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As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval- pupal metamorphosis. [ABSTRACT FROM AUTHOR]

Background: Silk has numerous unique properties that make it a staple of textile manufacturing for several thousand years. However, wider applications of silk in modern have been stalled due to limitations of traditional silk produced by Bombyx mori. While silk is commonly produced by B. mori, several wild non-mulberry silkmoths--especially members of family Saturniidae--produce silk with superior properties that may be useful for wider applications. Further utilization of such silks is hampered by the non-domestication status or limited culturing population of wild silkworms. To date there is insufficient basic genomic or transcriptomic data on these organisms or their silk production. Results: We sequenced and compared the transcriptomes of silk glands of six Saturniidae wild silkmoth species through next-generation sequencing technology, identifying 37758 ~ 51734 silkmoth unigenes, at least 36.3% of which are annotated with an e-value less than 10-5. Sequence analyses of these unigenes identified a batch of genes specific to Saturniidae that are enriched in growth and development. Analyses of silk proteins including fibroin and sericin indicate intra-genus conservation and inter-genus diversification of silk protein features among the wild silkmoths, e.g., isoelectric points, hydrophilicity profile and amino acid composition in motifs of silk H-fibroin. Interestingly, we identified p25 in two of the silkmoths, which were previously predicted to be absent in Saturniidae. There are rapid evolutionary changes in sericin proteins, which might account for the highly heterogeneity of sericin in Saturniidae silkmoths. Within the six sikmoths, both colored-cocoon silkmoth specific transcripts and differentially expressed genes between the colored-cocoon and non-colored-cocoon silkmoths are significantly enriched in catalytic activity, especially transferase activity, suggesting potentially viable targets for future gene mining or genetic manipulation. Conclusions: Our results characterize novel and potentially valuable gene resources of saturniid silkmoths that may facilitate future genetic improvement and modification of mulberry silkworms. Our results suggest that the disparate features of silk- -coloration, retention, strength, etc. --are likely not only due to silk proteins, but also to the environment of silk assembly, and more specifically, that stable silk coloration exhibited by some Saturniidae silkmoths may be attributable to active catalytic progress in pigmentation. [ABSTRACT FROM AUTHOR]

Lepidoptera, the most diverse group of insects, exhibit female heterogamy (Z0 or ZW), which is different from most other insects (male heterogamy, XY). Previous studies suggest a single origin of the Z chromosome. However, the origin of the lepidopteran W chromosome remains poorly understood. Here, we assemble the genome from females down to the chromosome level of a model insect (Bombyx mori) and identify a W chromosome of approximately 10.1 megabase using a newly developed tool. In addition, we identify 3593 genes that were not previously annotated in the genomes of B. mori. Comparisons of 21 lepidopteran species (including 17 ZW and four Z0 systems) and three trichopteran species (Z0 system) reveal that the formation of Ditrysia W involves multiple mechanisms, including previously proposed canonical and noncanonical models, as well as a newly proposed mechanism called single-Z turnover. We conclude that there are multiple independent origins of the W chromosome in the Ditrysia (most moths and all butterflies) of Lepidoptera. [ABSTRACT FROM AUTHOR]

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor of dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermal features in some insects. [ABSTRACT FROM AUTHOR]

