Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Identification of a novel host protein SINAL10 interacting with GP64 and its role in Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important pathogen of Bombyx mori, silkworm and causes severe losses in the silk industry. During the virus infectious cycle, budded virus (BVs) and occlusion-derived virus (ODVs) particles, which have identical genetic content but different phenotypes, are produced. The envelope glycoprotein GP64, specific in BVs, is involved in host cell receptor binding and is sufficient to mediate membrane fusion during the viral entry. However, the host cell factors, interacting with GP64 to mediate BVs infection, are still unknown. In this study, a cDNA library of Bombyx mori cells (BmN) was constructed and yeast two-hybrid screening was used to identify the host cell factors interacting with GP64. One of the eight candidate proteins encoded the E3 ubiquitin-protein ligase SINA-like 10 (SINAL10), was further confirmed through coimmunoprecipitation assays as novel GP64 binding protein. Moreover, overexpression of SINAL10 significantly enhances viral reproduction, and conversely, silencing its expression by small interfering RNAs showed significant inhibitory effects. Collectively, we demonstrated that SINAL10 is a novel GP64-binding protein that stimulates BmNPV proliferation.

Genetic variation in ecoraces of tropical tasar silkworm, Antheraea mylitta using SSR markers.

The tropical tasar silkworm, Antheraea mylitta, polyphagous sericigenous insect mostly found in the tropical areas of India. It is found in these regions as ecotypes or ecoraces. It feeds primarily on plants, a variety of secondary plants like Terminalia arjuna and T. tomentosa. Tasar culture is a traditional livelihood for lakhs of tribal populace in the areas of Jharkhand, Chhatisgarh, Orissa, Maharashtra, Andhra Pradesh, West Bengal and Uttar Pradesh. In the present study, the genetic diversity of these ecoraces is identified by DNA markers, namely simple sequence repeats (SSRs), most of which produced polymorphic bands.

Molecular cloning, characterization and expression analysis of cathepsin O in silkworm Bombyx mori related to bacterial response.

Cathepsins are the main members of the cysteine family and play important roles in immune response in vertebrates. The Cathepsin O of Bombyx mori (BmCathepsin O) was cloned from the hemocytes by the rapid amplification of cDNA ends (RACE). The genomic DNA was 6131bp long with a total of six exons and five introns. Its pre-mRNA was spliced to generate two spliceosomes. By comparisons with other reported cathepsins O, it was concluded that the identity between them ranged from 29 to 39%. Expression analysis indicated that BmCathepsin O was specific-expressed in hemocytes, and highly expressed at the 4th molting and metamorphosis stages. Immunofluorescence assay and qRT-PCR showed that BmCathepsin O was expressed in granulocytes and plasmatocytes. Interestingly, BmCathepsin O was significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo, which suggested that BmCathepsin O may be regulated by 20E. Moreover, activation of BmCathepsin O was also observed in hemocytes challenged by Escherichia coli, indicating its potential involvement in the innate immune system of silkworm, B. mori. In summary, our studies provide a new insight into the functional features of Cathepsin O.

Ação de inseticidas sobre os ovos e lagartas da broca-pequena-dofruto do tomate, em bioensaio de laboratório / Action of insecticides on the eggs and larvae of the borer small-fruit tomato in laboratory bioassay

A broca-pequena-do-fruto é praga-chave na cultura do tomate por causar danos significativos às partes reprodutivas. Devido a isto, foi objetivo deste trabalho avaliar a eficiência de inseticidas sobre ovos e lagartas recém-emergidas, com e sem a adição de óleo vegetal (0,25%), em bioensaios de laboratório. Frutos com ovos foram coletados em cultivo de tomate estaqueado na quinzena posterior à última aplicação de agrotóxicos, sendo selecionados os frutos com ovos de coloração variável de branco a marrom claro, com 1 e 4 dias de incubação, e imersos em 1 L da calda inseticida por 5 segundos. O delineamento estatístico utilizado foi o inteiramente casualizado, com média de 18 frutos nos tratamentos e de, aproximadamente, 4 ovos/fruto. A avaliação da ação inseticida de 24 produtos foi realizada após a imersão na calda inseticida, observando-se, sob microscópio estereoscópico, os ovos quanto à integridade do córion, textura e coloração, lagartas emergidas, bem como os orifícios de entrada e de saída das larvas (aos 7 e 21 dias). Os produtos testados diferiram da testemunha quanto à densidade de lagartas eclodidas, bem como quanto à redução populacional de lagartas, podendo-se destacar Trebon 100 SC (etofenprox; 200 mL do produto comercial/100 L), Lannate BR (methomil; 100 mL), Thiobel 500 (cartap; 250 g) e Vertimec 18 CE (abamectin; 100 mL). A adição de óleo vegetal resultou em incremento na eficiência dos produtos.

