Deciphering the Genetic Basis of Silkworm Cocoon Colors Provides New Insights into Biological Coloration and Phenotypic Diversification.

The genetic basis of phenotypic variation is a long-standing concern of evolutionary biology. Coloration has proven to be a visual, easily quantifiable, and highly tractable system for genetic analysis and is an ever-evolving focus of biological research. Compared with the homogenized brown-yellow cocoons of wild silkworms, the cocoons of domestic silkworms are spectacularly diverse in color, such as white, green, and yellow-red; this provides an outstanding model for exploring the phenotypic diversification and biological coloration. Herein, the molecular mechanism underlying silkworm green cocoon formation was investigated, which was not fully understood. We demonstrated that five of the seven members of a sugar transporter gene cluster were specifically duplicated in the Bombycidae and evolved new spatial expression patterns predominantly expressed in silk glands, accompanying complementary temporal expression; they synergistically facilitate the uptake of flavonoids, thus determining the green cocoon. Subsequently, polymorphic cocoon coloring landscape involving multiple loci and the evolution of cocoon color from wild to domestic silkworms were analyzed based on the pan-genome sequencing data. It was found that cocoon coloration involved epistatic interaction between loci; all the identified cocoon color-related loci existed in wild silkworms; the genetic segregation, recombination, and variation of these loci shaped the multicolored cocoons of domestic silkworms. This study revealed a new mechanism for flavonoids-based biological coloration that highlights the crucial role of gene duplication followed by functional diversification in acquiring new genetic functions; furthermore, the results in this work provide insight into phenotypic innovation during domestication.

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.

Molecular basis of the silkworm mutant rel causing red egg color and embryonic death.

The coloration and hatchability of insect eggs can affect individual and population survival. However, few genetic loci have been documented to affect both traits, and the genes involved in regulating these two traits are unclear. The silkworm recessive mutant rel shows both red egg color and embryo mortality. We studied the molecular basis of the rel phenotype formation. Through genetic analysis, gene screening and sequencing, we found that two closely linked genes, BGIBMGA003497 (Bm-re) and BGIBMGA003697 (BmSema1a), control egg color and embryo mortality, respectively. Six base pairs of the Bm-re gene are deleted in its open reading frame, and BmSema1a is expressed at abnormally low levels in mutant rel . BmSema1a gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats (CRISPR)/CRISPR-associate protein 9. Deficiency of the BmSema1a gene can cause the death of silkworm embryos. This study revealed the molecular basis of silkworm rel mutant formation and indicated that the Sema1a gene is essential for insect embryo development.

Flight Muscle and Wing Mechanical Properties are Involved in Flightlessness of the Domestic Silkmoth, Bombyx mori.

Flight loss has occurred in many winged insect taxa. The flightless silkmoth Bombyx mori, is domesticated from the wild silkmoth, Bombyx mandarina, which can fly. In this paper, we studied morphological characteristics attributed to flightlessness in silkmoths. Three domestic flightless B. mori strains and one B. mandarina population were used to compare morphological components of the flight apparatus, including wing characteristics (shape, forewing area, loading, and stiffness), flight muscle (weight, ratio, and microscopic detail) and body mass. Compared with B. mandarina, B. mori strains have a larger body, greater wing loading, more flexible wings and a lower flight muscle ratio. The arrangement in microscopy of dorsal longitudinal flight muscles (DLFMs) of B. mori was irregular. Comparative analysis of the sexes suggests that degeneration of flight muscles and reduction of wing mechanical properties (stiffness) are associated with silkmoth flightlessness. The findings provide important clues for further research of the molecular mechanisms of B. mori flight loss.

Excess melanin precursors rescue defective cuticular traits in stony mutant silkworms probably by upregulating four genes encoding RR1-type larval cuticular proteins.

Melanin and cuticular proteins are vital cuticle components in insects. Cuticular defects caused by mutations in cuticular protein-encoding genes can obstruct melanin deposition. The effects of changes in melanin on the expression of cuticular protein-encoding genes, the cuticular and morphological traits, and the origins of these effects are unknown. We found that the cuticular physical characteristics and the expression patterns of larval cuticular protein-encoding genes markedly differed between the melanic and non-melanic integument regions. By using four p multiple-allele color pattern mutants with increasing degrees of melanism (+p, pM, pS, and pB), we found that the degree of melanism and the expression of four RR1-type larval cuticular protein-encoding genes (BmCPR2, BmLcp18, BmLcp22, and BmLcp30) were positively correlated. By modulating the content of melanin precursors and the expression of cuticular protein-encoding genes in cells in tissues and in vivo, we showed that this positive correlation was due to the induction of melanin precursors. More importantly, the melanism trait introduced into the BmCPR2 deletion strain Dazao-stony induced up-regulation of three other similar chitin-binding characteristic larval cuticular protein-encoding genes, thus rescuing the cuticular, morphological and adaptability defects of the Dazao-stony strain. This rescue ability increased with increasing melanism levels. This is the first study reporting the induction of cuticular protein-encoding genes by melanin and the biological importance of this induction in affecting the physiological characteristics of the cuticle.

