| Schistosomiasis,caused by parasitic trematodes of the genus Schistosoma,remains a devastating public health problem,with over 250 million people infected and 779 million people at risk worldwide,especially in developing countries.It is estimated that the annual death rate due to schistosomiasis is at 20,000 globally.The freshwater amphibious snail Oncomelania hupensis is the obligate intermediate host of Schistosoma japonicum.Additionally,O.hpensis also serves as the first intermediate host of Exorchis sp.,a trematode which belongs to the family Cryptogonimidae and uses the carnivorous fish Parasilurus asoyus as definitive host,but not humans.It has been demonstrated that when O.hupensis was pre-infected with larvae of Exorchis sp.at different time intervals,and then the development of all S.japonicum larvae was blocked and inhibited.However,the molecular mechanisms involved in this process were not clear and require further investigation.In this study,the preliminary investigations of biological control resources were conducted in Poyang Lake schistosomiasis epidemic region in Xingzi County,Jiangxi Province.The results showed the total number of collected P.asoyus fishes were 494 from 2012 to 2016,the total infection rate by Exorchis sp.was 65.79%,and the average intensity of infection was 14.21.Additionally,the collected snails were screened individually at least three times for any natural infections using the shedding method.The tested total number of O.hupensis snails were 19528,and the infection rate by Exorchis sp.and S.japonicum was 1.11%and 0.06%,respectively.In order to further study on the prevention and control of schistosomiasis,more investigations on the interactions of parasites in O.hupensis snails are needed.In recent years,continuous studies on snail,schistosome and Exorchis interactions have achieved remarkable advances,while little information is available about the mechanism involved in the interaction among Oncomelania,Schistosoma and Exorchis.Therefore,we compared the transcriptomes of DNA purified from O.hupensis infected with different parasites to identify some relevant molecules of the infected snails involved in this biological control process.And we obtained some crucial molecules including MIF(Macrophage migration inhibitory factor),TRX(Thioredoxin),etc.Next,based on the Digital Gene Expression Profiling(DGE)analysis and validation by qPCR,we chose the O.hupensis MIF(OhMIF)as the target molecule for detailed studies.MIF,a pleiotropic immunoregulatory cytokine of innate immunity,plays an important role in the host's antimicrobial defense system and stress response that promotes the pro-inflammatory functions of immune cells.In this study,we have successfully identified and characterized MIF from O.hupensis(OhMIF),and then analysed the biological activities of OhMIF.The results showed rOhMIF displayed the conserved D-dopachrome tautomerase activity depending on the N-terminal Proline(Pro2),and this enzymatic activity can be significantly inhibited by mammalian MIF antagonist ISO-1,implying the binding sites of MIF are evolutionarily conserved throughout species.Next,the enzymatic kinetics for D-dopachrome tautomerase activity of rOhMIF and rHsMIF(recombinant Homo sapiens MIF)were measured and compared,and the results indicated the reaction rate,catalytic efficiency and substrate affinity of rOhMIF are significantly lower than those of rHsMIF.Moreover,rOhMIF had the oxidoreductase activity which can utilize DTT as reductant to reduce insulin,while mammalian MIF use GSH as reductant.Additionally,we also demonstrated rOhMIF and its mutant rOhMIFP2G can promote the activation and phosphorylation of ERK1/2,implying that tautomerase activity of rOhMIF is not required for this biological function.Next,the involvement of OhMIF in the snail host immune response to the parsite S.japonicum was studied in this research.We found OhMIF was expressed in various snail tissues,and immunolocalized in the cytoplasm of hemocytes.Our results also showed that the expression of OhMIF was significantly increased in O.hupensis following challenge with S.japonicum,implying OhMIF was responsive to the parasite invasion.Additionally,we successfully conducted the dsRNA-mediated knockdown of OhMIF in snails by oral feeding,and found that knockdown of OhMIF reduced significantly the percentage of phagocytic cells of circulating hemocytes in O.hupensis.Moreover,the results also indicated that OhMIF is not only implicated in maturity and differentiation of three major hemocyte subpopulations of different sizes,but also closely linked to the migration and recruitment of phagocytic hemocytes towards infected sites.Owing to the limited information regarding cDNA sequences of O.hupensis genes,we next used combined proteomics and transcriptomics to obtain more information of crucial factors involved in the internal molecular mechanism of biological control on S.japonicum.We have obtained 46162 unigenes from the transcriptome of O.hupensis,and identified 3811 O.hupensis proteins by iTRAQ quantitative proteomics.And then the functions of all the identified genes and proteins were annotated and classified.Next the differentially expressed proteins among OhN-1,OhSj-1,OhE-1 and OhES-1 were identified and analysed.We chose the identified proteins in two more interested groups(Common and Only)for further investigation,and found some important factors involved in the snail host immune reponse to the parasite S.japonicum.In conclusion,we identified many differentially expressed proteins which may be involved in the process of biological control on S.japonicum,analysed the biological activities of OhMIF,and confirmed the involvement of OhMIF in the snail host innate immune response to the parasite S.japonicum.This work provided the first known functional evidence in exploring the molecular mechanisms of interaction between Oncomelania and Schistosoma,the tools and methods developed in this study will provide a template for the research of other Oncomelania related proteins. |
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