Background: In contrast to wild species, which have typically evolved phenotypes over long periods of natural selection, domesticates rapidly gained human-preferred agronomic traits in a relatively short-time frame via artificial selection. Under domesticated conditions, many traits can be observed that cannot only be due to environmental alteration. In the case of silkworms, aside from genetic divergence, whether epigenetic divergence played a role in domestication is an unanswered question. The silkworm is still an enigma in that it has two DNA methyltransferases (DNMT1 and DNMT2) but their functionality is unknown. Even in particular the functionality of the widely distributed DNMT1 remains unknown in insects in general. Results: By embryonic RNA interference, we reveal that knockdown of silkworm Dnmt1 caused decreased hatchability, providing the first direct experimental evidence of functional significance of insect Dnmt1. In the light of this fact and those that DNA methylation is correlated with gene expression in silkworms and some agronomic traits in domesticated organisms are not stable, we comprehensively compare silk gland methylomes of 3 domesticated (Bombyx mori) and 4 wild (Bombyx mandarina) silkworms to identify differentially methylated genes between the two. We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication. Further study of more domesticated and wild silkworms narrowed down the domesticates’ epimutations, and we were able to identify a number of differential genes. One such gene showing demethyaltion in domesticates correspondently displays lower gene expression, and more interestingly, has experienced selective sweep. A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a possible correlation with the enlargement of silk glands in domesticated silkworms. Conclusions: Our results imply epigenetic influences at work during domestication, which gives insight into long time historical controversies regarding acquired inheritance. [ABSTRACT FROM AUTHOR]

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. We propose, therefore, that insect wing development occurs in an Antp-dependent manner. [ABSTRACT FROM AUTHOR]

Wild type silkworm larvae have opaque white skin, whereas the mutants Sel (Sepialumazine) and Xan (Xanthous) are yellow-skinned. Previous genetic analysis indicated that Sel and Xan are on established linkage groups 24 (0.0) and 27 (0.0), respectively. However, in constructing a molecular linkage map using simple sequence repeat (SSR) loci, we found that the two mutations were linked. To confirm this finding, we developed a set of SSR markers and used them to score reciprocal backcross populations. Taking advantage of the lack of crossing-over in female silkworms, we found that the progeny of backcrosses between F1 females and males of the parental strains (BC1F) of the two visible mutations had the same inheritance patterns linked to the same SSR markers. This indicated that the two visible mutations belonged to the same chromosome. To confirm this finding, we tested for independent assortment by crossing Sel and Xan marker strains with each other to obtain F1 and F2 populations. Absence of the expected wild type class among 5000 F2 progeny indicated that the two visible mutations were located on the same linkage group. We carried out recombination analysis for each mutation by scoring 190 progeny of backcrosses between F1 males and parental females (BC1M) and constructed a linkage map for each strain. The results indicated that the Sel gene was 12 cM from SSR marker S2404, and the Xan gene was 7.03 cM from SSR marker S2407. To construct a combined SSR map and to avoid having to discriminate the two similar dominant mutations in heterozygotes, we carried out recombination analysis by scoring recessive wild type segregants of F2 populations for each mutation. The results showed that the Sel and Xan genes were 13 cM and 13.7 cM from the S2404 marker, respectively, consistent with the possibility that they are alleles of the same locus, which we provisionally assigned to SSR linkage group 24. We also used the F2 recessive populations to construct two linkage groups for the Sel and Xan genes. [ABSTRACT FROM AUTHOR]

The traditional Chinese medicine Astragalus polysaccharide (APS) has been widely used to improve glucose homeostasis and immunoregulator properties. In recent years, it has also been shown to extend the lifespan of Caenorhabditis elegans; however, the underlying molecular mechanisms are not fully understood. Here, our study shows that APS could significantly extend adult stage, mean, and maximum lifespan of the silkworm, Bombyx mori and increase body weight without affecting food intake and fecundity. Meanwhile, the activities of glutathione S-transferase and superoxide dismutase are significantly enhanced, and the reaction oxygen species content is reduced concomitantly. Moreover, the activity of lysozyme is increased dramatically. In addition, APS rescues the shortened lifespan by Bacillus thuringiensis infection in silkworm. Furthermore, the transcription of the crucial genes involved in endoplasmic reticulum stress is upregulated upon the endoplasmic reticulum stress stimulation. APS also significantly ameliorates endoplasmic reticulum stress in silkworm cell line and in vivo. Together, the results of this study indicate that APS can prolong the silkworm lifespan by mitigating endoplasmic reticulum stress. This study improves our understanding of the molecular mechanism of APS-induced lifespan extension and highlights the importance of the silkworm as an experimental animal for evaluating the effects and revealing the mechanisms in lifespan extension of traditional Chinese medicine. [ABSTRACT FROM AUTHOR]