Ara-c induces cell cycle G1/S arrest by inducing upregulation of the INK4 family gene or directly inhibiting the formation of the cell cycle-dependent complex CDK4/cyclin D1.

Cytosine arabinoside (Ara-c) is a pyrimidine anti-metabolite that is capable of interfering with cellular proliferation by inhibiting DNA synthesis. Each inhibitor of cyclin-dependent kinase 4 (INK4) family member has the ability to bind to cyclin-dependent kinase 4 (CDK4) and inhibit the formation of the cell cycle-dependent CDK4/cyclin D1 complex, subsequently leading to cell cycle arrest in the G1/S phase. In this study, the expression of INK4 family genes in kidney cancer and the impact of these genes on patient prognosis were examined. Additionally, the effects of INK4 family genes and Ara-c on cell proliferation and tumor formation and development were examined. Finally, a potential association between Ara-c-induced cell cycle arrest and INK4-associated gene expression was evaluated. An upregulation of INK4 family genes was found to be positively correlated with the prognosis of patients with kidney cancer. Both the INK4 family genes and Ara-c were shown to induce cell cycle arrest and inhibit tumor formation and development. Moreover, Ara-c-induced cell cycle arrest was found to be associated with an Ara-c-induced upregulation of INK4 family gene expression, which ultimately inhibited the formation of the CDK4/cyclin D1 complex. These findings suggested that an upregulation of INK4 family genes has a positive effect on kidney cancer prognosis and can inhibit the formation and development of tumors. Moreover, Ara-c was shown to promote the upregulation of INK4 family genes, at the same time, Ara-c could directly regulate the cell cycle-dependent genes CDK4 and cyclin D1 (CCND1), independent of the INK4 family genes.

Genome-Wide Identification and Characterization of Tyrosine Kinases in the Silkworm, Bombyx mori.

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.

Genome-Wide Identification and Characterization of WD40 Protein Genes in the Silkworm, Bombyx mori.

WD40 proteins are scaffolding molecules in protein-protein interactions and play crucial roles in fundamental biological processes. Genome-wide characterization of WD40 proteins in animals has been conducted solely in humans. We retrieved 172 WD40 protein genes in silkworm (BmWD40s) and identified these genes in 7 other insects, 9 vertebrates and 5 nematodes. Comparative analysis revealed that the WD40 protein gene family underwent lineage-specific expansions during animal evolution, but did not undergo significant expansion during insect evolution. The BmWD40s were categorized into five clusters and 12 classes according to the phylogenetic classification and their domain architectures, respectively. Sequence analyses indicated that tandem and segmental duplication played minor roles in producing the current number of BmWD40s, and domain recombination events of multi-domain BmWD40s might have occurred mainly after gene duplication events. Gene Ontology (GO) analysis revealed that a higher proportion of BmWD40s was involved in processes, such as binding, transcription-regulation and cellular component biogenesis, compared to all silkworm genes annotated in GO. Microarray-based analysis demonstrated that many BmWD40s had tissue-specific expression and exhibited high and/or sex-related expression during metamorphosis. These findings contribute to a better understanding of the evolution of the animal WD40 protein family and assist the study of the functions of BmWD40s.

Comparative Analysis of Transcriptomes among Bombyx mori Strains and Sexes Reveals the Genes Regulating Melanic Morph and the Related Phenotypes.

