Changes Study Of Structure And Function In The Silkworm Involving Peritrophic Matrix Degradation And Formation

The PM is a non-cellular,semipermeable membrane-like structure that encloses food groups in the digestive tract of most insects.It extends from the anterior end of the midgut to the hindgut and consists mainly of chitin and proteins,which protects the midgut epithelial cells,promotes digestion,has selective permeability,and has the resistance to pathogenic microorganisms,is a natural barrier for the body's resistance to pathogenic microorganisms and undesirable chemical factors.However,the current research on PM mainly focuses on the destruction of the PM by the pathogenic microorganisms,physical and chemical reagents,and gene interference,so as to achieve the purpose of controlling pests.However,the study of morphological structure and composition of the PM during different developmental stage is less and less comprehensive.In order to further explore the degradation and formation mechanism of PM of silkworm and to further study the secretion,morphological components of the PM.The biomechanics of tissue sections,scanning electron microscopy,the analysis of enzyme activity and proteomics were used to study the morphological structure of the PM,changes of relevant enzyme activities and the comparative analysis of PM proteins in the silkworm at different developmental stages.According to these studies,we mainly got the following experimental results:1.The results of paraffin sections in different periods showed that PM was present during the whole period of sleep,after molting stage,and feeding stage;in the feeding stage,PM had a multi-layered dense structure,wrapped foods,and PM had a small outer space and closely adhered to the midgut epithelial cells;After entering sleep stage,in the 4th instar sleep 6h-18 h period,PM structure becaome loose,gradually shifted in,and the PM outer space gradually increased;midgut secretory activity existed in all periods,in the 4th instar sleep 24 h and 5th instar molting stage,the secretion activity of the midgut epithelial cells was stronger;at 2 h after the 5th instar and 10 h after the molting,the PM was already relatively densed,and the midgut secretion activity was also significantly weakened.The above results indicated that the degradation of the peritrophic membrane of silkworm larvae mainly occured in the sleeping stage,which was a slow,continuous,and dynamic process.In the late sleep period,that is,4 instar sleep for 24 hours,the secretion activity of the midgut epithelial cells was intense,and it was speculated that the PM degradation severely at the 4th instar sleep 24 h,the synthesis and maturation of the PM were mainly after molting,and food stimulation also promoted the secretion of PM.2.Scanning electron microscopy of the inner surface of PM at different stages showed that the PM inner surface of the silkworm larvae was a complete,smooth and compact structure.After entering the sleeping period,the PM gradually degraded.When the 4th instar sleep was 6h,the PM surface became clear and the regular fiber network structurerelatively was clear;During the 4h instar sleep 12h-18 h,PM inner surface gradually became rough,a few areas appeared granular structure;In the 4th instar sleep24 h,the PM inner surface was rough and the whole grainy,even micropores could be seen in some areas and was not complete.After the 5th instar moltig stage,PM was not yet completely formed,and the inner surface was rough,and there were still areas in granular form;when after 5th instar moltig stage was 2h-10 h,the inner surface of the PM was smooth and dense,relatively complete,and the fiber network was relatively rule.In summary,the silkworm PM will gradually degraded after entering the sleep period,especially in the post-sleep phase such as 4 instar sleep for 24 h,the most severe degradation;its synthesis process was relatively fast,and the synthesis and maturation of PM were mainly occured after molting stages.3.The results of enzymatic activity measurement of midgut and intestinal fluid in different periods showed that the degradation of the chitin was also directly degraded by chitinase and converted into chitosan and then degraded by chitin deacetylase.In the pathway,and the chitin deacetylase started earlier,in the midgut tissue,once it entered sleeping stage,at 4h instar sleep 6h,the enzyme activity reached its peak value,and then gradually declined,and in 5th-0-10 h,the value was the lowest;chitinase activity gradually increased during sleeping stage,which reached the highest at 4th instar sleep24 h,gradually decreased after molting;?-N-acetylglucosamine glucosidase activity increased significantly at 4th instar sleep 6h;in the 4th instar sleep 24 h,the enzyme active enzyme activity was the strongest;after molting,the enzyme activity gradually decreased to the level of feeding stage.The total protease activity was low at the time of sleeping stage,and increased after molting and the enzyme activity peaked at 2h after molting;the trehalase peaked at 4th instar sleep 24 h and gradually decreased after molting.The tendency of these several enzymes in intestinal fluid was the same as to the midgut tissue,but the peak value of enzyme activity laged behind.It was presumed that these enzymes were secreted by the midgut tissue and then released into the intestinal fluid to work,so that it showed hysteresis.4.Using iTRAQ combined with LC-MS/MS analysis of PM proteins in the silkworm M(4 instar sleep 24 h),V(after molting)and T(10 h after molting)results showed that a total of 1482 proteins were identified.The relative molecular mass of the identified proteins was mostly below 110 kDa,and the PI of most proteins was between5-10.There were 182 differentially expressed proteins in the M/T group were up-regulated,and 135 differentially expressed proteins were down-regulated;There were 188 differentially expressed proteins in the M/V group were up-regulated,and 293 differentially expressed proteins were down-regulated;and There were 268 differentially expressed proteins in the V/T group was up-regulated,126 proteins expressed proteins were down-regulated.5.GO annotation and KEGG metabolic pathway analysis were performed on the raw data of quantitative results of proteins detected by LC-MS/MS.The paired GO annotations indicate that differential proteins were mainly involved in metabolic processes,cellular processes,and catalytically active,binding functions.The results of the KEGG pathway showed that in the M/T group,oxidative phosphorylation,protein processing in the endoplasmic reticulum,lysosomes,RNA transpor,and ribosome pathways were significantly changed;in the M/V group,carbon metabolism,ribosome,Oxidative phosphorylation and lysosomal pathways were significantly changed;in the V/T group,oxidative phosphorylation,processing of proteins in the endoplasmic reticulum,lysosomes,and carbon metabolism pathways were significantly changed.After comprehensive analysis,it was speculated that the RNA transpor pathway,carbon metabolism,glycosaminoglycan degradation pathway and lysosomal pathway were related to the degradation of the PM,while the protein processing pathway in the endoplasmic reticulum and the ribosome pathway were involved in thre synthesis ofPM.In this study,through the paraffin sections,scanning electron microscopy,enzyme activity analysis and proteomic analysis,the dynamic changes of the matrix structure,the related enzymatic activity and the PM proteins during the PM degradation and formation of silkworm larvae were studied.We can further understand of the PM and know the foundation for the physiological and physiological basis for the degradation and formation mechanism of the PM.