Positional Cloning And Molecular Mechanism Of Body Shape And Coloration Mutant Bamboo (Bo) In Silkworm, Bombyx Mori

The highly diverse body shape and coloration of insects are fundamental traits for survival.Studying the occurrence of these two morphological characteristics is not only helpful to understand the adaptability and genetic basis of insects to environment,which is of great significance for us to understanding the nature,but also lay the foundation for the utilization genes,such as the preparation of eco-friendly insecticides for pest control and create the material of resistance to stress in economic insects.Although some studies have reported factors that control body shape and coloration patterns of insects and advance our understanding of the diversity of these traits,they focused on the formation of each trait independently.It is possible that a common factor may control body shape and coloration pattern simultaneously.The molecular characterization of such factors,however,remains elusive.The silkworm,Bombyx mori,an important economic insect,is also a model organism with unique advantages in life science research.As a representative of Lepidoptera,hundreds of mutants related to body shape and coloration are preserved in the silkworm gene bank,which provides us with material support.Bamboo(Bo)is a mutant that causes variation in both body shape and coloration.Here we performed linkage mapping and mutation screening,to determine the mechanical Bo gene product that affects body shape and coloration simultaneously.The results of this study are as follows:1.Bo mutant has epidermal defectsDominant mutant Bo exhibit a bamboo-like body shape with a dilated thorax and a slim but hard abdomen;the segmental boundaries bulge outward from the body,and the main area of each segment is narrow and constricted.Bo mutants also display reduced markings and pigmentation on their cuticle.The introduction of the Bo mutation,via genetic crosses,into two other strains,L and U,with large amounts of pigmentation on their larval body,led to reduced pigmentation in these strains.We further analyzed the epidermis morphology of wild type and Bo.First of all,in wild-type larvae,the length/width(L-W)ratio of the internode is significantly higher than the L-W ratio of the intersegmental fold.In contrast,the L-W ratio of Bo mutants was variable,with the L-W ratio of the internode being comparable,or even lower,than that of the intersegmental fold.The ratio of epidermal development is defective compared with the wild type.In addition,the epidermal scanning electron microscopy showed that the surface of the epidermal internodes of Bo was more ruffled,rugged and abnormal in bristles.These results indicate that the phenotype of the Bo mutant could result from defects in the larval cuticle during development.2.The Bo locus is a low-complexity cuticular protein gene named Bmor CPH24To identify the gene responsible for the Bo phenotype,we performed positional cloning using the BC1 M progeny.Using nine polymorphic markers and 1045 BC1 M individuals,we pinpointed the Bo locus to a 143-kb genomic region which contains 15 predicted genes.Then the expression and sequence differences of these genes in the wild type and Bo were analyzed,and two genes were found to be different.The gene BGIBMGA011765 was significantly down-regulated in Bo,and the deletion of 5bp nucleotide sequence in the ORF that resulted in the frame shift mutation indicating the loss-of-function of BGIBMGA011765 in Bo mutants.The other gene BGIBMGA011766 has no significant difference in wild type and mutants,but the ORF was inserted 48-bp in Bo.Further,multi-strain comparisons showed that the 5-bp deletion in the BGIBMGA011765 gene was unique to Bo and was not found in other non-Bo phenotype strains.However,no Bo mutant-specific mutation was found in the BGIBMGA011766 gene.These results collectively suggest that BGIBMGA011765 is the most likely gene responsible for the Bo mutant phenotype.BGIBMGA011765 is a low-complexity cuticular protein(CP)gene without the typical CP domain,previously named Bmor CPH24.3.Bmor CPH24 is mainly expressed in the larval epidermis and is associated with changes in body shape and coloration of Bo.We investigated the temporal and spatial expression patterns of Bmor CPH24 in wild type by q RT-PCR.The expression analysis of the tissues from day 3,fifth instar larvae showed that Bmor CPH24 was highly expressed in the epidermis but not in other tissues,and the expression pattern of Bmor CPH24 was investigated by using the epidermis developed in different stages.It was found that the expression of Bmor CPH24 was mainly fluctuant in larval stage,showing peak expression during mulberry feeding period,but decreased rapidly to trough in molting stage.To further elucidate the molecular mechanism of Bo,we tested whether the expression of Bmor CPH24 correlated with finer scale variation in body shape and pigmentation markings in Bo and wild type larvae.First,we found that the gene was highly expressed in the pigmented areas by q RT-PCR,indicating that Bmor CPH24 was required for the development of larval markings.Furthermore,in situ hybridizations showed that Bmor CPH24 was mainly expressed at the internode(in)of each segment and was below detection level at the posterior margin of each segment,in the intersegmental fold(in-f).This expression pattern perfectly coincided with the shorter internodes and longer intersegmental folds of Bo mutants.q RT-PCR also revealed that Bmor CPH24 was expressed higher levels in the dorsal vs.ventral part of the epidermis,and at gradually higher levels toward the posterior end of the larval body.Thus,these results indicate a strong association between the expression pattern of Bmor CPH24 and the Bo phenotype.4.Bmor CPH24 produces the Bo phenotype in silkworm.To test whether Bmor CPH24 caused the Bo phenotype,we used a previously established functional tool,electroporation-mediated RNAi.According to the expression pattern of Bmor CPH24,double stranded RNA(ds RNA)targeting Bmor CPH24 sequence was injected at the fourth instar U strain using electroporation.The results showed that the phenotype with pigments inhibited and epidermal extension