Permeability Of Peritrophic Membrane Of Bombyx Mori And Its Influencing Factors

The peritrophic membrane(PM)is a non-cellular semi-permeable membrane which enclosing food particles in the digestive tract of most insects.It composed of chitin,proteoglycans and proteins.As the first physical barrier for insects to prevent pathogenic microorganisms in the digestive tract,the PM needs to be integrity and compactness to protect the intestinal epithelial cells and prevent the invasion of pathogens;on the other hand,it must maintain a certain permeability to facilitate Material circulation and ensure the normal physiological activities of insects.It suggests that the PM can be a significant structural target for insect control in many study,and various synergistic factors that could increase the insecticidal rate by destroying the PM have been found.However,the basic research on the permeability of PM is still not comprehensive.The effects,mechanisms and degree of destruction of the factors affecting the permeability of the PM are still unclear.In this study,The Ussing chamber was used to measure the permeability of the PM to dextrans of different molecular weights.The surface structure of the PM was observed using an environmental scanning electron microscope.SDS-PAGE gel electrophoresis and mass spectrometry was used to research the relationship between PM protein and permeability.According to these studies,we mainly got following results:1.The permeability of different parts of the PM is discrepant,it is gradually reduced from anterior to posterior.And the permeability of PM of silkworms at different stages is discrepant,it is gradually reduced from 3th to 5th stage.the permeability during sleep is significantly increased and restored to normal after molting.It is speculated that there is a process of degradation and replacement of PM in each sleeping period.In next intar,the peritrophic membrane is denser and thicker,resulting in decreased permeability.After starvation treatment from the 5th-molting,the PM permeability was not significantly different from the control group(normal feeding)at 5th-1d,suggesting that the formation of PM was not related to feeding activities.There is no significant change in PM permeability during 48 Hours post-operation,and it began to increase after that.there was a significant difference between control and treatment group at 96 h.It was speculated that the PM components of the silkworm were partially degraded due to prolonged starvation.2.Many synergistic factors(Fluorescent brighteners,Lectins,Chitin synthesis inhibitors,Chitinase,Protease)have effects on the structure and function of PM of silkworm,but the effect on the degree of damage is different from some other Lepidoptera Insects.In the feeding experiments with synergists,fluorescent brightener FB28 disrupted the PM most intensely and rapidly,and the permeability of the PM increased significantly at 2–4 h after feeding.At 4 h,it was permeable to 2000 kDa dextrans,environmental scanning electron micrographs showed that the surface of the PM was not smooth,and circular protrusions of about 1.4?m appeared on the surface.At 6 h,the PM almost disappeared.Wheat germ agglutinin(WGA)also had a certain effect on the silkworm PM.The PM was still relatively intact at 48 h,but the thickness of the membrane was not even.At the same time,"amorphous,spherical,electron-dense structure" was observed under electron microscopy.After feeding diflubenzuron,the surface of the PM became rough,but permeability was reduced.Congo red staining of the PM revealed that the multi-layer structure of the PM was abnormal;its layers became loose and wrinkled.Electron micrographs showed that's the surface structure of the PM was rough and uneven.It was presumed that the inhibition of chitin synthesis led to abnormal protein binding and abnormal lamellar structure,resulting in slow passage of the substance,thereby lowering the permeability of the PM.3.Both chitinase and protease disrupted the PM in a short time,resulting in increased permeability,However,electron micrographs of the surface morphology of the PM showed that the effects of the two enzymes were different.Protease provoked a rough surface of the PM,and the surface proteins of the matrix decomposed forming bulges.Chitinase decomposed the chitin skeleton of PM,resulting in the formation of grooves and slits.4.Among the four kinds of PM proteins(saline-soluble protein,surfactant-soluble protein,denaturant-soluble protein and non-extractable protein).the third type of extractant(urea in this experiment)has the greatest impact on PM permeability.In addition to protein extractants,some emulsifiers and dispersants in pesticide adjuvants can also increase the permeability of PM.5.SDS-PAGE and mass spectrometry analysis showed that FB28 had a significant effect on PM proteins.In the period of drastic morphological changes(2-4 hours after feeding),the high molecular weight proteins decreased and a clearly visible protein band with a molecular mass of approximately 17 kDa in the SDS-PAGE gel.This band disappeared and the high molecular weight protein reappeared after the restoration of normal diet.After 12 hours,the protein basically recovered.Mass spectrometry analysis of the 17 kDa bands showed that there were new proteins associated with immunity(Apolipophorin-III,Beta-1,3-glucanase),insect stress resistance(Methuselah-like protein 5)and protein binding(60s ribosomal export protein NMD3)compared with the control group.6.Saline can elute a large amount of protein(at least 10 peptides)on the PM,but those protein has no significant effect on the permeability of the PM,suggesting that these proteins are probably enzymes and intestinal residues attached to the PM surface.Mass spectrometry analysis of urea-eluted proteins revealed that most of them were chitin-binding proteins and membrane-binding proteins,which affected the permeability.