High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation.

The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

A multi-motion bionic soft hexapod robot driven by self-sensing controlled twisted artificial muscles.

Artificial muscles have unique advantages for driving bionic robots because their driving mode is similar to biological muscles. However, there is still a big gap between the existing artificial muscle and biological muscle in performance. The twisted artificial muscles (TAMs) from nylon 6,6 provides a low-cost, high integration, low hysteresis driving method. But as a soft actuator, the control of the TAM is so complex that the advantage of excellent embeddedness has not been brought into play. This work presents a self-sensing control method for the TAM by monitoring the real-time resistance of the heating wire which realizes the accurate controlling of the TAM temperature. The simultaneous control of 18 TAMs is realized by using the self-sensing control method. By using a new step walking method based on the principle of insect bionics, a bionic soft hexapod robot with both multi-motion and load capacity is realized. Besides, due to the excellent environmental adaptability of the TAM, the bionic robot can realize amphibious motion both on land and underwater conditions, and the corresponding maximum load capacities are 300 g and 1 kg, respectively. This work not only provides a reliable self-sensing control method of the TAMs but also promotes the development of bionic soft robots.

Whole-genome resequencing reveals loci under selection during silkworm improvement.

Breeding or genetic improvement refers to the process of artificial selection following domestication; as such, it has had a major influence on modern agriculture and animal production. Improvement generally focuses on traits that greatly affect the economic performance. Therefore, understanding the genetic basis underlying improvement will contribute to the identification of genes controlling economic traits and will facilitate future crop and animal breeding. However, genome-wide study of the molecular basis underlying improvement remains rare. The silkworm is a unique, entirely domesticated economically important invertebrate; genetic improvement has had a huge effect on the silkworm regarding silk-related traits. Herein, we performed whole-genomic sequencing on local and genetically improved silkworm lines to identify the genomic regions under strong selection in silkworm breeding/improvement. By genomic-wide selective sweeping analysis, we identified 24 genomic regions with strong selection signals, eight of which contained 13 candidate genes underlying silkworm breeding. Interestingly, six of these genes were annotated with functions related to neural signal response. Among the six genes, BGIBMGA004050 encodes silkworm CREB-regulated_transcription_coactivator_1 (BmCRTC1), which was reported to be involved in energy-sensing pathways. These results suggested that improvement may have affected the nervous system of the silkworm. This research will provide new insights into the genetic basis underlying the genetic improvement of silkworms and possibly of other species.

Identification and effect of Zf-AD-containing C2H2 zinc finger genes on BmNPV replication in the silkworm (Bombyx mori).

Zf-AD-containing C2H2 zinc -finger genes (ZAD) are uniquely present and have lineage-specific expansion in arthropods. Arthropods are also the hosts of Baculoviruses. We studied the possible relationship between the lineage-specific expansion of ZAD genes and arthropod-Baculovirus co-evolution. We used the silkworm (Bombyx mori) as a model. We identified 73 ZAD genes (BmZAD) in the silkworm. Sequence-based similarity analysis showed that nine clusters involving 28 BmZADs may have undergone species-specific expansion in the silkworm. Expression pattern analysis showed that the BmZADs were divided into five groups. Group I comprised 10 genes with high expression in multiple tissues, suggesting that BmZADs may play roles in the development of various tissues. We identified six BmZADs that could be induced by the Nucleopolyhedrovirus (BmNPV). Among them, BmZAD69 expression is capable of responding to BmNPV infection, and the ZAD domain is indispensable for the function of BmZAD69 in BmNPV replication. We also detected a 3 bp deletion at 1.7 kb upstream of BmZAD69, which may make it more sensitive to BmNPV infection, and thus elevate the BmNPV resistance in Qiufeng_N, a strain with strong virus resistance. These data suggest that BmZADs may be involved in BmNPV infection and that ZAD genes may play a role in arthropod-Baculovirus co-evolution.

The beta-1, 4-N-acetylglucosaminidase 1 gene, selected by domestication and breeding, is involved in cocoon construction of Bombyx mori.