Action of insecticides on tomato fruit borer eggs and larvae using laboratory bioassay. The tomato fruit borer is a key tomato pest in light of its damages to the plants’ reproductive parts. Therefore, the present study was aimed to evaluate the effectiveness of insecticides on the eggs and newly hatched larvae, when applied alone or associated with vegetable oil (0.25%), in laboratory bioassays. Fruits with eggs were collected in staked tomato crops fifteen days after the last application of agro-chemicals, selecting the fruits with eggs colored from white to light brown, which had between 1 and 4 days of incubation, followed by immersion of the fruit for 5 seconds in 1 L of the insecticide solutions. The experimental design was completely randomized, with an average of 18 fruits per treatment (24 insecticides and a control) and about 4 eggs per fruit. The evaluations were performed 7 and 21 days after immersion, using a stereomicroscope to observe the corion integrity, egg color, number of larvae emerged, and larvae fruit holes (entry and exit). The insecticides differed significantly from the control, most notably Trebon SC 100 (etofenprox; 200 mL of the commercial product/100 L), Lannate BR (methomil; 100 mL), Thiobel 500 (cartap; 250 g) and Vertimec 18 CE (abamectin; 100 mL). The addition of vegetable oil increased the insecticides’ effectiveness.

Invited review nonmulberry silk biopolymers.

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.

Comparative genetic diversity and genetic structure of three chinese silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini donovan (Lepidoptera: Saturniidae)

The genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L., Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan were comparatively assessed based on RAPD markers. At the species level, A. pernyi and B. mori showed high levels of genetic diversity, whereas S. cynthia ricini showed low level of genetic diversity. However, at the strain level, A. pernyi had relatively highest genetic diversity and B. mori had lowest genetic diversity. Analysis of molecular variance (AMOVA) suggested that 60 percent and 72 percent of genetic variation resided within strains in A. pernyi and S. cynthia ricini, respectively, whereas only 16 percent of genetic variation occurred within strains in B. mori. In UPGMA dendrogram, individuals of A. pernyi and B. mori formed the strain-specific genetic clades, whereas those of S. cynthia ricini were distributed in a mixed way. The implications of these results for the conservation and utilization in breeding programs of three silkworm species are discussed.

Genome-based identification of spliceosomal proteins in the silk moth Bombyx mori.

Pre-messenger RNA splicing is a highly conserved eukaryotic cellular function that takes place by way of a large, RNA-protein assembly known as the spliceosome. In the mammalian system, nearly 300 proteins associate with uridine-rich small nuclear (sn)RNAs to form this complex. Some of these splicing factors are ubiquitously present in the spliceosome, whereas others are involved only in the processing of specific transcripts. Several proteomics analyses have delineated the proteins of the spliceosome in several species. In this study, we mine multiple sequence data sets of the silk moth Bombyx mori in an attempt to identify the entire set of known spliceosomal proteins. Five data sets were utilized, including the 3X, 6X, and Build 2.0 genomic contigs as well as the expressed sequence tag and protein libraries. While homologs for 88% of vertebrate splicing factors were delineated in the Bombyx mori genome, there appear to be several spliceosomal polypeptides absent in Bombyx mori and seven additional insect species. This apparent increase in spliceosomal complexity in vertebrates may reflect the tissue-specific and developmental stage-specific alternative pre-mRNA splicing requirements in vertebrates. Phylogenetic analyses of 15 eukaryotic taxa using the core splicing factors suggest that the essential functional units of the pre-mRNA processing machinery have remained highly conserved from yeast to humans. The Sm and LSm proteins are the most conserved, whereas proteins of the U1 small nuclear ribonucleoprotein particle are the most divergent. These data highlight both the differential conservation and relative phylogenetic signals of the essential spliceosomal components throughout evolution.

