QTL analysis to identify genes involved in the trade-off between silk protein synthesis and larva-pupa transition in silkworms.

BACKGROUND: Insect-based food and feed are increasingly attracting attention. As a domesticated insect, the silkworm (Bombyx mori) has a highly nutritious pupa that can be easily raised in large quantities through large-scale farming, making it a highly promising source of food. The ratio of pupa to cocoon (RPC) refers to the proportion of the weight of the cocoon that is attributed to pupae, and is of significant value for edible utilization, as a higher RPC means a higher ratio of conversion of mulberry leaves to pupa. In silkworm production, there is a trade-off between RPC and cocoon shell ratiao(CSR), which refers the ratio of silk protein to the entire cocoon, during metamorphosis process. Understanding the genetic basis of this balance is crucial for breeding edible strains with a high RPC and further advancing its use as feed. RESULTS: Using QTL-seq, we identified a quantitative trait locus (QTL) for the balance between RPC and CSR that is located on chromosome 11 and covers a 9,773,115-bp region. This locus is an artificial selection hot spot that contains ten non-overlapping genomic regions under selection that were involved in the domestication and genetic breeding processes. These regions include 17 genes, nine of which are highly expressed in the silk gland, which is a vital component in the trade-off between RPC and CSR. These genes are annotate with function related with epigenetic modifications and the regulation of DNA replication et al. We identified one and two single nucleotide polymorphisms (SNPs) in the exons of teh KWMTBOMO06541 and KWMTBOMO06485 genes that result in amino acid changes in the protein domains. These SNPs have been strongly selected for during the domestication process. The KWMTBOMO06485 gene encodes the Bombyx mori (Bm) tRNA methyltransferase (BmDnmt2) and its knockout results in a significant change in the trade-off between CSR and RPC in both sexes. CONCLUSIONS: Taken together, our results contribute to a better understanding of the genetic basis of RPC and CSR. The identified QTL and genes that affect RPC can be used for marker-assisted and genomic selection of silkworm strains with a high RPC. This will further enhance the production efficiency of silkworms and of closely-related insects for edible and feed purposes.

The development of silk glands and transcriptome aberration induced by cyantraniliprole in Bombyx mori.

Bombyx mori is an insect species of great economic importance, and its silk gland is a vital organ for the synthesis and secretion of silk protein. However, long-term artificial domestication of B. mori has resulted in high sensitivity to chemical toxins, especially insecticides. Cyantraniliprole (Cya), a second-generation ryanodine receptor modulator insecticide, is widely utilized in agriculture for pest control. In this study, the impact of Cya toxicity on the development of silk glands in the 5th instar larvae of B. mori was assessed using Cya LC5, LC10 and LC20, as well as a starvation treatment group for comparison. Short-term exposure (24 h) to different concentrations of Cya resulted in delayed development of silk glands in B. mori. Meanwhile, the body weight, silk gland weight, silk gland index and cocoon quality were significantly reduced in a concentration-dependent manner, except for the Cya LC5 treatment. Histopathological and ultrastructural analysis revealed that Cya LC10 induced disruption of the nuclear membrane and endoplasmic reticulum in the posterior silk gland (PSG) cells, leading to the formation of intracellular vacuoles. Transcriptome sequencing of PSGs identified 2152 genes that were differentially expressed after exposure to Cya LC10, with 1153 down-regulated genes and 999 up-regulated genes. All differentially expressed genes were subjected to functional annotation using gene ontology and Kyoto encyclopedia of genes and genomes database, and it was found that protein synthesis-related pathways were significantly enriched, with the majority of genes being down-regulated. Furthermore, the transcription levels of genes involved in "protein processing in endoplasmic reticulum", "protein export", "proteasome" and "DNA replication" were quantified using qRT-PCR. Our findings suggested that short-term exposure to Cya LC10 resulted in disruption of DNA replication, as well as protein transport, processing and hydrolysis in the PSG cells of B. mori. The results of this study provide a theoretical foundation for the safe utilization of Cya in sericulture production.

Multiple independent origins of the female W chromosome in moths and butterflies.

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.

Cyclin B3 plays pleiotropic roles in female reproductive organogenesis and early embryogenesis in the silkworm, Bombyx mori.

