Study On Transcriptome Of Manila Clam (Ruditapes Philippinarum) Infected By Digenetic Trematode

The Manila clam(Ruditapes philippinarum)is one of the four famous cultivated shellfish in China.However,the deterioration of the culture environment and the disease of clam breeding is becoming more and more serious in recent years.A large number of Manila clam died in Dalu island in June 2015.By dissecting the clam,10% of the gonads were orange red,and a large number of parasites were found in the orange red gonads.In this study,the molecular identification of the species of clam parasites,the morphological observation of parasites and the transcriptional analysis of the infected parasite clam were carried out.The amplified fragment of the parasite 18 S rRNA gene(1787bp)was obtained.The parasite was 92% similar to the trematode Steringophorus margolisiin GenBank(AJ287578.1.1934)by BLAST.It was found that there were two periods of digenetic trematode(sporocyst and cercariae)in the infected gonad.The mature sporocyst was cylindrical,3900–4250 ?m long,and 370–470 ?m wide.It contained dark brown granular substance,an embryo cell,an embryo ball,and a large number of cercariae.he heads of the cercariae were circular or elliptical,with length of 230–26 0?m and width of 185–228 ?m.The midgut was very short,and its end was connected to the middle section of the "V" excretory cystic duct.The tail size was 254–410×38–60 ?m,and the two sides of the tail had tailhairs.The RNA of gill,mantle and gonad tissue of infected and uninfected clams was extracted to explore the mechanism of infection of digenetic trematode in Manila clam.27652 transcripts were obtained from the sequencing results,in which there were 460 different genes,up 416 and 44 down.Among these differentially expressed genes,272 genes were successfully annotated to GO classification,and were significantly enriched in transmembrane transport,transportation,location,establishment,location,single organic transport and redox process of GO term(p<0.05).Deferentially expressed genes main enriched in trans-membrane transport,establishment of localization,single-organism transport and oxidoreductase-reduction process in Biological Process(p<0.05).While in MF,Transport activity and oxidoreductase activity were the most significant(p<0.05).The significant enrichment of up-regulated genes was the same as that of the total difference gene,and the down regulated gene had no significant enrichment of GO terms.Total of 331 differentially expressed genes were successfully annotated to 195 KEGG pathways.these pathway are associated with disease,receptor recognition and metabolism of amino acids,vitamins and other substances.ECM-receptor interaction was the pathway that most differentially expressed genes enriched and there are 14 genes on it.The 14 pathways of the up-regulated genes mostly are associated with the metabolic processes of various substances in the body such as tthe pentose and glucuronate interconversions,glycine,serine and threonine metabolism as well as peroxisome and CYP450 enzyme metabolic pathway.The down regulated genes are significantly enriched in 16 Pathway,and their functions are mostly related to human disease and pathogen infection.On this basis,11 genes(SGLT1,CRY1,CYP1A1,CC1Q2,SGST,CTL,HSP70,COL4,IAP,17-?HSD,TITI1)were selected from the differentially expressed genes and verified the transcriptome results with fluorescent quantitative PCR to explored the difference in expression of these genes between infected group,control group,and different tissues(gills,mantle and gonad).The result show that the expression of all candidate gene in gill of uninfected clam was significantly higher than the mantle and gonad(p<0.05).In infected clams,SGLT1 in three tissues showed up-regulated expression trend(p<0.05)and,the expression in the gonads were higher than gill(p<0.05).CYP1A1 was significantly down-regulated in the gills(p<0.05)but significantly up-regulated in the mantle and gonad(p<0.05).LCRY1 were increased significantly in the gills and gonad.In addition,the expression was no significant differences between gonad and gills.However,from the expression of immune related genes point of view,expression of SGST and HSP70 were increased significantly in three tissues(p<0.05)and the expression of SGST in gonad was significantly higher than that of gill(p<0.05).The expression of CC1Q2 was decreased significantly in the gills(p<0.05)and there was no significant difference of CTL expression in gills(p>0.05),but the expression of both were increased significantly in the mantle and gonad(p<0.05).In the case of genes related to reproductive damage,IAP was increased significantly in three tissues(p<0.05)and the others expression were decreased significantly in the gills(p<0.05).The expression of COL4 A increased significantly(p<0.05)in gonads and was significantly higher than gill and mantle(p<0.05).The expression of 17?-HSD and TITI1 both were significantly up-regulated in the mantle(p<0.05).In addition,TITI1 is the only one gene which is down-regulated significantly in the gonads(p<0.05).By studying the functions of these differentially expressed genes,it is found that in the process of parasitism,digenetic trematode can not only cause immune and reproductive damage,but also impede glucose transport and fat accumulation.This paper lays a theoretical foundation for the study of the molecular mechanism of the response of Manila clam infected with digenetic trematode.