Anisakidae Nematodes On Digestive Tract Tissue Damage And The Effect Of Immune Response Of Coilia Nasus

Coilia nasus grows and fattens in the coastal waters.It often infects Anisakidae nematodes due to the ingestion of infect bait such as fish and shrimp.Because Anisakidae nematodes mainly infect in the digestive tract and abdominal cavity of C.nasus.They are less affected by the changes of external water environment.Anisakidae nematodes are infected for a long time in the process of reproductive migration of C.nasus.In this paper,the way of Anisakidae nematodes are entered into the abdominal cavity from the digestive tract of C.nasus and the tissue damage caused by Anisakidae parasites.Further,the effects of Anisakidae nematodes on the expression levels of various immune molecules of C.nasus were detected by ELISA.Finally,based on proteomics and metabolomics,the effects of Anisakidae nematodes on proteins and metabolites of C.nasus were studied.The immune pathway of C.nasus to Anisakidae nematodes was expounded.This study provides basic data for the immune interaction mechanism between Anisakidae nematodes and C.nasus,and provides a theoretical basis for the follow-up study of the impact of Anisakidae nematodes on the reproductive migration and population supplement of C.nasus.The main research contents are as follows:1.To observe the damage of Anisakidae nematodes to the digestive tract tissue of C.nasus: larges number of digestive tract tissue of C.nasus infected on Anisakidae nematodes were collected in the Yangtze Estuary during the flood season,and the digestive tract tissue of C.nasus not infected on Anisakidae nematodes was collected as the control group.Dissect examination,scanning electron microscopy and HE staining.The results showed thant: Anisakidae nematodes could pierce the stomach body and intestinal tissue of C.nasus and enter the abdominal cavity.At present,the phenomenon has not been found in the pyloric caecum.Anisakidae nematodes destroyed the mucosal folds of stomach tissue,intestinal tissue and pyloric caecum tissue of C.nasus,resulted in the injury and abscission of columnar epithelial cells.Anisakidae nematodes cause the separation between the mucosa and submucosa of the intestinal tissue,and accumulation of a large number of lymphocytes.The results showed that Anisakidae nematodes was not possibly conducive to the health of C.nasus.2.To study the levels of seven immune indices(TH1,MIF,IL-10,TNF-α,Ig M,Ig T and Ig D)expression of C.nasus: The expression levels of seven immune indices in spleen,head kidney and serum of control group and parasitic group C.nasus were measured by ELISA.and influenced by Anisakidae nematodes.The expression of seven immune indices in the spleen,head kidney and serum were measured by ELISA.The results showed that:(1)For blank group,the level of immune indices in spleen,head kidney and serum showed the same following trend: Ig D(P<0.01)> Ig M(P<0.001)> Ig T(P<0.001)>MIF(P<0.001)> TH1(P>0.05)> TNF-α(P>0.05)> IL-10,and the expression of seven immune indices in serum were significantly higher than those in spleen and head kidney(P<0.001).(2)For parasitic group,the level of seven immune indices in spleen of C.nasus were significantly higher than those of blank group(P<0.01).The level of six immune indices except Ig M in head kidney were significantly higher than those in the blank group(P<0.05).The level of IL-10 in serum was significantly higher than the blank group(P<0.001).(3)Overall,the IL-10 level of parasitic group in C.nasus decreased with the increase of parasitic number.The TH1 cell level of parasitic group in C.nasus decreased firstly but then increased with the parasitic number increasing,while MIF,TNF-α,Ig D,Ig M and Ig T showed the opposite tendency.Moreover,there was a significant correlation between the level of immune molecules in spleen(Ig T,TNF-α,TH1 cell,MIF,Ig M),serum(TH1 cell,Ig D,IL-10,TNF-α),head kidney(Ig T,TNF-α)and the parasites number.The results showed that Anisakidae parasites could induce the changes of inflammatory indexes and adaptive immune indexes of C.nasus.There was a significant correlation between the level of some immune molecules in spleen,head kidney and serum and the number of Anisakidae parasites.3.To analyze the Proteomic of C.nasus in liver: using i TRAQ technology to analyze the protein expression of C.nasus in liver after infected Anisakidae nematodes,a total of393 differential proteins were screened,of which 193 were up-regulated and 146 were down regulated.There were 37 differential proteins related to immunity,of which 9 were up-regulated and 28 were down regulated.Through GO analysis found that most of the differential proteins have lysosomal and complement functions,and pathway enrichment analysis found that 10 immune related pathways were screened,such as phagosome,lysosome,endocytosis,autophagy animal,complement and coagulation cascade and toll like receptor signaling pathway etc.Seven immune related differential proteins were identified,such as complement C3,C7 and C1 q,MHC-Ⅰ,MHC-Ⅱ,Ig and Rab-7a.And these seven immune related proteins were significantly down regulated in the parasitic group.It is speculated that Anisakidae nematodes were possibly inhibited the complement cascade reaction and phagocytosis process of C.nasus,which may be conducive to the survival of Anisakidae nematodes.4.To analyze metabolomic of C.nasus in liver: using LC/MS technology to analyze the metabolites in the liver of C.nasus after infected Anisakidae nematodes,a total of 88 differentially expressed metabolites were screened,of which 60 were significantly upregulated and 28 were significantly down regulated.Multivariate statistical analysis(PLS-DA and OPLSD-DA)showed that Anisakidae pnematodes produced a significant effect on liver metabolites of C.nasus(P<0.05).Through pathway enrichment analysis.The significant signal pathways were identified,including protein digestion and absorption,central carbon metabolism(cancer),linoleic acid metabolism,aminoacyl t RNA biosynthesis,unsaturated fatty acid biosynthesis,phenylalanine metabolism,glycerol phosphatide metabolism,ABC transports,Amino acid biosynthesis and purine metabolism.Interestingly,most metabolites in the above pathways was significantly upregulated,but most metabolites in glycerol phospholipid metabolism were significantly down regulated.but most of these metabolites were significantly down regulated.It was reflected the complex interaction mechanism between C.nasus and Anisakidae nematodes,which needs to be further studied.