Comprehensive silk gland multi-omics comparison illuminates two alternative mechanisms in silkworm heterosis.

Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms. Here, we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis. We found significant differences in the silk gland transcriptomic landscape between the two systems, including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents. In the quaternary hybrid system, hybrid vigor was primarily due to up-regulated genes and the parent-dominant up-regulated expression pattern, involving multiple transport processes, cellular nitrogen compound catabolism, glucose metabolism, and tricarboxylic acid cycle. In the binary system, hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern, mainly involving basic nitrogen synthesis metabolism and body function. We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes. Thus, this study revealed two alternative mechanisms that may contribute to silkworm heterosis, both of which facilitate the efficient utilization of energy and nitrogen for silk production.

Bmmp influences wing morphology by regulating anterior-posterior and proximal-distal axes development.

Insect wings are subject to strong selective pressure, resulting in the evolution of remarkably diverse wing morphologies that largely determine flight capacity. However, the genetic basis and regulatory mechanisms underlying wing size and shape development are not well understood. The silkworm Bombyx mori micropterous (mp) mutant exhibits shortened wing length and enlarged vein spacings, albeit without changes in total wing area. Thus, the mp mutant comprises a valuable genetic resource for studying wing development. In this study, we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp. Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in noncoding regions. These mutations resulted in decreased Bmmp messenger RNA levels and changes in transcript isoform composition. Bmmp null mutants were generated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 and exhibited changed wing shape, similar to mp mutants, and significantly smaller total wing area. By examining the expression of genes critical to wing development in wildtype and Bmmp null mutants, we found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axes development. We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila. The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity. Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.

Sob gene is critical to wing development in Bombyx mori and Tribolium castaneum.

The development of insect appendages requires the expression of multiple genes in a strict spatial and temporal order. The odd-skipped family genes are vital transcriptional factors involved in embryonic development. The development and morphogenesis of the insect wing requires multiple transcription factors to regulate the expression of wing patterning genes at the transcriptional level. However, the function of odd-related genes in insect wing morphogenesis and development during postembryonic stages is unclear. We focused on the roles of the sister of odd and bowl (sob) gene, a member of odd-skipped family genes, during the wing morphopoiesis in Bombyx mori using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 system and in Tribolium castaneum by RNA interference. The results showed that the wings were significantly smaller and degenerated, and wing veins were indistinct in the sob gene loss-of-function group in both B. mori and T. castaneum. Quantitative real-time polymerase chain reaction revealed that the Tcsob gene regulated the expression of wing development genes, such as the cht 7 and the vg gene. The findings suggest the importance of sob gene in insect wing morphology formation during postembryonic stages.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.

BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome. RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in 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.

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.

Artemisinin is highly soluble in polyethylene Glycol 4000 and such solution has multiple biological effects.

Artemisinin has a significant role in treatment of malaria, as well as effective anti-inflammatory and anti-cancer activities. However, such problems as poor water solubility and easy recrystallization limit its application. In this study, polyethylene glycol, a solvent which is widely used in pharmaceutics, was introduced to prepare an artemisinin dissolution. Under the action of hydrogen bonding in 12% polyethylene glycol 4000 solvent, the maximum solubility of artemisinin could reach up to 1 mg/mL. Meanwhile, biological functions of such artemisinin solution were evaluated. The obtained artemisinin solution had a significant inhibitory effect on Gram-positive bacteria, Gram-negative bacteria and fungi. As for the anti-inflammatory property, 0.031 mg/mL artemisinin solution had an obvious inhibitory effect on nitric oxide release in inflammatory cells, and the survival rate of cells was greater than 50%. Low concentration of the obtained artemisinin solution (0.031 mg/mL) had no significant cytotoxicity, while it displayed selective inhibition in cancer cells. IC50 for human hepatoma cells BEL-7404, SMMC7721 and Hep G2 is 0.0016 mg/mL, 0.0084 mg/mL and 0.0541 mg/mL, respectively. In conclusion, the 12% PEG4000-assisted artemisinin solution has a good biological effect and it can be further applied in pharmaceutics, biomaterials and medicine.

Fibroblast growth factor 21 prolongs lifespan and improves stress tolerance in the silkworm, Bombyx mori.

