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.

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.

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a major pathogen of the economic insect silkworm,Bombyx mori.Virus‐encoded microRNAs(miRNAs)have been proven to play important roles in host–pathogen interactions.In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA,BmCPV‐miR‐1,from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR(qPCR)and investigated its functions with qPCR and lentiviral expression systems.Bombyx mori inhibitor of apoptosis protein(BmIAP)gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′untranslated region.It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae.At the same time,it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics.Furthermore,BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm.In the midgut of BmCPV‐infected larvae,BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene.With the viral genomic RNA segments S1 and S10 as indicators,BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm.These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression,providing the virus with a better cell circumstance for its replication.

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.