and moltinism classes

We characterized the nucleotide sequences of PCR-amplified mitochondrial COI fragments of 147 silkworm (Bombyx mori) strains that have been maintained in the National Institute of Agrobiological Sciences. Coding sequences (714 bp) of the 147 COI fragments were classified into eight haplotypes based on nucleotide differences at eight segregating sites. No length variation was identified in this region. The 5'-noncoding region showed different features, wherein changes in the number of Ts in the T-stretch, together with two base substitutions, were observed. As a result, the 147 COI noncoding sequences were classified into six haplotypes. Combining the coding and noncoding regions, we identified 14 haplotypes. One of the 14 haplotypes, Hap1A was exclusively abundant in the Japanese native strain class, while this haplotype was less frequent in the other three native strain classes. This finding suggests that the Japanese strain class underwent significant genetic differentiation from the Chinese, European, and moltinism classes, when the each class is regarded as a population. Comparison of the nucleotide sequences to those of B. mandarina (which inhabits Japan) revealed changes that are significantly larger than those within either B. mori or B. mandarina. Furthermore, we detected no common haplotypes between them, which suggests the concept of suppressed gene flow between the two species.     

野桑蚕卵黄原蛋白的鉴定及cDNA序列分析

利用SDS-PAGE和Western blot方法分析鉴定了野桑蚕Bombyx mandarina Moore卵黄原蛋白,发现该蛋白由大小两个亚基组成,分子量分别为175 kD和42 kD。利用昆虫卵黄原蛋白进化上的保守性,根据家蚕的卵黄原蛋白cDNA序列设计特异性引物在野桑蚕的总RNA进行RT-PCR扩增,对于3′和5′端进行RACE扩增,解析获得了野桑蚕卵黄原蛋白cDNA全序列(GenBank登录号AY 309967)。该序列含有5.754个碱基,由一个开放阅读框组成,编码1.780个氨基酸,卵黄原蛋白的氨基酸序列与家蚕的同源性达到97.6%。在特定的酶切位点(RSRR)处,即第364～367个氨基酸位置,卵黄原蛋白前体被酶切为大小两个亚基,根据氨基酸推算的相对分子质量分别为161.571 kD和40.794 kD,如果考虑到翻译后的修饰,这与SDS-PAGE的结果是吻合的。同源性分析表明,昆虫卵黄原蛋白一级结构分化基本上局限在同一目内,具有较高的保守性。     

M chromosome of the wild silkworm, Bombyx mandarina (n = 27), corresponds to two chromosomes in the domesticated silkworm, Bombyx mori (n = 28).

Chromosomes of Bombyx mori (n = 28) and of Bombyx mandarina (n = 27) were studied cytogenetically to resolve the origin of the large M chromosome in the Japaneses type of B. mandarina. In the F1 progeny from the reciprocal cross between B. mandarina and B. mori, the mitotic chromosome number was 2n = 55, and a chromosome configuration of 26 bivalents plus 1 trivalent was observed at metaphase I of germ cells. The trivalent chromosome consisted of the M chromosome from B. mandarina and two chromosomes from B. mori. When males of B. mori were mated to the F1 females, nuclei with two types of chromosome number (2n = 55 and 2n = 56) and two sets of chromosome pairs (26 bivalents plus 1 trivalent versus 28 bivalents) were observed in the metaphase I stage. Linkage analysis showed that the 14th chromosome of B. mori was involved in these two types of chromosome segregation. This result indicates that the M chromosome in B. mandarina arose from a fusion between a chromosome corresponding to the 14th linkage group and another, yet unidentified linkage group.     

Geographic dimorphism of the wild silkworm, Bombyx mandarina, in the chromosome number and the occurrence of a retroposon-like insertion in the arylphorin gene.

Individuals of the wild silkworm, Bombyx mandarina, collected in South Korea (Taegu City) and Japan (Tsushima Islands and Fukuoka City) had the chromosome number of 2n = 54, while those collected in China (Hangzhou City) had the chromosome number of 2n = 56. Analysis by PCR (polymerase chain reaction) showed that the 66-bp-long retroposon-like insertion known in the arylphorin gene was present in the B. mandarina specimens with 2n = 54, but not in those with 2n = 56. Thus, dimorphism in the chromosome number coincided with the occurrence of the insertion. It is likely that the boundary dividing the two geographic B. mandarina populations lies somewhere in the northern part of the Korean Peninsula.     

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.     

Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.     