Comparison of Phenotypic and Genetic Performance of Local Silkworm Groups and Two Commercial Lines

Five Iranian native silkworm groups: Baghdad, Khorasan Orange, Guilan Orange, Khorasan Pink, Khorasan Lemon, and 107 and 110 commercial lines (12 families from each breed) were randomly selected and reared during 2003-2005 (five generations in spring and autumn). In each family, 30 male and 30 female cocoons were individually recorded for weight, shell weight and shell ratio. From among the native groups, the highest average in all three traits belonged to Baghdad and Khorasan Pink, and the lowest to Khorasan Orange and Khorasan Lemon. From among the commercial lines, the highest average in all three traits belonged to 107. In comparing heritabihty for cocoon weight in native groups, the highest heritabihty belonged to Guilan Orange (0.5147) and Khorasan Orange (0.5036) and the lowest heritabihty belonged to Khorasan Pink (0.0967). In the two other traits, the highest heritabihty belonged to Khorasan Orange and Baghdad and the lowest to Khorasan Pink. In the commercial lines, linellO had higher heritabihty than linel07 for cocoon weight and cocoon shell weight. In all the groups, genetic correlations between cocoon weight and cocoon shell weight were high, expect for the Baghdad group. There was médium or low genetic correlation among cocoon weight, cocoon shell weight and cocoon shell ratio.

Evaluation of economically important traits from sixteen parental strains of the silkworm Bombyx mori L (Lepidoptera: Bombycidae) / Avaliação de características economicamente importantes de dezesseis linhagens do bicho-da-seda Bombyx mori L (Lepidoptera: Bombycidae)

The classification and characterization of silkworm strains are important for sericulture, which is supported by the constant development of new hybrids. In this study, 16 parental strains of Bombyx mori L from the germplasm banks of the Universidade Estadual de Maringá - UEM, and Associação dos Criadores de Bicho-da-Seda de Nova Esperança e Regiões Sericícolas do Paraná - ACESP, were evaluated regarding biological and productive traits economically important. The Chinese C122-B and C121-A, and the Japanese HA-A and HA-B strains yielded the highest cocoon weight, which is related to the raw silk percentage. Our data will be useful in breeding programs for the production of superior silkworm strains and hybrids.

A classificação e caracterização de linhagens de Bombyx mori L é importante para a sericicultura, uma vez que essa atividade é sustentada pelo constante desenvolvimento de novos híbridos da espécie. Neste trabalho, 16 linhages parentais de B. mori do banco de germoplasma da Universidade Estadual de Maringá - UEM, e da Associação dos Criadores de Bicho-da-Seda de Nova Esperança e Regiões Sericícolas do Paraná - ACESP, foram avaliadas em relação às características biológicas e produtivas consideradas economicamente importantes. As linhagens C122-B e C121-A, de origem Chinesa, e as HA-A e HA-B, Japonesas, apresentaram o maior peso de casulo (CW), o qual é associado ao teor de seda (RSP). Os resultados apresentados neste trabalho podem ser utilizados em vários programas de melhoramento visando à produção de linhagens e híbridos geneticamente superiores.

A serine protease in the midgut of the silkworm, Bombyx mori: protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form and activation during midgut remodeling.

We identified a serine protease with a molecular mass of 37kDa in the midgut of the silkworm, Bombyx mori. The activity of this protease (37-kDa protease: p37k) appears after pupation, when the metamorphic remodeling of the midgut is under progress. The sequence analysis of the purified protease and its cDNA revealed that p37k is a trypsin-type serine protease, which is highly similar to serine proteases of other insects, including CG4386 of Drosophila melanogaster. In our molecular phylogenetic analysis, these proteases are grouped together with CG4386-like serine proteases of other insects to form an isolated cluster. The p37k protein and its putative orthologs present in this cluster have two unique sequence motifs, CxxCxC and FIDWLxxLLG, in the N-terminal side of the catalytic region. The gene for p37k is expressed in the midgut on day 2 of the silk-spinning larva, and the p37k polypeptide becomes detectable with a specific antibody at this stage of the midgut. On the other hand, p37k activity is not detectable until pupation, indicating that p37k is present in the larval midgut as an inactive precursor, which then is activated after pupation. A recombinant p37k produced using a baculovirus system is also inactive in its intact form. However, the recombinant p37k can be converted to an active protease when incubated in the homogenate of the midgut, suggesting that some unidentified midgut factor(s) are involved in the activation of p37k.

Molecular phylogeny of silk producing insects based on internal transcribed spacer DNA1.

Silk moths are the best studied silk secreting insects and belong to the families Bombycidae and Saturniidae. The phylogenetic relationship between eleven silk producing insects was analyzed using the complete DNA sequence of the internal transcribed spacer DNA 1 locus. The PCR amplification and sequence analysis showed variation in length ranging from 138 bp (Antheraea polyphemus) to 911 bp (Hyalopora cecropia). Microsatellite sequences were found and was be used to distinguish Saturniidae and Bombycidae members. The nucleotide sequences were aligned manually and used for construction of phylogenetic trees based on Maximum parsimony and Maximum likelihood methods. The topology in both the approaches yielded a similar tree that supports the ancestral position of the Antheraea assama.

Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori.

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.