BACKGROUND: Fibroblast growth factor 21 (FGF21), an FGF family member, is an atypical hormone and pro-longevity factor. METHODS: To better understand of the effects of exogenous administration of FGF21 on lifespan and stress tolerance, and the underlying molecular basis, we used the silkworm, Bombyx mori, as an experimental animal model to evaluate FGF21's pharmaceutical effects. RESULTS: Lifespan was significantly prolonged in female silkworms with FGF21 replenishment, whereas no effect was observed in the male silkworms. FGF21 replenishment also significantly improved the activity of antioxidant systems such as glutathione-S-transferase (GST) and superoxide dismutase (SOD) and significantly decreased malondialdehyde (MDA) content. Moreover, FGF21 was found to play a critical role in enhancing stress resistance, including ultraviolet (UV) irradiation tolerance and thermotolerance. Furthermore, AMPK, FoxO, and sirtuins were activated by FGF21 and may be responsible for the prolonged lifespan and enhanced antioxidant activity observed in silkworms. CONCLUSIONS: Collectively, the results suggest the molecular pathways underlying of FGF21-induced longevity and stress tolerance, and support the use of silkworms as a promising experimental animal model for evaluating the pharmaceutical effects of small molecules.

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.

BmBlimp-1 gene encoding a C2H2 zinc finger protein is required for wing development in the silkworm Bombyx mori.

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the most common class of transcription factors. These factors have great potential for the management of developmental progression by regulating the specific spatiotemporal expression of genes. In this study, we cloned one C2H2-ZF protein gene of Bombyx mori, BGIBMGA000319, that is orthologous to B-lymphocyte-induced maturation protein-1 (Blimp-1); we thus named it as Bombyx mori Blimp-1 (BmBlimp-1). In the silkworm, the BmBlimp-1 gene is specifically upregulated during day 2 of the pupal to adult stage and is highly expressed in wing discs on day 3 of the pupa. Knockdown of its expression level in the pupal stage results in a crumpled-winged silkworm moth. Using the predicted DNA-binding sequences of BmBlimp-1 to search the silkworm genome to screen target genes of BmBlimp-1, 7049 genes were identified to have at least one binding site of BmBlimp-1 on their 1 kb upstream and downstream genome regions. Comparisons of those genes with a reported pupal wing disc transcriptome data resulted in 4065 overlapping genes being retrieved. GO enrichment analysis of the overlapping genes showed that most of the genes were enriched in the binding term. Combining functional annotation and real-time quantitative PCR, 15 genes were identified as the candidate target genes of BmBlimp-1, including several wing cuticular protein genes, chitin synthase A, and wing disc development genes, such as Wnt1, cubitus interruptus (ci) and engrailed (en). Moreover, the amino acid sequence of the zinc finger motif of Blimp-1 gene was highly conserved among the 15 insect species. We propose that BmBlimp-1 is an important regulatory factor in silkworm wing development.

The Association of D-dimer and Spontaneous Hemorrhagic Ascites with Outcomes in Cirrhosis: a Retrospective Study.

BACKGROUND: Cirrhotic patients with hemorrhagic ascites have significant morbidity and mortality. This study aims to determine the relationship between D-dimer values and hemorrhagic ascites in cirrhotic patients and analyze its predictive value. METHODS: This retrospective study screened 572 consecutive cirrhotic patients with ascites and hemorrhagic ascites (defined as red blood cells (RBC) in ascitic fluid ≥ 10,000/µL) during a 72-month period. The overall patient survival rate was measured by Kaplan-Meier analysis method. The relationship between D-dimer and hemorrhagic ascites was also examined. A multivariate Cox proportional hazard analysis was performed to assess the indepen-dent risk factors related to mortality. RESULTS: Both control group and hemorrhagic ascites patients had obvious hepatic dysfunction as determined by Model for End-Stage Liver Disease (MELD) scores of 6.37 ± 1.05 and 11.82 ± 2.86, respectively (p < 0.001). There was a higher prevalence of patients with significant ascites in those with spontaneous hemorrhagic ascites than in the control group (p = 0.003). There were significant differences in D-dimer levels between both groups (9.44 ± 5.11 vs. 26.83 ± 5.35, p < 0.001). Hemorrhagic ascites was significantly and positively correlated with D-dimer levels (r = 0.692, p < 0.0001). The area under the receiver operating characteristic (ROC) curve was 0.9838. Using Cox proportional hazard model for multivariate prognostic analysis, MELD, D-dimer and presence of spontaneous hemorrhagic ascites were independent predictors of 3-year mortality. CONCLUSIONS: Patients with hemorrhagic ascites had a significantly higher MELD score, D-dimer, and mortality than patients with ascites alone. D-dimer was associated with the appearance of hemorrhagic ascites and was found to be a marker of advanced liver disease and poor outcomes.

[Applications of the CRISPR/Cas9 system in insects].