BACKGROUND: The reproductive system plays a crucial role in insect survival, reproduction and species specificity. Understanding the molecular mechanisms underlying reproductive organogenesis contributes to improving the efficiency of sterile insect technique marked by an eco-friendly pest management strategy. Lepidoptera is one of the largest orders of insects, most of which are major pests in agriculture and forestry. Our study aimed to screen the genes responsible for reproductive organogenesis and unravel the mechanism underlying female reproductive organ defects. RESULTS: Morphological investigation of female reproductive organs showed a defective connection between oviductus geminus and oviductus communis on the second day of pupa (P2) in Speckled mutant silkworm. RNA_Seq identified a total of 18 049 transcripts that were expressed in the P2 female internal reproductive organs without ovary in Spc/+ compared to +Spc /+Spc . Differential expression analysis identified 312 up-regulated genes and 221 down-regulated genes in Spc/+. KEGG analysis identified 44 significantly enriched pathways. The results of qRT-PCR performed on 33 genes significantly matched the outcomes of the RNA_Seq. Dysfunction of Cyclin B3 resulted in a defective connection of the oviductus communis with the ovariole, dysfunction of oogenesis, and a petite body. Moreover, homozygous recessive lethality of Cyclin B3/Cyclin B3 occurred during early embryogenesis. CONCLUSION: Our results suggest that Cyclin B3 is a pleiotropic functional gene that regulates early embryogenesis, oogenesis, development, and female reproductive organogenesis. These results showed that Cyclin B3 has significant effects on lepidopteran mortality, growth, and reproductive physiology, which might be considered a novel and potentially eco-friendly target for lepidopteran pest management. © 2023 Society of Chemical Industry.

SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data.

The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.

Maternal dominance contributes to subgenome differentiation in allopolyploid fishes.

Teleost fishes, which are the largest and most diverse group of living vertebrates, have a rich history of ancient and recent polyploidy. Previous studies of allotetraploid common carp and goldfish (cyprinids) reported a dominant subgenome, which is more expressed and exhibits biased gene retention. However, the underlying mechanisms contributing to observed 'subgenome dominance' remains poorly understood. Here we report high-quality genomes of twenty-one cyprinids to investigate the origin and subsequent subgenome evolution patterns following three independent allopolyploidy events. We identify the closest extant relatives of the diploid progenitor species, investigate genetic and epigenetic differences among subgenomes, and conclude that observed subgenome dominance patterns are likely due to a combination of maternal dominance and transposable element densities in each polyploid. These findings provide an important foundation to understanding subgenome dominance patterns observed in teleost fishes, and ultimately the role of polyploidy in contributing to evolutionary innovations.

Mutation Rate and Spectrum of the Silkworm in Normal and Temperature Stress Conditions.

Mutation rate is a crucial parameter in evolutionary genetics. However, the mutation rate of most species as well as the extent to which the environment can alter the genome of multicellular organisms remain poorly understood. Here, we used parents-progeny sequencing to investigate the mutation rate and spectrum of the domestic silkworm (Bombyx mori) among normal and two temperature stress conditions (32 °C and 0 °C). The rate of single-nucleotide mutations in the normal temperature rearing condition was 0.41 × 10-8 (95% confidence interval, 0.33 × 10-8-0.49 × 10-8) per site per generation, which was up to 1.5-fold higher than in four previously studied insects. Moreover, the mutation rates of the silkworm under the stresses are significantly higher than in normal conditions. Furthermore, the mutation rate varies less in gene regions under normal and temperature stresses. Together, these findings expand the known diversity of the mutation rate among eukaryotes but also have implications for evolutionary analysis that assumes a constant mutation rate among species and environments.

Population Structure, Demographic History, and Adaptation of Giant Honeybees in China Revealed by Population Genomic Data.

There have been many population-based genomic studies on human-managed honeybees (Apis mellifera and Apis cerana), but there has been a notable lack of analysis with regard to wild honeybees, particularly in relation to their evolutionary history. Nevertheless, giant honeybees have been found to occupy distinct habitats and display remarkable characteristics, which are attracting an increased amount of attention. In this study, we de novo sequenced and then assembled the draft genome sequence of the Himalayan giant honeybee, Apis laboriosa. Phylogenetic analysis based on genomic information indicated that A. laboriosa and its tropical sister species Apis dorsata diverged ∼2.61 Ma, which supports the speciation hypothesis that links A. laboriosa to geological changes throughout history. Furthermore, we re-sequenced A. laboriosa and A. dorsata samples from five and six regions, respectively, across their population ranges in China. These analyses highlighted major genetic differences for Tibetan A. laboriosa as well as the Hainan Island A. dorsata. The demographic history of most giant honeybee populations has mirrored glacial cycles. More importantly, contrary to what has occurred among human-managed honeybees, the demographic history of these two wild honeybee species indicates a rapid decline in effective population size in the recent past, reflecting their differences in evolutionary histories. Several genes were found to be subject to selection, which may help giant honeybees to adapt to specific local conditions. In summary, our study sheds light on the evolutionary and adaptational characteristics of two wild giant honeybee species, which was useful for giant honeybee conservation.

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.

Mutation of a lepidopteran-specific PMP-like protein, BmLSPMP-like, induces a stick body shape in silkworm, Bombyx mori.

