Domestic Silkworm Research Articles

The complete mitochondrial genome of Yao silkworm (Bombyx mori)

Here, we describe the first complete mitochondrial genome of Yao silkworm, a unique silkworm resource native at Guangxi, China. This circular molecule is 15,656 bp long and contains the typical set of 37 genes (13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes) and one non-coding A + T-rich region of 494 bp long. The genome organization and gene arrangement are identical to those observed in all available Bombyx mori strains. The phylogenetic tree inferred from Bayesian inference provides a molecular evidence that Yao silkworm belongs to the domestic silkworm (B. mori), rather than a novel silkworm species.

Complete mitochondrial genome and phylogenetic analysis of Bombyx mandarina strain Shiquan

Bombyx mandarina is generally thought to be the wild ancestor nearest to the domesticated silkworm, Bombyx mori. Here, we report the complete mitochondrial genome (mitogenome) of B. mandarina strain Shiquan. The mitogenome contains 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one A + T-rich region. Phylogenetic analysis provides solid evidence that B. mandarina Shiquan belongs to Chinese B. mandarina, rather than Japanese B. mandarina. The new mitogenome provides useful information to further explore the origin and domestication of this species.

Physiological and Proteomic Responses of Mulberry Trees (Morus alba. L.) to Combined Salt and Drought Stress.

Intensive investigations have been conducted on the effect of sole drought or salinity stress on the growth of plants. However, there is relatively little knowledge on how plants, particularly woody species, respond to a combination of these two stresses although these stresses can simultaneously occur in the field. In this study, mulberry, an economically important resource for traditional medicine, and the sole food of domesticated silkworms was subjected to a combination of salt and drought stress and analyzed by physiological methods and TMT-based proteomics. Stressed mulberry exhibited significant alteration in physiological parameters, including root/shoot ratio, chlorophyll fluorescence, total carbon, and ion reallocation. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively. Through KEGG analysis, these DEPs were assigned to multiple pathways, including carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism. Among these pathways, the sucrose related metabolic pathway was distinctly enriched in both stressed leaves and roots, indicating an important contribution in mulberry under stress condition. The results provide a comprehensive understanding of the adaptive mechanism of mulberry in response to salt and drought stress, which will facilitate further studies on innovations in terms of crop performance.

Genome-wide annotation and comparative analysis of cuticular protein genes in the noctuid pest Spodoptera litura

Insect cuticle is considered an adaptable and versatile building material with roles in the construction and function of exoskeleton. Its physical properties are varied, as the biological requirements differ among diverse structures and change during the life cycle of the insect. Although the bulk of cuticle consists basically of cuticular proteins (CPs) associated with chitin, the degree of cuticular sclerotization is an important factor in determining its physical properties. Spodoptera litura, the tobacco cutworm, is an important agricultural pest in Asia. Compared to the domestic silkworm, Bombyx mori, another lepidopteran whose CP genes have been well annotated, S. litura has a shorter life cycle, hides in soil during daytime beginning in the 5th instar and is exposed to soil in the pupal stage without the protection of a cocoon. In order to understand how the CP genes may have been adapted to support the characteristic life style of S. litura, we searched its genome and found 287 putative cuticular proteins that can be classified into 9 CP families (CPR with three groups (RR-1, RR-2, RR-3), CPAP1, CPAP3, CPF, CPFL, CPT, CPG, CPCFC and CPLCA), and a collection of unclassified CPs named CPH. There were also 112 cuticular proteins enriched in Histidine residues with content varying from 6% to 30%, comprising many more His-rich cuticular proteins than B. mori. A phylogenetic analysis between S. litura, M. sexta and B. mori uncovered large expansions of RR-1 and RR-2 CPs, forming large gene clusters in different regions of S. litura chromosome 9. We used RNA-seq analysis to document the expression profiles of CPs in different developmental stages and tissues of S. litura. The comparative genomic analysis of CPs between S. litura and B. mori integrated with the unique behavior and life cycle of the two species offers new insights into their contrasting ecological adaptations.