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system guides Cas9 to specific genomic locations by a short RNA search string. This technology enables the systematic interrogation of mammalian genome editing, repairing damaged genes, silencing harmful genes and improving quality traits. In recent years, with the introduction of the CRISPR/Cas9 system for easy, fast and efficient genetic modification, it has been possible to conduct meaningful functional studies in a broad array of insect species, such as Drosophila, Bombyx mori, Aedes aegypti and butterflies et al. In this review, we summarize the application of CRISPR/Cas9 in different insect species, discuss methods for its promotion, and consider its application for future insect studies.

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.

[Progress and perspective of silkworm as a model of human diseases for drug screening].

As a typical representative of Lepidopteran insects, the silkworm, Bombyx mori, has numerous advantages, such as simple husbandry,highly prolific nature, short generation time, easily handled to be operated with moderate body size, clear genetic background and abundant mutation resources. Silkworm has not only been studied by the geneticists, but also been used as a new laboratory animal model of human disease and drug screening. There is a plenty of genetic resources in silkworm, some of which could be used as models of human genetic diseases, such as Phenylketonuria, Parkinson's disease, Hermansky-Pudlak syndrome and so on. Silkworm has also played a significant role in the study of pathogenesis of human pathogenic microorganisms. Moreover, silkworm could be used to evaluate the pharmacokinetic/pharmacodynamics properties and safety of a new drug comprehensively and systematically. At the same time, it can be used in the high throughput drug screening assays to shorten the period of the new drug research and development. This review summarizes that the silkworm is an excellent model in the drug screening assays, and has a potential in application to the large-scale drug screening.

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.

A single origin for nymphalid butterfly eyespots followed by widespread loss of associated gene expression.

Understanding how novel complex traits originate involves investigating the time of origin of the trait, as well as the origin of its underlying gene regulatory network in a broad comparative phylogenetic framework. The eyespot of nymphalid butterflies has served as an example of a novel complex trait, as multiple genes are expressed during eyespot development. Yet the origins of eyespots remain unknown. Using a dataset of more than 400 images of butterflies with a known phylogeny and gene expression data for five eyespot-associated genes from over twenty species, we tested origin hypotheses for both eyespots and eyespot-associated genes. We show that eyespots evolved once within the family Nymphalidae, approximately 90 million years ago, concurrent with expression of at least three genes associated with early eyespot development. We also show multiple losses of expression of most genes from this early three-gene cluster, without corresponding losses of eyespots. We propose that complex traits, such as eyespots, may have originated via co-option of a large pre-existing complex gene regulatory network that was subsequently streamlined of genes not required to fulfill its novel developmental function.

Mutations of an arylalkylamine-N-acetyltransferase, Bm-iAANAT, are responsible for silkworm melanism mutant.

Coloration is one of the most variable characters in animals and provides rich material for studying the developmental genetic basis of pigment patterns. In the silkworm, more than 100 gene mutation systems are related to aberrant color patterns. The melanism (mln) is a rare body color mutant that exhibits an easily distinguishable phenotype in both larval and adult silkworms. By positional cloning, we identified the candidate gene of the mln locus, Bm-iAANAT, whose homologous gene (Dat) converts dopamine into N-acetyldopamine, a precursor for N-acetyldopamine sclerotin in Drosophila. In the mln mutant, two types of abnormal Bm-iAANAT transcripts were identified, whose expression levels are markedly lower than the wild type (WT). Moreover, dopamine content was approximately twice as high in the sclerified tissues (head, thoracic legs, and anal plate) of the mutant as in WT, resulting in phenotypic differences between the two. Quantitative reverse transcription PCR analyses showed that other genes involved in the melanin metabolism pathway were regulated by the aberrant Bm-iAANAT activity in mln mutant in different ways and degrees. We therefore propose that greater accumulation of dopamine results from the functional deficiency of Bm-iAANAT in the mutant, causing a darker pattern in the sclerified regions than in the WT. In summary, our results indicate that Bm-iAANAT is responsible for the color pattern of the silkworm mutant, mln. To our knowledge, this is the first report showing a role for arylalkylamine-N-acetyltransferases in color pattern mutation in Lepidoptera.

[Construction of linkage localization lines containing genes with maternal effects].

Since the phenotypes of hybrid progenies involving genes with maternal effects are affected by the genotype of female parent, they cannot reflect the genotypes of individuals. This makes it difficult to develop test cross parents (triple or double recessive lines) for linkage localization and, consequently, hinders the progress in localization researches for these types of genes. In this study, we designed a set of hybridization schemes, the key of which was to make the "maternal-effect" genes homozygous at first, and then the non-maternal-effect genes. Using this scheme, we successfully produced a triple recessive line for genes ch (chocolate), nlw (non-lepis wing) and b-t (maternal brown egg of Tsujitan) on the 13th linkage group and a double recessive line for genes nb (narrow breast) and ki-2 (kidney-shaped egg 2) on the 19th linkage group of Bombyx mori.