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene or by defects in the tetrahydrobiopterin (BH4) synthesis pathway. Here, by positional cloning, we report that the 6-pyruvoyl-tetrahydropterin synthase (PTPS) gene, encoding a key enzyme of BH4 biosynthesis, is responsible for the alc (albino C) mutation that displays pale body color, head shaking, and eventually lethality after the first molting in silkworm. Compared to wild type, the alc mutant produced more substrates (phenylalanine (Phe) and tyrosine (Tyr)) and generated less DOPA and dopamine. Application of 2,4-diamino-6-hydroxypyrimidine (DAHP) to block BH4 synthesis in the wild type effectively produced the alc-like phenotype, while BH4 supplementation rescued the defective body color and lethal phenotype in both alc and DAHP-treated individuals. The detection of gene expressions and metabolic substances after drugs treatments in alc and normal individuals imply that silkworms and humans have a high similarity in the drugs metabolic features and the gene pathway related to BH4 and the dopamine biosynthesis. We propose that the alc mutant could be used as an animal model for drug evaluation for BH4-deficient PKU. [ABSTRACT FROM AUTHOR]

The tyrosine kinases (TKs) are important parts of metazoan signaling pathways and play significant roles in cell growth, development, apoptosis and disease. Genome-wide characterization of TKs has been conducted in many metazoans, however, systematic information about this family in Lepidoptera is still lacking. We retrieved 33 TK-encoding genes in silkworm and classified them into 25 subfamilies by sequence analysis, without members in AXL, FRK, PDGFR, STYK1 and TIE subfamilies. Although domain sequences in each subfamily are conserved, TKs in vertebrates tend to be remarkably conserved and stable. Our results of phylogenetic analysis supported the previous conclusion for the second major expansion of TK family. Gene-Ontology (GO) analysis revealed that a higher proportion of BmTKs played roles in binding, catalysis, signal transduction, metabolism, biological regulation and response to stimulus, compared to all silkworm genes annotated in GO. Moreover, the expression profile analysis of BmTKs among multiple tissues and developmental stages demonstrated that many genes exhibited stage-specific and/or sex-related expression during embryogenesis, molting and metamorphosis, and that 8 BmTKs presented tissue-specific high expression. Our study provides systematic description of silkworm tyrosine kinases, and may also provide further insights into metazoan TKs and assist future studies addressing their functions. [ABSTRACT FROM AUTHOR]

The genetic basis of body shape and coloration patterns on caterpillars is often assumed to be regulated separately, but it is possible that common molecules affect both types of trait simultaneously. Here we examine the genetic basis of a spontaneous cuticle defect in silkworm, where larvae exhibit a bamboo-like body shape and decreased pigmentation. We performed linkage mapping and mutation screening to determine the gene product that affects body shape and coloration simultaneously. In these mutant larvae we identified a null mutation in BmorCPH24, a gene encoding a cuticular protein with low complexity sequence. Spatiotemporal expression analyses showed that BmorCPH24 is expressed in the larval epidermis postecdysis. RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout of BmorCPH24 produced the abnormal body shape and the inhibited pigment typical of the mutant phenotype. In addition, our results showed that BmorCPH24 may be involved in the synthesis of endocuticle and its disruption-induced apoptosis of epidermal cells that accompanied the reduced expression of R&R-type larval cuticle protein genes and pigmentation gene Wnt1. Strikingly, BmorCPH24, a fast-evolving gene, has evolved a new function responsible for the assembly of silkworm larval cuticle and has evolved to be an indispensable factor maintaining the larval body shape and its coloration pattern. This is the first study to identify a molecule whose pleiotropic function affects the development of body shape and color patterns in insect larvae. [ABSTRACT FROM AUTHOR]