Study of gut bacterial diversity of Bombyx mandarina and Bombyx mori through 16S rRNA gene sequencing

Abstract The wild silkworm B. mandarina is living in the natural environment has a strong stress resistance and adaptability after harsh natural selection. The indoor rearing or domestication of the wild silkworm under artificial custody for long period deteriorates stress resistance and ecological adaptability. Therefore, we aimed to investigate the effects of artificial domestication and evolutionary pressure on the gut bacterial diversity of B. mandarina and B. mori. The intestinal content of 6th day of fifth instar B. mandarina and B. mori larvae were analyzed by sequencing of the 16S rRNA gene through Illumina miseq sequencing technology. The outcome of the study revealed that abundance of predominant bacteria of phylum Firmicutes were respectively 81.40% and 81.85% in the late fifth instar silkworm larvae (6th day) of B. mandarina and B. mori. In Firmicutes, abundance of predominant bacterial genus Enterococcus in B. mandarina (69.73%) was comparatively higher than B. mori (48.99%). The genus Advenella belongs to phylum Proteobacteria was recorded only in B. mandarina (11.54%). The abundance of Unclassified_Peptostreptococcaceae, Methanobrevibacter, Ignatzschineria, Petrimonas and Proteiniphilum in B. mandarina were between 0.12 and 0.17%, nevertheless, these bacterial genera were not detected in B. mori. The abundance of genera Lactococcus, Bacillus and Pseudomonas in B. mori (17.73%, 5.02%, and 1.61%) were remarkably higher than B. mandarina (0.15%, 0.54% and 0.45%). These results indicated that substantial difference was observed between the intestinal bacteria of B. mori and B. mandarina population, and structure of the intestinal bacteria could be affected by the artificial domestication and evolutionary pressure.

Comparative mitochondrial genomes provide new insights into the true wild progenitor and origin of domestic silkworm Bombyx mori.

The domestication of domestic silkworm Bombyx mori, the only truly domesticated insect, is a distinctive event in agricultural history. The domestication and origin of domestic silkworm remains unclear, although it has connected with human for ~5500 years. In the present study, we would like to highlight our evidence from whole mitochondrial genome for the presence of two genetically distinctive subtypes in Chinese B. mandarina populations, corresponding to northern Chinese B. mandarina and southern Chinese B. mandarina, respectively. The mitochondrial genomes and mitochondrial phylogenetic tree provide a solid molecular evidence that the true wild ancestor of domestic silkworm is northern Chinese B. mandarina, rather than southern Chinese B. mandarina, thus implying that the early domestication event may have occurred in northern China. Our finding provides new insights into the origin and evolution of domestic silkworm.

A determining factor for insect feeding preference in the silkworm, Bombyx mori.

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.

High-quality genome assembly of the silkworm, Bombyx mori

In 2008, the genome assembly and gene models for the domestic silkworm, Bombyx mori, were published by a Japanese and Chinese collaboration group. However, the genome assembly contains a non-negligible number of misassembled and gap regions due to the presence of many repetitive sequences within the silkworm genome. The erroneous genome assembly occasionally causes incorrect gene prediction. Here we performed hybrid assembly based on 140 × deep sequencing of long (PacBio) and short (Illumina) reads. The remaining gaps in the initial genome assembly were closed using BAC and Fosmid sequences, giving a new total length of 460.3 Mb, with 30 gap regions and an N50 comprising 16.8 Mb in scaffolds and 12.2 Mb in contigs. More RNA-seq and piRNA-seq reads were mapped on the new genome assembly compared with the previous version, indicating that the new genome assembly covers more transcribed regions, including repetitive elements. We performed gene prediction based on the new genome assembly using available mRNA and protein sequence data. The number of gene models was 16,880 with an N50 of 2154 bp. The new gene models reflected more accurate coding sequences and gene sets than old ones. The proportion of repetitive elements was also reestimated using the new genome assembly, and was calculated to be 46.8% in the silkworm genome. The new genome assembly and gene models are provided in SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp).

Artificial selection on storage protein 1 possibly contributes to increase of hatchability during silkworm domestication.

Like other domesticates, the efficient utilization of nitrogen resources is also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources and to advance human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's evolutionary theory on domestication. Storage proteins (SPs), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. In this study, through blast searching on the silkworm genome and further screening of the artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e., the methionine-rich storage protein 1 (SP1), which is clearly divergent from other storage proteins and exhibits increased expression in the ova of domestic silkworms. Knockout of SP1 via the CRISPR/Cas9 technique resulted in a dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the effect in the wild silkworm compared with the domestic type. Larval development and metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated significant higher expression of genes encoding vitellogenin, chorions, and structural components in the extracellular matrix (ECM)-interaction pathway, enzymes in folate biosynthesis, and notably hormone synthesis in the domestic silkworm, compared to both the SP1 mutant and the wild silkworm. Moreover, compared with the wild silkworms, the domestic one also showed generally up-regulated expression of genes enriched in the structural constituent of ribosome and amide, as well as peptide biosynthesis. This study exemplified a novel case in which artificial selection could act directly on nitrogen resource proteins, further affecting egg nutrients and eggshell formation possibly through a hormone signaling mediated regulatory network and the activation of ribosomes, resulting in improved biosynthesis and increased hatchability during domestication. These findings shed new light on both the understanding of artificial selection and silkworm breeding from the perspective of nitrogen and amino acid resources.