Holometabolous insects have distinct larval, pupal, and adult stages. The pupal stage is typically immobile and can be subject to predation, but cocoon offers pupal protection for many insect species. The cocoon provides a space in which the pupa to adult metamorphosis occurs. It also protects the pupa from weather, predators and parasitoids. Silk protein is a precursor of the silk used in cocoon construction. We used the silkworm as a model species to identify genes affecting silk protein synthesis and cocoon construction. We used quantitative genetic analysis to demonstrate that ß-1,4-N-acetylglucosaminidase 1 (BmGlcNase1) is associated with synthesis of sericin, the main composite of cocoon. BmGlcNase1 has an expression pattern coupled with silk gland development and cocoon shell weight (CSW) variation, and CSW is an index of the ability to synthesize silk protein. Up-regulated expression of BmGlcNase1 increased sericin content by 13.9% and 22.5% while down-regulation reduced sericin content by 41.2% and 27.3% in the cocoons of females and males, respectively. Genomic sequencing revealed that sequence variation upstream of the BmGlcNase1 transcriptional start site (TSS) is associated with the expression of BmGlcNase1 and CSW. Selective pressure analysis showed that GlcNase1 was differentially selected in insects with and without cocoons (ω1 = 0.044 vs. ω2 = 0.154). This indicates that this gene has a conserved function in the cocooning process of insects. BmGlcNase1 appears to be involved in sericin synthesis and silkworm cocooning.

[Cloning and function analysis of elongase of very long chain fatty acid gene Bmelo424 in silkworm].

Genes belonging to the elongases of very long chain fatty acid (ELOVL) family affect many physiological functions in organism. In this paper, Bmelo424 gene, a member of the ELOVL family in silkworm, was cloned and its ORF was 558 bp. Its protein sequence was predicted to have four transmembrane domains, six serine phosphorylation sites, eight threonine phosphorylation sites and four tyrosine phosphorylation sites, and its subcellular localization was in the endoplasmic reticulum. Secondary structure analysis showed that the percentage of alpha-helix and beta-strand was 26.7% and 20% respectively. The results of fluorescence quantitative PCR showed that Bmelo424 gene was expressed in all tissues of silkworm, especially with the highest expression in head. By heterologous expression of Bmelo424 gene in Saccharomyces cerevisiae, the effect of Bmelo424 gene on fatty acid elongation was studied. GC-MS results indicated that the fatty acid content of C16:1n-7 in S. cerevisiae with pYES2-Bmelo424 recombinant plasmid increased significantly, whereas the content of C16:0, C18:0 and C18:1n-9 decreased. The results of temperature stress revealed that Bmelo424 gene could improve the low temperature adaptability of S. cerevisiae, but its high temperature adaptability decreased. This provides a reference for exploring the function of Bmelo424 gene in silkworm.

Genome-wide identification and characterization of myosin genes in the silkworm, Bombyx mori.

Myosins are a large family of actin filament-based motor proteins with a broad range of functions such as intracellular membrane trafficking, endocytosis, exocytosis, organellar transport, growth cone motility, cytokinesis, and cell locomotion. They are found in many organisms from fungi to humans. The myosin gene family in Bombyx mori is poorly studied, even though the molecular functions of these genes in vertebrates and insects, such as Drosophila, are well known. We identified 16 myosin genes from B. mori and identified the myosin genes in 12 vertebrates, eight insects, three nematodes, and seven protozoa. The number of myosin genes in vertebrates is double the number in invertebrates. The number of myosin isoforms in classes I and II is larger in vertebrates compared to invertebrates. B. mori myosin genes can be classified into 11 classes. Compared to B. mori, some myosin classes are not present in other insects. Classes I, II, XVIII, and XXI appear to be important for insect survival because they are conserved among nine insects. The relatively large sizes of B. mori myosin genes are due to their longer introns. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis demonstrated that many B. mori myosin genes have tissue-specific expression and exhibit temporal-specific activity during metamorphosis. These data provide insights into evolutionary and functional aspects of B. mori myosin genes that could be useful for the study of homologous myosins in other Lepidoptera species.

