SSR based linkage and mapping analysis of C, a yellow cocoon gene in the silkworm, Bombyx moil

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.

Functional characterization of a low-density lipoprotein receptor in the lepidopteran model,Bombyx mori

The growth and development of metabolous insects are mainly regulated by ecdysone and juvenile hormone.As a member of the low-density lipoprotein receptor(LDLR)family,megalin(mgl)is involved in the lipoprotein transport of cholesterol which is an essential precursor for the synthesis of ecdysone.Despite extensive studies in mammals,the function of mgl is still largely unknown in insects.In this study,we characterize the function of mgl in the silkworm Bombyx mori,the model species of Lepidoptera.We find that mgl is broadly present in the genomes of lepidopteran species and evolved with divergence between lepidopterans and Drosophila.The expression pattern suggests a ubiquitous role of mgl in the growth and development in the silkworm.We further perform clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-based mutagenesis of Bmmgl and find that both the development and the silk production of the silkworm are seriously affected by the disruption of Bmmgl.Our results not only explore the function of mgl in Lepidoptera but also add to our understanding of how cholesterol metabolism is involved in the development of insects.

Fumarate mitigates disruption induced by fenpropathrin in the silkworm Bombyx mori(Lepidoptera):A metabolomics study

The silkworm Bombyx mori L.is a model organism of the order Lepidoptera.Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control.In this study,comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6,12,and 24 h after feeding with fenpropathrin.Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways:glycerophospholipid metabolism,sulfur metabolism,glycolysis,amino acid metabolism,the urea cycle,and the tricarboxylic acid(TCA)cycle.After analyzing the percentage changes in the metabolic pathways at the 3 time points,sulfur metabolism,glycolysis,and the TCA cycle showed significant responses to fenpropathrin.Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways.The combination of iron(II)fumarate+folic acid(IF-FA)enhanced fenpropathrin resistance in silkworms 6.38 fold,indicating that the TCA cycle is the core pathway associated with resistance.Furthermore,the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro.Therefore,IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.

BmSd gene regulates the silkworm wing size by affecting the Hippo pathway

Insect wings are developed from the wing disc during metamorphosis.Bombyx mori,a model lepidopteran insect,loses flight ability after long-term domestication from the wild silkworm,Bombyx mandarina.The mw mutant(ul 1 strain)shows minute wings compared to wild type(e.g.,p50 strain)wings.RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the ull and p50strains.The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals.In this study,the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development.We found that mats,warts,and hippo expression levels were higher in u11 compared to p50 wing discs.BmSd(scalloped)expression,which encodes a prominent transcriptional partner to Yorkie(Yki),gradually decreased during the wandering stage in ull,but exhibited the opposite expression pattern in p50.When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain,57.9%of the individuals showed minute wings.Additionally,ex,kibras and wingless expression levels decreased in the BmSd knockdown mutant.Further,BmSd deletion mediated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 induced 50%of individuals with minute wings,a phenotype similar to the mw mutant.This result demonstrates that BmSd plays pivotal roles in silkworm wing development.Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development,and our findings provide a basis for further research on B.mori wing development.

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.

CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori

Circadian rhythms,which are ubiquitous and adaptive,occur across all species,from microbes to humans,in which they organize and modify behavior and physiology.timeless(im)is a canonical clock gene.The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated;however,in lepidopteran insects,including Bombyr mori,the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities.To arrive at a comprehensive understanding of the role of tim in the B.mori endogenous circadian clock,we exploited the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 gene editing system.We attermpted to elucidate the functions of tim in the circadian clock of B.mori using Bmtim mutants.The knockouts affected two circadian behavioral activities:adult emergence and embryo hatching rhythms.Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels,and thereby the oscillation amplitudes,of Bmper and Bmclk messenger RNAs during both the photophase and scotophase.Additionally,the daily rhythmic expression of Bmndbt was up-regulated in the photophase and downregulated in the scotophase in a tim-knockout.Our study reveals that tim is integral to the B.mori circadian clock and may be involved in regulating eclosion and hatching rhythms.

Cloning and characterization of the gene encoding an ubiquitin-activating enzyme E1 domain-containing protein of silkworm, Bombyx moil

Bombyx mori cytoplasmic polyhedrosis virus （BmCPV） is one of the major viral pathogens for the silkworm. To date, the molecular mechanism of BmCPV invasion has been unclear. We cloned the full length complementary （c）DNA which encodes the ubiquitin-activating enzyme El-domain containing proteinl （UbE1DC1） ofBombyx mori by using suppression subtraefive hybridization （SSH） and rapid amplification of com- plementary （c）DNA ends （RACE）. The full-length eDNA of UbE1DClgene is 1 919 bp, consisting of a 100 bp 5＇ untranslated region, a 637 bp 3＇ untranslated region and an 1 182 bp open reading frame （ORF）, encoding a 393 amino acid protein. The protein contained the THiF_MoeB_hesA_family domain, an adenosine triphosphate binding site, which belongs to the family of ubiquitin-activating enzyme El. Reverse transcription - polymerase chain reaction analysis from the silkworm tissues, namely silk gland, hemo- cyte, fat body, gonad and midgut revealed that UbE1DC1 was expressed in all the five tissues. The real-time quantitative polymerase chain reaction analysis indicated that the relative expression of UbE1DC1 in the normal midgut was approximately 9.78-fold of that in the BmCPV-infected midgut. It is implicated that UbEIDCI may play an important role in the interaction between the host and BmCPV invasion.