Functional characterization of the horizontally transferred 4,5-DOPA extradiol dioxygenase gene in the domestic silkworm, Bombyx mori.

4,5-DOPA dioxygenase (DODA) is a crucial enzyme in the biosynthetic pathway of betalain. Previous studies have shown that DODA is present in plants, fungi and bacteria. Using updated data, here we show that DODA genes (BmDODA) in the domestic silkworm (Bombyx mori) and other lepidopteran insects are most likely to be horizontally transferred from fungi. A synteny analysis indicated that BmDODA1 is orthologous to other lepidopteran DODAs and that BmDODA2 is a paralogous gene. To explore the function of DODA in Lepidoptera, we first examined the expression patterns of BmDODA1. BmDODA1 showed high transcriptional and translational levels in the midgut and head. Then, we exogenously expressed the BmDODA1 gene, detected 4,5-DOPA ring-cleaving activity and calculated the kinetic parameters of the recombinant BmDODA1. We found that the transcription levels of BmDODA1 were significantly induced by the pathogens Bacillus bombyseptieus and Escherichia coli. Thus, the horizontal transfer of the BmDODA gene in the silkworm may be involved in dopa metabolism and contribute to antimicrobial activity in this species. Our results provide a documented example of functional horizontal gene transfer (HGT) between fungi and animals and expand our knowledge of HGT amongst eukaryotes.

Complete mitochondrial genome and phylogenetic analysis of Bombyx mandarina (Lepidoptera: Bombycidae)

Bombyx mandarina is generally thought to be the wild ancestor nearest to the domesticated silkworm, B. mori. Here, we sequenced and assembled a complete mitochondrial genome (mitogenome) of B. mandarina. The mitogenome contains 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and one A + T-rich region. Phylogenetic analysis confirmed that B. mori is more closely related to the Chinese B. mandarina than the Japanese B. mandarina. The new mitogenome provides useful information to further explore the origin and domestication of this species.

SGID: a comprehensive and interactive database of the silkworm.

Although the domestic silkworm (Bombyx mori) is an important model and economic animal, there is a lack of comprehensive database for this organism. Here, we developed the silkworm genome informatics database (SGID). It aims to bring together all silkworm-related biological data and provide an interactive platform for gene inquiry and analysis. The function annotation in SGID is thorough and covers 98% of the silkworm genes. The annotation details include function description, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, subcellular location, transmembrane topology, protein secondary/tertiary structure, homologous group and transcription factor. SGID provides genome-scale visualization of population genetics test results based on high-depth resequencing data of 158 silkworm samples. It also provides interactive analysis tools of transcriptomic and epigenomic data from 79 NCBI BioProjects. SGID will be extremely useful to silkworm research in the future.

Genetically engineered bi-functional silk material with improved cell proliferation and anti-inflammatory activity for medical application

Functional silk is a promising material for future medical applications. These include fabrication of diverse silk fiber and silk protein-regenerated biomaterials such as silk sutures, hydrogel, films, and 3D scaffolds for wound healing and tissue regeneration and reconstruction. Here, a novel bi-functional silk with improved cell proliferation and anti-inflammatory activities was created by co-expressing the human basic fibroblast growth factor (FGF2) and transforming growth factor-β1 (TGF_β1) genes in silkworm. First, both FGF2 and TGF_β1 genes were confirmed to be successfully expressed in silk thread. The characterization of silk properties by SEM, FTIR, and mechanical tests showed that this new silk (FT silk) had a similar diameter, inner molecular composition, and mechanical properties as those of normal silk. Additionally, expressed FGF2 and TGF_β1 proteins were continuously and slowly released from FT silk for one week. Most importantly, the FGF2 and TGF_β1 contained in FT silk not only promoted cell proliferation by activating the ERK pathway but also significantly reduced LPS-induced inflammation responses in macrophages by mediating the Smad pathway. Moreover, this FT silk had no apparent toxicity for cell growth and caused no cell inflammation. These properties suggest that it has a potential for medical applications. Statement of SignificanceSilk spun by domestic silkworm is a promising material for fabricating various silk protein regenerated biomaterials in medical area, since it owes good biocompatibility, biodegradability and low immunogenicity. Recently, fabricating various functional silk fibers and regenerated silk protein biomaterials which has ability of releasing functional protein factor is the hot point field. This study is a first time to create a novel bi-functional silk material with the improved cell proliferation and anti-inflammatory activity by genetic engineered technology. This novel silk has a great application potential as new and novel medical material, and this study also provides a new strategy to create various functional or multifunctional silk fiber materials in future.

