Structure And Function Of Peritrophic Membrane In Intestine Of Eriocheir Sinensis And Its Response To Dietary Non-starch Polysaccharides

Peritrophic membrane(PM) is an important part in insect gastrointestinal tract, which wascomposed of chitin fibrils and protein. It is a semi-permeable barrier between the chyme and the cells lining the gut to protect the underlying epithelial cells from food mechanical damage, chemical toxins and pathogens infection. In addition, the PM separates the gut into the endoperitrophic space and the ectoperitrophic space, which facilitates the nutrients absorption. So far, the PM had been studied deeply in insects and been a potential target for biocontrol of pest insects. PM was also observed in crustaceans, however, its formation and biological characteristics had not been studied enough. Chinese mitten crab is one of important aquatic animals. In the present study, we studied the structure and function of the PM in Chinese mitten crab firstly, and then studied the effect of non-starch polysaccharides on peritrophic membrane.Experment one. The aim of this study was to investigate the secretion, structure and function of peritrophic membrane(PM) in gastrointestinal tract of Eriocheir sinensis. The gastrointestinal tract was sampled from the Chinese mitten crab of 30 g and in state of intermolt. Histology and electron microscopy techniques were employed to observe the intestinal and PM morphology of E. sinensis. Chitinase dissolution test and SDS-PAGE gel electrophoresis were used to study the composition of the PM, and the LC-ESI-MS/MS was used to identify the function of PM protein. Under the microscope it was observed the surface of whole gastrointestinal tract of E. sinensis was covered by PM. Compartmentalization of PM was observed in intestine except midgut. When PM was detached from the epithelial cells, the columnar epithelial cells were becoming irregular round with the nucleus disappeared. A large number of mitochondria and secretory vesicles were observed in cytoplasm under the electron microscope. PM was able to be dissolved by chitinase. SDA-PAGE map showed that there were 7 clearer electrophoresis bands and their molecular weight was mainly distributed in more than 40 KD. Results from LC- ESI- MS/MS analysis showed that the PM proteins included sodium-potassium ATPase, ATP synthetase, actin and tubulin, thioredoxin, oxygenase, etc. These results indicated that the PM of E. sinensis is secreted by epithelial cells. it improves absorption by compartmentalization, and may participate in physiological processes of immune, osmotic regulation and nutrients transportation.Experiment two. The aim of this experiment was to study the identification and molecular characterisation of PM protein. Two full-length c DNA sequences of peritrophic membrane proteins was obtained by the rapid amplification of c DNA ends, and named Es-PL44 and Es-PP1 respectively. Es-PL44 was 855 bp and encoded 267 amino acids, and Es-PP1 was 1611 bp and encoded 468 amino acids. The results of the amino acid sequence analysis shown that there was a signal peptide sequence in the N-terminal in both Es-PL44 and Es-PP1, and all belonged to secreted protein.Protein domain analysis showed the Es-PL44 and Es-PP1 had three and one chitin binding domains(CBD) respectively. The pairwise percentage identities of the deduced amino acid sequences for Es-PL44 showed the highest similarity of 56% to the peritrophins 3-B precursor of Acyrthosiphonpisum, and the Es-PP1 has the highest similarity of 70% to theperitrophins 1-H precursor of Triboliumcastaneum, however, the percentage identities of amino acid sequence was low when compared Es-PL44 and Es-PP1 with that of Penaeusmonodon and Penaeussemisulcatus, which were 16.32% and 10.71%, 14.02% and 7.49%, 17.01% and 10.28%, 18.58% and 9.42%, respectively.Phylogenetic analysis showed that two kinds of peritrophic membrane proteins had a close relative with insects, but in different branches with aquatic animals, and Es- peritrophin1 and Es- peritrophin2 had distant relatives. The full-length c DNA sequences were submitted to the NCBI gene library, and two Gen Bank accession numbers, KU041138 and KU041139, were obtained:. The results suggested that Es-PL44 and Es-PP1 were two new were two kinds of new type of genes of peritrophic membrane protein, which contained Chitin binding domain structure and mucins domain structure, and it was the foundation for revealing the digestive physiological characteristics of eriocheir sinensis.Experment three. The expression pattern of Es- peritrophin1 and Es- peritrophin2 in the digestive tract and other tissues were studied. The digestive tract from Chinese mitten crab(weight 50g) was been taken, and paraffin slice were made and then the expression of the two peritrophic membrane protein genes by in situ hybridization technique. The results shown that Es-PL44 and Es-PP1 gene in the stomach, midgut, intestinal ball were expressed on epithelial cells, two gene expression in the stomach of different sites, and Es-PL44 gene compared with the Es-PP1 gene signal is stronger which were different from in insect. In order to understand more of the function of peritrophic membrane,three experiment groups of high salinity, high Ph and starvation were set, and crabs, whose initial weight was 30 g, were fed diet for 10 days. The expression of the peritrophinm RNA in digestive tract were analysed by RT-q PCR. In order to knowing the function of the further peritrophic membrane, we studied The results shown showed that Es-PL44 and Es-PP1 genes expressed in intestinal tract were significantly lower comparing with the control group under different stimulating conditions. The results suggested that Es-PL44 and Es-PP1 played different roles in peritrophic membrane, taking part in the osmotic adjustment, immune adjustment and digestive physiological process.Experment four. The aim of this study was to explore the response of PM to dietary non-starch polysaccharides. Four experimental diets, SP, ISP, SX and ISX, were formulated to contain 12% of soluble pectin, insoluble pectin, soluble xylan and insoluble xylan respectively. Crabs, whose initial weight was 1.6 ± 0.7g, were fed test diets for 13 weeks, and then the effect of solubility of NSPs on the permeability of the PM was studied by administrating blue Dextran 2000 solution. No blue Dextran 2000 were overflowed from intestine wall in all groups. The expression activity of the peritrophin gene in different group was compared by the RT-q PCR technology, and result showed the expression activity of the Es-PL44 was lowest in the ISP group, and was not significantly different between SP、ISP and ISX group. The expression activity of Es-PP1 gene in SP group was significantly higher than in the ISP, SX and ISX group, the expression activity of Es-PP1 gene in SX group was significantly lower than in the ISP. Results suggested that dietary NSP no significant effect of NSP on the permeability of the PM; had a potential effect on intestine secretion and PM formation.