Identification of Genes that Control Silk Yield by RNA Sequencing Analysis of Silkworm (Bombyx mori) Strains of Variable Silk Yield.

Silk is an important natural fiber of high economic value, and thus genetic study of the silkworm is a major area of research. Transcriptome analysis can provide guidance for genetic studies of silk yield traits. In this study, we performed a transcriptome comparison using multiple silkworms with different silk yields. A total of 22 common differentially expressed genes (DEGs) were identified in multiple strains and were mainly involved in metabolic pathways. Among these, seven significant common DEGs were verified by quantitative reverse transcription polymerase chain reaction, and the results coincided with the findings generated by RNA sequencing. Association analysis showed that BGIBMGA003330 and BGIBMGA005780 are significantly associated with cocoon shell weight and encode uridine nucleosidase and small heat shock protein, respectively. Functional annotation of these genes suggest that these play a role in silkworm silk gland development or silk protein synthesis. In addition, we performed principal component analysis (PCA) in combination with wild silkworm analysis, which indicates that modern breeding has a stronger selection effect on silk yield traits than domestication, and imply that silkworm breeding induces aggregation of genes related to silk yield.

Genome-wide identification and analysis of elongase of very long chain fatty acid genes in the silkworm, Bombyx mori.

Very long chain fatty acids (VLCFAs), such as sphingolipids, are components of cellular lipids, which are essential for cell proliferation. Mutations in the genes that encode proteins participating in VLCFA biosynthesis may cause inherited diseases, such as macular degeneration. Elongases of very long chain fatty acid (ELOVL) are enzymes that are involved in the biosynthesis of VLCFAs. Here, a total of 13 ELOVL genes, distributed across three chromosomes, were identified in the silkworm genome; all the ELOVL members contain a distinct ELO domain and a conserved HXXHH motif. Phylogenetic reconstruction was performed to analyze the evolutionary relationships among different species and to predict gene functions. The 13 ELOVL genes were assigned to the ELOVL3/6, ELOVL1/7, and ELOVL4 clades. Microarray and semiquantitative PCR analyses indicated that these genes are differentially expressed among various tissues, in turn suggesting functional divergence in the growth and development of each tissue. Further investigation showed that the expression level of the BGIBMGA000424 gene is significantly negatively correlated with the cocoon-shell weight among different silkworm strains. Taken together, the present study is the first comprehensive analysis of ELOVL genes in silkworm, and the results may serve as a foundation for further analysis of the physiological functions of ELOVL genes in silkworm.

BmYki is transcribed into four functional splicing isoforms in the silk glands of the silkworm Bombyx mori.

Yorkie (Yki), the Drosophila homolog of vertebrate yes-associated protein (YAP), is a key effector of the Hippo pathway, which modulates organ size via the transcriptional regulation of downstream targets involved in cell proliferation and survival. YAP has been shown to be expressed as multiple splicing isoforms in mammals, but thus far, no evidence of alternatively spliced Yki isoforms has been reported in insects. Here, we confirmed that the Yki protein of the silkworm Bombyx mori, BmYki, is transcribed in the silk gland into at least four splicing isoforms, named BmY1329, BmY1314, BmY1188, and BmY1173. Further analysis revealed that BmY1329 and BmY1314 each contain two WW domains, whereas BmY1188 and BmY1173 each contain only one WW domain. Each BmYki isoform functions in regulating expression of Yki target genes in cultured B. mori embryonic cells, and exhibits a few different effects on the expression of Yki targets. Interestingly, the expression of silk fibroin protein genes could also be influenced by each of the BmYki isoforms, suggesting that BmYki is involved in the regulation of silk protein-coding genes. This study provides novel insights into the role of BmYki. The contribution of each BmYki isoform to the modulation of gene expression will be of great interest for further study.

QTL analysis of cocoon shell weight identifies BmRPL18 associated with silk protein synthesis in silkworm by pooling sequencing.