He—Ne激光辐照野桑蚕生物学效应研究

采用一定量He-Ne激光辐照野桑蚕催青卵及蚕肾,并与家蚕杂交,对其子代的蛋白质和酯酶同工酶进行电泳分析,结果发现谱带数目及活性有变化。另外,茧质调查和丝质调查与对照相比也有差异,并发现丝长和纤度特好的变异个体,显示He-Ne激光辐照对野桑蚕生理生化性状有一定影响。     

基于amy基因的中国野桑蚕遗传多样性及其与家蚕的系统发育关系

为探索中国野桑蚕Bombyx mandarina的遗传多样性及其与家蚕B. mori的系统发育关系, 采用PCR产物直接测序法（少数样本克隆测序）获得34个家蚕和野桑蚕样本淀粉酶基因amy序列片段（715 bp）。分析发现56个多态性位点, 鉴定出28种单倍型（haplotype）; 核苷酸多样性π=0.01390±0.00103, 单倍型多样度Hd=0.988±0.011。核苷酸不配对分析（mismatch analysis）和Fu’s Fs 检测表明中国野桑蚕曾发生过种群扩张。分子方差分析（AMOVA）表明, 遗野桑蚕传差异主要在种群内, 种群间和地理组群间差异不显著。聚类树上34个样本聚为3枝/3蔟, 野蚕和家蚕都不按地理区域或系统（类型）聚类, A枝由来自不同地区的野蚕和不同类型的家蚕混合构成, 并且进一步分成3个亚枝, 每一亚枝也同时包含家蚕和野蚕, B枝由3个家蚕和1个野蚕混合构成, C枝全部由来自不同地区的野蚕构成。网络分析没有发现“祖先单倍型”和优势单倍型。结果提示, 淀粉酶基因是一个多态性丰富的分子标记, 中国野桑蚕遗传多样性十分丰富, 据此推测家蚕起源于多种生态类型混杂的野桑蚕。     

中国野桑蚕遗传多样性的AFLP分析

采用AFLP技术对我国具有代表性的7个省市的10个野桑蚕(Bombyx mandarina)种群和2个家蚕（Bombyx mori)品种的遗传多样性进行了研究。结果表明：杭州、陕西和重庆3个地区的野桑蚕种群间及种群内个体间的遗传距离都比家蚕品种大。野桑蚕存在广泛的变异,7个省市的10个野桑蚕种群之间的遗传距离为0．164－0．444（平均值为0．3826）,平均杂合度为0．7061,表明野桑蚕自然群体的遗传多样性十分丰富。     

Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA

Molecular phylogeny of some of the economically important silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and the control region (CR). Maximum likelihood (ML) analyses showed two distinct clades, one consisting of moths from Bombycidae family and the other from Saturniidae family. The mitochondrial CR showed length polymorphisms with indels. The ML analyses for complete mitochondrial genome sequences of Bombyx mori (strains Aojuku, C108, Backokjam, and Xiafang), Japanese and Chinese strains of B. mandarina (Japanese mandarina and Chinese mandarina) and, Antheraea pernyi revealed two distinct clades, one comprising of B. mori strains and the other with B. mandarina, and A. pernyi forming an outgroup. Pairwise distances revealed that all of the strains of B. mori studied are closer to Chinese than to Japanese mandarina. Phylogenetic analyses based on whole mitochondrial genome sequences, the finding of a tandem triplication of a 126bp repeat element only in Japanese mandarina, and chromosome number variation in B. mandarina suggest that B. mori must have shared its recent common ancestor with Chinese mandarina. Another wild species of the Bombycidae family, Theophila religiosa, whose phylogenetic status was not clear, clustered together with the other bombycid moths in the study. Analysis of the interspecific hybrid, A. proylei gave evidence for paternal inheritance of mitochondrial DNA.     

Tissue-/stage-dependent expression of a cloned Bombyx mandarina QM homologue

QM, a novel gene that was firstly isolated as a putative tumor suppressor gene from Wilms' tumor cell line. Although it is well known that the QM gene product plays an important role within the tumor cells, the precise role of QM in the non-tumor cells has remained elusive. With in this mind we isolated a cDNA encoding QM homologue from Bombyx mandarina to understand the function of QM. The 596 bp cDNA has an open reading frame of 219 amino acids and a predicted mol. wt. of 25 kDa. The protein has more than 88% amino acid sequence identity to the QM protein from Drosophila melanogaster. mRNA expression gradually increased from 1-2 days after egg laying to 2 days of finial instar, while very low expressions were detected for either the pupae and the moth stages. The organs, posterior/middle division of silkgland, midgut, fat body and malpighian tubes, also show relatively high mRNA expression levels, respectively. The high degree of conservation and expression of the B. mandarina QM homologous suggest that it has a selectively conserved amino acid sequence due, presumably, to an important biological role which is associated with pupae formation.     

家蚕滞育的分子机理2.蛹期滞育激素基因的表达与滞育决定

滞育激素是由食道下神经节分泌的重要昆虫神经肽,诱导昆虫的滞育。选择具有ＲＮＡ聚合酶能够识别的启动子的质粒载体,将滞育激素ｃＤＮＡ克隆进去,在体外大量合成单一的滞育激素ｃＲＮＡ为参照,测定食道下神经节分泌滞育激素ｍＲＮＡ量来确定滞育激素的分泌量。结果证明食道下神经节分泌的滞育激素的数量决定昆虫的滞育。     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.