BACKGROUND: Lepidoptera is one of the largest orders of insects, some of which are major pests of crops and forests. The cuticles of lepidopteran pests play important roles in defense against insecticides and pathogens, and are indispensable for constructing and maintaining extracellular structures and locomotion during their life cycle. Lepidopteran-specific cuticular proteins could be potential targets for lepidopteran pest control. But information on this is limited. Our research aimed to screen the lepidopteran-specific cuticular proteins using the lepidopteran model, the silkworm, to explore the molecular mechanism underlying the involvement of cuticular proteins in body shape construction. RESULTS: Positional cloning showed that BmLSPMP-like, a gene encoding a lepidopteran-specific peritrophic matrix protein (PMP) like protein which includes a peritrophin A-type chitin-binding domain (CBM_14), is responsible for the stick (sk) mutation. BmLSPMP-like is an evolutionarily conserved gene that exhibits synteny in Lepidoptera and underwent purifying selection during evolution. Expression profiles demonstrated that BmLSPMP-like is expressed in chitin-forming tissues, testis and ovary, and accumulates in the cuticle. BmLSPMP-like knockout, generated with CRISPR/Cas9, resulted in a stick-like larval body shape phenotype. Over-expression of BmLSPMP-like in the sk mutant rescued its abnormal body shape. The results showed that BmLSPMP-like may be involved in assemblage in the larval cuticle. CONCLUSION: Our results suggested that the dysfunction of BmLSPMP-like may result in a stick body shape phenotype in silkworm, through the regulation of the arrangement of the chitinous laminae and cuticle thickness. Our study provides new evidence of the effects of LSPMP-likes on lepidopteran body shape formation, metamorphosis and mortality, which could be an eco-friendly target for lepidopteran pest management. © 2022 Society of Chemical Industry.

A Blueprint of Microstructures and Stage-Specific Transcriptome Dynamics of Cuticle Formation in Bombyx mori.

Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

The Landscapes of Full-Length Transcripts and Splice Isoforms as Well as Transposons Exonization in the Lepidopteran Model System, Bombyx mori.

The domesticated silkworm, Bombyx mori, is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm.

Comparative Transcriptome Analysis Reveals bmo-miR-6497-3p Regulate Circadian Clock Genes during the Embryonic Diapause Induction Process in Bivoltine Silkworm.

Diapause is one of the survival strategies of insects for confronting adverse environmental conditions. Bombyx mori displays typical embryonic diapause, and offspring diapause depends on the incubation environment of the maternal embryo in the bivoltine strains of the silkworm. However, the molecular mechanisms of the diapause induction process are still poorly understood. In this study, we compared the differentially expressed miRNAs (DEmiRs) in bivoltine silkworm embryos incubated at diapause- (25 °C) and non-diapause (15 °C)-inducing temperatures during the blastokinesis (BK) and head pigmentation (HP) phases using transcriptome sequencing. There were 411 known miRNAs and 71 novel miRNAs identified during the two phases. Among those miRNAs, there were 108 and 74 DEmiRs in the BK and HP groups, respectively. By the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the predicted target genes of the DEmiRs, we found that aside from metabolism, the targets were also enriched in phototransduction-fly and insect hormone biosynthesis in the BK group and the HP group, respectively. Dual luciferase reporter assay illustrated that bmo-miR-6497-3p directly regulated Bmcycle and subsequently regulated the expression of circadian genes. These results imply that microRNAs, as vitally important regulators, respond to different temperatures and participate in the diapause induction process across species.

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, expression, and artificial selection of silkworm epigenetic modification enzymes.

BACKGROUND: Understanding the genetic basis of phenotype variations during domestication and breeding is of great interest. Epigenetics and epigenetic modification enzymes (EMEs) may play a role in phenotypic variations; however, no comprehensive study has been performed to date. Domesticated silkworm (Bombyx mori) may be utilized as a model in determining how EMEs influence domestication traits. RESULTS: We identified 44 EMEs in the genome of silkworm (Bombyx mori) using homology searching. Phylogenetic analysis showed that genes in a subfamily among different animals were well clustered, and the expression pattern of EMEs is constant among Bombyx mori, Drosophila melanogaster, and Mus musculus. These are most highly expressed in brain, early embryo, and internal genitalia. By gene-related selective sweeping, we identified five BmEMEs under artificial selection during the domestication and breeding of silkworm. Among these selected genes, BmSuv4-20 and BmDNMT2 harbor selective mutations in their upstream regions that alter transcription factor-binding sites. Furthermore, these two genes are expressed higher in the testis and ovary of domesticated silkworm compared to wild silkworms, and correlations between their expression pattern and meiosis of the sperm and ova were observed. CONCLUSIONS: The domestication of silkworm has induced artificial selection on epigenetic modification markers that may have led to phenotypic changes during domestication. We present a novel perspective to understand the genetic basis underlying animal domestication and breeding.

Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.

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.

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.