Mechanisms that regulate silk protein synthesis provide the basis for silkworm variety breeding and silk gland bioreactor optimization. Here, using the pooling sequencing-based methodology, we deciphered the genetic basis for the varied silk production in different silkworm strains. We identified 8 SNPs, with 6 on chromosome 11 and 1 each on chromosomes 22 and 23, that were linked with silk production. After conducting an association analysis between gene expression pattern, silk gland development and cocoon shell weight (CSW), BMGN011620 was found to be regulating silk production. BMGN011620 encodes the 60S ribosomal protein, L18, which is an indispensable component of the 60S ribosomal subunit; therefore we named it BmRPL18. Moreover, the clustering of linked SNPs on chromosome 11 and the analysis of differentially expressed genes reported in previous Omics studies indicated that the genes regulating silk protein synthesis may exhibit a clustering distribution in the silkworm genome. These results collectively advance our understanding of the regulation of silk production, including the role of ribosomal proteins and the clustered distribution of genes involved in silk protein synthesis.

Cloning and expression analysis of BmYki gene in silkworm, Bombyx mori.

The transcriptional coactivator Yorkie(Yki), is a critical downstream effector of the Hippo(Hpo) signaling pathway that controls organ size through the regulation of cell proliferation and apoptosis. During the past ten years the biological function of Yki has been studied extensively in Drosophila and a few other insects, however, little is known about it in the silkworm, Bombyx mori, a major research model of lepidopteran insect. Here, we describe the isolation, characterization and expression of the B. mori Yki ortholog, BmYki. The coding sequence of the BmYki was 1314 bp in length, encoding a protein of 437 amino acids containing two conserved WW domains. BmYki transcripts were ubiquitous but not abundant in all detected tissues and developmental stages. Comparatively, it was expressed at pretty high level in silk glands and at the stage of fifth-instar day-3 larvae. Overexpression of BmYki in cultured B. mori embryonic cells significantly promoted transcription of genes associated with cell proliferation and apoptosis, indicating that BmYki functions in the regulation of organ growth-related biological processes. Interestingly, transcription of silk protein-coding genes and transcription factors regulating the synthesis of silk proteins was downregulated remarkably, suggesting that BmYki was involved in the regulation of silk protein synthesis. This study provides new insights into the role of BmYki in Hpo pathway regulation in silkworm.

[Screening, identification and optimization of fermentation conditions of an antagonistic endophyte to mulberry bacterial blight].

OBJECTIVE: Antagonistic endophytic strains with strongly inhibitory activity to mulberry bacterial blight (P. syringae pv. mori) were isolated from mulberry endophytes, we identified the antagonistic endophyte and optimized the fermentation conditions. METHOD: Streak plate method was used to separate the endophytes from healthy mulberry tissues after strict surface disinfection. Antagonistic endophytes were screened out through inhibition zone method. Strain SWg2 was identified by morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis. The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment. RESULT: In total 77 endophytic strains have been isolated from healthy mulberry. SWg2 showed strong and stable antagonistic activity to mulberry bacterial blight. The morphological, cultural, physiological, and biochemical characteristics assays indicated that SWg2 belongs to Pantoea sp. The 16S rDNA sequence phylogenetic analysis reveals that SWg2 appeared a sister lineage to P. agglomerans. The optimized culture conditions of strain SWg2 were liquid volume 20 mL in 100 mL flask, 170 r/min at 28 degrees C, inoculation size of 4% for 5 d with a medium of 2.0% glycerol, 2.0% NH4NO3, 0.1% KH2PO4, 0.15% MgSO4 x 7H2O at initial pH of 7.5. CONCLUSION: The antagonistic endophytic strain SWg2 to mulberry bacterial blight was identified as P. agglomerans. SWg2 strain shows stronger antagonistic action to mulberry bacterial blight under optimized fermentation conditions.

[Latest progress in postmortem interval estimation].

Accurate estimation of the postmortem interval (PMI) has been one of the most important and complicated issues in the forensic practice. In order to provide novel perspectives for the future research concerning PMI, the advantages and disadvantages of related traditional methods, postmortem degradation of nucleic acid and tissue, the componential change of vitreous humor and histological biochemistry since 2002 have been introduced and compared in this review.

Effect of organophosphate phoxim exposure on certain oxidative stress biomarkers in the silkworm.