limited in individuals of the interference group,and the expression of Bmor CPH24 gene was significantly lower than control group.To evaluate how a more extreme Bmor CPH24 loss-of-function mutation would affect the body shape and coloration of the wild-type Dazao strain,we disrupted the gene using CRISPR/Cas9-mediated gene editing.By in vitro transcription,we injected sg RNA and Cas9 m RNA into newly laid embryos,and obtained Bo-like mutant phenotypes(~200 larvae)in G1 generation larvae.Genomic sequencing of five mutant individuals identified four kinds of frame shift mutations all disrupting the Bmor CPH24 coding region.These results show that Bmor CPH24 is necessary for the development of a normal larval body shape and pigmentation during silkworm development.5.Disruption of Bmor CPH24 induces apoptosis in epidermal cells,leading to the down regulation of larval cuticular protein genes(LCPs)and pigment gene Wnt1 and ultimately causing abnormal body shape and coloration.We have confirmed the function of Bmor CPH24,so the molecular mechanism of controlling both body shape and pigmentation of silkworm larvae needs further study.On the one hand,what is the role of Bmor CPH24 as a CP in the process of cuticle assembly? We analyzed the structure of the Bmor CPH24-dependent cuticle,we found that Bmor CPH24 is very important for the development of larval endocuticle after ecdysis,and its defects can inhibit the deposition endocuticle of Bo.At the same time,it can affect the expression of the other four LCPs which are responsible for assembling the endocuticle.On the other hand,what is the relationship between Bmor CPH24 and pigmentation? By transgene overexpression and gene expression analysis,we indicated that presence of Bmor CPH24 protein is not sufficient to induce pigmentation,but its deficiency leads to the down-regulation of genes also required for pigment synthesis,such as Wnt1.As shown above,deficiency of Bmor CPH24 caused the downregulation of several LCPs as well as of Wnt1.However,since Bmor CPH24 is a cuticle structural protein,it is unable to directly regulate the expression of these genes.We,thus,investigated whether deficiency of Bmor CPH24 led to epidermal cell death and,thus,to lower levels of these other genes.The results indicated that Bmor CPH24 deficiency may induce apoptosis through the Caspase-dependent apoptosis pathway.Collectively,these results suggest that mutations in Bmor CPH24 appear to induce cell apoptosis,which inhibits the differentiation and development of epidermal cells,which disrupts the expression of LCPs and Wnt1 and leads to abnormal body shape and coloration.6.Bmor CPH24 is a novel gene that has evolved a new biological function,after undergoing gene duplication.To understand the evolutionary history of Bmor CPH24,we investigated its evolutionary history.We performed phylogenetic analysis using the genes encoding cuticular protein with low complexity sequence from four different insect species(B.mori,Dendrolimus punctatus,Manduca sexta,and Anopheles gambiae),and we directly used Bmor CPH24's sequence as a query to perform BLASTP and TBLASN searches against the non-redundant(nr)protein database in NCBI,and against the genomes or transcriptomes of six additional lepidopteran insects(M.sexta,Spodoptera frugiperda,Chilo suppressalis,Danaus plexippus,Papilio polytes and Plutella xylostella).No orthologs of Bmor CPH24 were found,but one paralogous gene,Bmor CPH26(BGIBMGA011767),with 77% amino acid sequence similarity,was obtained from silkworm and was found in M.sexta,and we named it Msex CPH26.These observations indicate that Bmor CPH24 was likely produced by gene duplication from Bmor CPH26 after the split from its sister clade M.sexta.Further analysis revealed that Bmor CPH24 is a novel gene that has evolved a new biological function over a short evolutionary time scale,after undergoing gene duplication.7.Bmor CPH24-null is sensitive to environmental stresses.The importance of the insect cuticle as a primary protective barrier against environment stresses has long been noted.Previous reports have shown that the components of the cuticle,such as cuticular hydrocarbon,chitin and melanin,play a role in the adaptability or resistance of insects to environmental stresses.Similarly,structural cuticular proteins(CPs)are crucial components of the cuticle.Whether CPs can also affect the ability of cuticle to defend against environmental stresses is little known.Here,we report the mechanical defense function of a structural cuticular protein,Bmor CPH24,to environmental stresses using Bo mutant.Ultraviolet(UV)irradiation and topical application of an acetone insecticide were used as environmental stresses to determine the differences in susceptibility between Bo and wild type larvae.UV irradiation resulted in a sunburn phenotype in the Bo strains earlier than the wild-type indicating the sensitivity of Bo.Higher MDA content and a lower survival ratio were also observed in the Bo strains.Treatment with deltamethrin revealed that Bo larvae were more sensitive to insecticides than the wild type.Furthermore,cuticle analysis by microsection revealed thinner cuticle and a significant decrease in the endocuticle layer(?64.0%)in Bo.These results suggest that Bmor CPH24 mutation can lead to deficiency in resources required to construct the cuticle in Bo resulting in thin cuticle and reduced resistance to UV and insecticides.Together,the CP Bmor CPH24,as a key component of the cuticle,contributes to UV and insecticides resistance in silkworm larvae by affecting the cuticle thickness.In the present study,we identified a fast-evolving gene,Bmor CPH24,that is responsible for the silkworm Bo mutant.This gene has undergone neofunctionalization after having duplicated and evolved a new function in constructing and pigmenting larval cuticle.Furthermore,we explored the role of the CP in maintaining the adaptability or resistance of silkworm/insect to environmental stress.In conclusion,these findings not only contribute to our understanding of the regulation and evolution of insect larval cuticle morphology,but also provide a target gene for the creation of resistant silkworm and the control of Lepidoptera pests.