Preliminary Study Of Imaginal Disc Growth Factor In Silkworm,Bombyx,Mori

The cuticle composed of chitin and cuticular proteins is a multifunctional device that protects its carriers against dehydration,arms them against predators,constitutes a physical barrier to prevent pathogen entry and serves as an exoskeleton allowing locomotion.Imaginal disc growth factors(IDGF)that belonged to rarely known glyco-18-domain hydrolase family worked as structural protein to maintain the extracellular-matrix scaffold against chitinolytic degradation and stable the natural cuticle.In addition,it also involved in innate immunity,detoxification and energy balance as well as regulating temperature adaptation and development,which is essential for the control of inst growth and development as well as environmental stress response.We found that lepidopteran insts have only one IDGF protein and Drosophila has evolved six IDGFs.In this study,we performed comprehensive analysis to investigate the function of BmIDGF.The main findings are listed as follows:1.First,we analyzed evolutionary relationship of IDGF and chitinase-like protein.Phylogenetic analysis showed a highly conserved monophyly group for IDGF proteins among lepidopteran species and Glyco-18 domain(motif1-12)was conserved among the six distantly related species.These results suggested that all IDGF and chitinase-like proteins may evolve from the same ancestral sequence.2.We generated three Bombyx mori imaginal disc growth factor(BmIDGF)mutant lines using the CRISPR/Cas9 system.Interestingly,the disappearance of star spots and weak pigmentation of crescents were significantly observed in the homozygous BmIDGF-7bp mutant larvae after the fourth moulting.3.We performed the transmission electron microscopy(TEM)analysis using the larval epidermis of the fifth abdominal segment form the wild type and the mutants.the BmIDGF-7bp mutants showed more chitinous layers and uric acid particles than the wild type.4.Spatiotemporal expression profiles analysis of the melanin synthesis pathway genes and upstream regulatory elements showed that IDGF is suggested to control the expression levels of the ecdysone inducible transcription factor E75A and pleiotropic factors apt-like and Toll8/spz3,to further affect the expression levels of BmTH,BmDDC and Bmebony.5.To better understand the metabolic mechanism changes underlying the disruption of BmIDGF,we generated a dynamic metabolome profiles of hemolymph collected at three time points(fourth instar,fourth instar moulting and fifth instar).A total of 581 significantly differential accumulated metabolites were identified.The KEGG analysis comparing the wild type and mutant samples revealed significant enrichment for several pathways,including phenylalanine metabolism,beta-alanine metabolism,and tyrosine metabolism.which induced a metabolic shift from eumelanin to O stizolobate,leading to a decrease in accumulating melanin of the BmIDGF-7bp mutant.Taken together,our findings highlight on not only the universal function of BmIDGF with a potential link to the maintenance of normal cuticle structure,but also an underexplored space in gene function of affecting melanin pigmentation.Therefore,these findings provide important foundation for understanding the mechanisms of fine scale regulation of pigment metabolism and striped pattern divergence.