As a source of insect polymorphism, melanism plays an important role in ecological adaption and usually endows advantageous phenotypic-effects on insects. However, due to the mechanistic diversity, there are knowledge gaps in the molecular mechanisms underlying melanism and the related phenotypes. In silk moths, a recessive melanic mutant (sex-controlled melanism, sml) strain exhibits extended adult longevity. We took a transcriptome approach to perform a comparative analysis between this sml strain and a wild-type strain (Dazao). Our analysis resulted in the identification of 59 unique differentially expressed genes in the melanic mutant. Two key genes (laccase2 and yellow) involved in melanin formation were significantly up-regulated in melanic individuals. The laccase2 B-type isoform (BGIBMGA006746) was found to likely participate in the silkworm cuticular melanism process at late pupal stage. Moreover, we discovered 22 cuticular protein encoding genes with the possible function in melanin transport and/or maintenance. Based on our findings, we presume that the longer survival of the melanic sml male moths might be associated with the enhanced antioxidant defense systems and a reduction in the insulin/IGF-1 signaling pathway (IIS). These findings will facilitate the understanding of the molecular basis underlying melanism and the derived phenotypic-effects.

Microarray analysis of New Green Cocoon associated genes in silkworm, Bombyx mori.

Green cocoons in silkworm, Bombyx mori, are caused by flavonoids accumulation in the silk proteins, fibroin and sericin. Despite the economic value of natural green cocoon and medical value of flavonoids, there is limited understanding of the molecular mechanism regulating flavonoids uptake in silkworm, which is tightly associated with the trait of green cocoon. The purpose of this study is to perform a comprehensive analysis to understand the molecular mechanisms of flavonoids uptake in silkworm based on microarray analyses. The study subject was the New Green Cocoon from the silkworm strains, G200 and N100, a new spontaneous dominant green cocoon trait identified in the 2000s. The genes regulating this trait are independent of other green cocoon genes previously reported. Genome-wide gene expression was compared between the New Green Cocoon producing silkworm strains, G200 and N100, and the control sample, which is the white cocoon producing strain 872B. Among these strains, N100 and 872B are near-isogenic lines. The results showed that 130 genes have consistently changing expression patterns in the green cocoon strains when compared with the white cocoon strain. Among these, we focused on the genes related to flavonoids metabolism and absorption, such as sugar transporter genes and UDP-glucosyltransferase genes. Based on our findings, we propose the potential mechanisms for flavonoids absorption and metabolism in silkworm. Our results imply that silkworm might be used as an underlying model for flavonoids in pharmaceutical research.

Molecular mapping and characterization of the silkworm apodal mutant.

The morphological diversity of insects is important for their survival; in essence, it results from the differential expression of genes during development of the insect body. The silkworm apodal (ap) mutant has degraded thoracic legs making crawling and eating difficult and the female is sterile, which is an ideal subject for studying the molecular mechanisms of morphogenesis. Here, we confirmed that the infertility of ap female moths is a result of the degradation of the bursa copulatrix. Positional cloning of ap locus and expression analyses reveal that the Bombyx mori sister of odd and bowl (Bmsob) gene is a strong candidate for the ap mutant. The expression of Bmsob is down-regulated, while the corresponding Hox genes are up-regulated in the ap mutant compared to the wild type. Analyses with the dual luciferase assay present a declined activity of the Bmsob promoter in the ap mutant. Furthermore, we demonstrate that Bmsob can inhibit Hox gene expression directly and by suppressing the expression of other genes, including the BmDsp gene. The results of this study are an important contribution to our understanding of the diversification of insect body plan.

A novel laminin ß gene BmLanB1-w regulates wing-specific cell adhesion in silkworm, Bombyx mori.

Laminins are important basement membrane (BM) components with crucial roles in development. The numbers of laminin isoforms in various organisms are determined by the composition of the different α, ß, and γ chains, and their coding genes, which are variable across spieces. In insects, only two α, one ß, and one γ chains have been identified thus far. Here, we isolated a novel laminin ß gene, BmLanB1-w, by positional cloning of the mutant (crayfish, cf) with blistered wings in silkworm. Gene structure analysis showed that a 2 bp deletion of the BmLanB1-w gene in the cf mutant caused a frame-shift in the open reading frame (ORF) and generated a premature stop codon. Knockdown of the BmLanB1-w gene produced individuals exhibiting blistered wings, indicating that this laminin gene was required for cell adhesion during wing development. We also identified laminin homologs in different species and showed that two copies of ß laminin likely originated in Lepidoptera during evolution. Furthermore, phylogenetic and gene expression analyses of silkworm laminin genes revealed that the BmLanB1-w gene is newly evolved, and is required for wing-specific cell adhesion. This is the first report showing the tissue specific distribution and functional differentiation of ß laminin in insects.

Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori.

The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of ß-alanine and N-ß-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of ß-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori.

Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.

Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant.

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 ß-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 Bm-iAANAT, 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 cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln 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.