Evidence of peripheral olfactory impairment in the domestic silkworms: insight from the comparative transcriptome and population genetics

BackgroundThe insect olfactory system is a highly specific and sensitive chemical detector, which plays important roles in feeding, mating and finding an appropriate oviposition site. The ecological niche of Bombyx mori has changed greatly since domestication from B. mandarina, and its olfactory response to environmental odorants clearly decreased. However, the mechanisms that result in the olfactory impairment are largely unknown.ResultsThe antennal transcriptomes were compared between the domestic and wild silkworms. Comparison of the same sex between the domestic and wild silkworms revealed 1410 and 1173 differentially expressed genes (DEGs) in males and females, respectively. To understand the olfactory impairment, we mainly focused on the olfactory-related genes. In total, 30 olfactory genes and 19 odorant-degrading enzymes (ODEs) showed differential expression in the two comparisons, in which 19 and 14 were down-regulated in the domestic silkworm, respectively. Based on population genomic data, the down-regulated odorant receptors (ORs) showed a higher ratio of unique non-synonymous polymorphisms to synonymous polymorphisms (N/S ratio) in the domestic populations than that in the wild silkworms. Furthermore, one deleterious mutation was found in OR30 of the domestic population, which was located in transmembrane helix 6 (TM6).ConclusionsOur results suggested that down-regulation of the olfactory-related genes and relaxed selection might be the major reasons for olfactory impairment of the domestic silkworm reared completely indoor environment. Reversely, wild silkworm may increase expression and remove deleterious polymorphisms of olfactory-related genes to retain sensitive olfaction.

Comparative analysis of iTRAQ-based proteomes for cocoons between the domestic silkworm (Bombyx mori) and wild silkworm (Bombyx mandarina)

The domestic silkworm Bombyx mori was domesticated from wild silkworm B. mandarina about 5000 years ago. Long-term domestication and breeding selection have led to the weight of domestic silkworm cocoon being ten times greater than that of wild silkworm cocoon. However, we found that wild silkworm cocoon has significantly much more abundance of sericin proteins than domestic silkworm cocoon. Strikingly, we found that many protease inhibitors have been significantly down-regulated in domestic silkworm cocoon compared with its wild progenitor by using isobaric tag for relative and absolute quantitation (iTraq) approach. Both real-time qPCR experiment and transcriptome data in silk gland also verified these results. In addition, our data indicated that protease inhibitor activity of wild silkworm cocoon was stronger than that of domestic silkworm cocoon, implying that relatively up-regulated protease inhibitors in wild silkworm cocoon may play protection roles against microorganisms. Thus, these protease inhibitors up-regulated in wild silkworm cocoon may have potential values in sericulture and medical practice. SignificanceThis study revealed that there is a big difference in abundance of sericin proteins in cocoon between domestic and wild silkworms. Strikingly, many protease inhibitors have been significantly down-regulated in domestic silkworm cocoon compared with its wild progenitor. This may be due to different environments where the two silkworms live. In addition, the results also implied that relatively up-regulated protease inhibitors in wild silkworm cocoon may play protection roles against microorganisms. Thus, these protease inhibitors may have potential values in sericulture and medical practice.

The evolutionary road from wild moth to domestic silkworm

The Silk Road, which derives its name from the trade of silk produced by the domestic silkworm Bombyx mori, was an important episode in the development and interaction of human civilizations. However, the detailed history behind silkworm domestication remains ambiguous, and little is known about the underlying genetics with respect to important aspects of its domestication. Here, we reconstruct the domestication processes and identify selective sweeps by sequencing 137 representative silkworm strains. The results present an evolutionary scenario in which silkworms may have been initially domesticated in China as trimoulting lines, then subjected to independent spreads along the Silk Road that gave rise to the development of most local strains, and further improved for modern silk production in Japan and China, having descended from diverse ancestral sources. We find that genes with key roles in nitrogen and amino acid metabolism may have contributed to the promotion of silk production, and that circadian-related genes are generally selected for their adaptation. We additionally identify associations between several candidate genes and important breeding traits, thereby advancing the applicable value of our resources.