Miniature inverted-repeat transposable elements (MITEs) have attracted much attention due to their widespread occurrence and high copy numbers in eukaryotic genomes. However, the systematic knowledge about MITEs in insects and other animals is still lacking. In this study, we identified 6012 MITE families from 98 insect species genomes. Comparison of these MITEs with known MITEs in the NCBI non-redundant database and Repbase showed that 5701(~95%) of 6012 MITE families are novel. The abundance of MITEs varies drastically among different insect species, and significantly correlates with genome size. In general, larger genomes contain more MITEs than small genomes. Furthermore, all identified MITEs were included in a newly constructed database (iMITEdb) (http://gene.cqu.edu.cn/iMITEdb/), which has functions such as browse, search, BLAST and download. Overall, our results not only provide insight on insect MITEs but will also improve assembly and annotation of insect genomes. More importantly, the results presented in this study will promote studies of MITEs function, evolution and application in insects. [ABSTRACT FROM AUTHOR]

Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of b-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild- type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of BmiAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a crosssection of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazaomln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao- mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research. [ABSTRACT FROM AUTHOR]

Epigenetic regulation in insects may have effects on diverse biological processes. Here we survey the methylome of a model insect, the silkworm Bombyx mori, at single-base resolution using Illumina high-throughput bisulfite sequencing (MethylC-Seq). We conservatively estimate that 0.11% of genomic cytosines are methylcytosines, all of which probably occur in CG dinucleotides. CG methylation is substantially enriched in gene bodies and is positively correlated with gene expression levels, suggesting it has a positive role in gene transcription. We find that transposable elements, promoters and ribosomal DNAs are hypomethylated, but in contrast, genomic loci matching small RNAs in gene bodies are densely methylated. This work contributes to our understanding of epigenetics in insects, and in contrast to previous studies of the highly methylated genomes of Arabidopsis and human, demonstrates a strategy for sequencing the epigenomes of organisms such as insects that have low levels of methylation. [ABSTRACT FROM AUTHOR]

A single-base pair resolution silkworm genetic variation map was constructed from 40 domesticated and wild silkworms, each sequenced to approximately threefold coverage, representing 99.88% Of the genome. We identified ~16 million single-nucleotide polymorphisms, many indels, and structural variations. We find that the domesticated silkworms are dearly genetically differentiated from the wild ones, but they have maintained large levels of genetic variability, suggesting a short domestication event involving a large number of individuals. We also identified signals of selection at 354 candidate genes that may have been important during domestication, some of which have enriched expression in the silk gland, midgut, and testis. These data add to our understanding of the domestication processes and may have applications in devising pest control strategies and advancing the use of silkworms as efficient bioreactors. [ABSTRACT FROM AUTHOR]

Wound infection caused by multiantibiotic-resistant bacteria has become a serious problem, and more effective antibacterial agents are required. Herein, we report the preparation of wound dressings using the biocompatible chitosan (CS) as a reducing and stabilizing agent in the synthesis of 2-mercapto-1-methylimidazole (MMT)-capped gold nanocomposites (CS-Au@MMT), with efficient antibacterial effects. The synergistic effects of AuNPs, MMT, and CS led to the disruption of bacterial membranes. After blending with gelatin, crosslinking with tannin acid, and freeze-drying, CS-gelatin (CS-Au@MMT/gelatin) dressing was prepared. It had good mechanical properties as well as efficient water absorption and retention capacities. It exhibited outstanding biocompatibility both in vitro and in a cell-based wound infection model. Moreover, the in vivo rabbit wound healing model revealed that the CS-Au@MMT/gelatin dressing possesses significant antibacterial potential against methicillin-resistant Staphylococcus aureus-associated wound infection. Therefore, the CS-Au@MMT/gelatin dressing described in this study may have huge potential in biomedical applications. [ABSTRACT FROM AUTHOR]