Organophosphorus (OP) insecticides are widely used in agriculture, which are toxic to insect pests and nontarget organisms. The current study mainly assessed the effect of the pesticide phoxim on oxidative stress by certain biomarkers in the fat body and midgut of the silkworm, Bombyx mori (L.), after exposure to 50% lethal concentration (LC50) of phoxim for 2 h. Malondialdehyde (MDA) content, activity of glutathione transferase (GST), and expression of GST at transcriptional level were assayed. LC50 value of phoxim was 2.5 mg/liter at 2-h exposure for the day 3 of the fifth-instar larvae. After exposure of phoxim, MDA content in the fat body significantly increased at 4-20 h posttreatment (p.t.),the highest increase was approximately 4.11-fold from 0.451 +/- 0.053 to 1.854 +/- 0.113 nmol/mg protein compared with corresponding control. In the midgut, significant increase in the MDA content (from 1.40- to 3.16-fold) was observed at 8-42 h p.t. The activity of GSTs increased to 1.48-2.00-fold at 24-42 h p.t. and 1.33-1.48-fold at 20-24 h p.t. in the fat body and midgut, respectively. The peroxidase activity of GSTs also was induced, which increased to 1.46-2.06-fold and 1.31-1.50-fold in the fat body and midgut, respectively. BmGSTe8 showed a late up-regulation of transcripts at 24-42 h after exposure to phoxim, which might contribute to the improved phoxim tolerance of silkworm larvae. These results indicated that phoxim could trigger oxidative stress and that MDA content and GST activity might be used as biomarkers of OP insecticide exposure. In addition, activity of GSTs were more inducible in the fat body than in midgut.

Concentrations of heavy metals and polycyclic aromatic hydrocarbons in needles of Masson pine (Pinus massoniana L.) growing nearby different industrial sources.

Emissions from industrial activities pose a serious threat to human health and impose the need for monitoring both inorganic and organic pollutants in industrial areas. We selected Masson pine (Pinus massoniana L.) as potential biomonitor and collected the current (C) and previous year (C+1) needles from three industrial sites dominated by petrochemical, ceramics manufacturing, and iron and steel smelting plants and one remote site to determine heavy metals (Cu, Cd, Pb, Zn, Cr, Ni and Co) and polycyclic aromatic hydrocarbons (PAHs) in unwashed and water-washed needles. Both unwashed and washed C+1 needles showed generally higher concentrations of heavy metals and PAHs than C needles, although the washed needles more clearly spotlighted the accumulation effect of PAHs over exposure time. Water-washing resulted in a significant decrease in needle PAH concentrations with more significant effects shown in C needles. By contrast, needle heavy metal concentrations were much less affected by washing. Although heavy metals and PAHs might differ in adsorption and uptake strategies, their higher concentrations in the needles at the industrial sites indicated conspicuous contamination due to industrial emissions there. The PAH distribution patterns in pine needles accorded with the real types of energy consumption in the study sites and were efficiently used for pinpointing local pollutant sources.

Identification, genomic organization and expression pattern of glutathione S-transferase in the silkworm, Bombyx mori.

Glutathione S-transferases (GSTs) are a multifunctional supergene family and some play an important role in insecticide resistance. We have identified 23 putative cytosolic GSTs by searching the new assembly of the Bombyx mori genome sequence. Phylogenetic analyses on the amino acid sequences reveal that 21 of the B. mori GSTs fall into six classes represented in other insects, the other two being unclassified. The majority of the silkworm GSTs belong to the Delta, Epsilon, and Omega classes. Most members of each class are tandemly arranged in the genome, except for the Epsilon GSTs. Expressed sequence tags (ESTs) corresponding to 19 of the 23 GSTs were found in available databases. Furthermore RT-PCR experiments detected expression of all the GSTs in multiple tissues on day 3 of fifth instar larvae. Surprisingly, we found little or no expression of most Delta and Epsilon GSTs in the fat body, which is thought to be the main detoxification organ. This may explain the sensitivity of the silkworm to certain insecticides. Our data provide some insights into the evolution of the B. mori GST family and the functions of individual GST enzymes.