Construction of a Platform for the Development of Pharmaceutical and Medical Applications Using Transgenic Silkworms

We have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins. An innovative method for generating transgenic silkworms was established in 2000, and ever since this epoch-defining technological development, longstanding efforts have succeeded in developing novel silks that enable the manufacture of new textile materials for regenerative medical uses. Furthermore, we have succeeded in developing a new system of recombinant protein production. This recombinant protein production system is currently capable of producing a maximum of approximately 15 mg recombinant protein per silkworm larva. Transgenic silkworms have also been shown to produce a wide variety of useful proteins, including antibodies and membrane proteins. Some of these recombinant proteins have been in commercial use since 2011. In addition, we have been developing transgenic silkworms as a novel animal model for testing medicines based on metabolic similarities between silkworms and mammals. These applications show the suitability and potential of transgenic silkworms for medical use. Here, we will describe the challenges faced in creating a transgenic silkworm-based platform for pharmaceutical and medical applications.

Discovery of a New Species of Telenomus (Hymenoptera: Scelionidae) Parasitic on Eggs of Bombyx mandarina and Bombyx mori (Lepidoptera: Bombycidae) in Japan and Taiwan.

We reared a Telenomus species from eggs of Bombyx mandarina (Moore) (Lepidoptera: Bombycidae) and Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae) in Japan, and from eggs of B. mandarina in Taiwan. Morphological examination revealed that this Telenomus species is new to science. In this article, we describe it as Telenomus moricolus Matsuo et Hirose, sp. nov. Because B. mandarina is considered to be an ancestor of B. mori, a domestic insect, it is reasonable to assume that B. mandarina is an original host of T. moricolus. This is the second discovery of an egg parasitoid attacking wild and domesticated silkworms, following the first discovery of T. theophilae, a Chinese species. The significance of the discovery of T. moricolus is discussed in relation to examining the effects of host-insect domestication on egg parasitism.

Evolutionary dynamics of transposable elements during silkworm domestication.

Although there are some documented examples on population dynamics of transposable elements (TEs) in model organisms, the evolutionary dynamics of TEs in domesticated species has not been systematically investigated. The objective of this study is to understand population dynamics of TEs during silkworm domestication. In this work, using transposon-display we examined the polymorphism of seven TE families [they represent about 59% of silkworm (Bombyx mori) total TE content] in four domesticated silkworm populations and one wild silkworm population. Maximum likelihood (ML) was used to estimate selection pressure. Population differentiation and structure were performed by using AMOVA analysis and program DISTRUCT, respectively. The results of transposon-display showed that significant differentiation occurred between the domesticated silkworm and wild silkworm. These TEs have experienced expansions and fixation in the domesticated silkworm but not in wild silkworm. Furthermore, the ML results indicated that purifying selection of TEs in the domesticated silkworm were significantly weaker than that in the wild silkworm. Interestingly, an adaptation insertion induced by BmMITE-2 was found, and this insertion can reduce the polymorphism of the flanking regions of its neighboring COQ7 gene. Our results suggested that TEs expanded and were fixed in the domesticated silkworm might result from demographic effects and artificial selection during domestication. We concluded that the data presented in this study have general implication in animal and crop improvements as well as in domestication of new species.

Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives

Bombyx mori, the domesticated silkworm, is of great importance as a silk producer and as a powerful experimental model for the basic and applied research. Similar to other animals, abundant microorganisms live inside the silkworm gut; however, surprisingly, the microbiota of this model insect has not been well characterized to date. Here, we comprehensively characterized the gut microbiota of the domesticated silkworm and its wild relatives. Comparative analyses with the mulberry-feeding moths Acronicta major and Diaphania pyloalis revealed a highly diverse but distinctive silkworm gut microbiota despite thousands of years of domestication, and stage-specific signatures in both total (DNA-based) and active (RNA-based) bacterial populations, dominated by the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Most fungal sequences were assigned to the phyla Ascomycota and Basidiomycota. Environmental factors, including diet and human manipulation (egg production), likely influence the silkworm gut composition. Despite a lack of spatial variation along the gut, microbial community shifts were apparent between early instars and late instars, in concert with host developmental changes. Our results demonstrate that the gut microbiota of silkworms assembles into increasingly identical community throughout development, which differs greatly from those of other mulberry-feeding lepidopterans from the same niche, highlighting host-specific effects on microbial associations and the potential roles these communities play in host biology.