| Schistosomiasis caused by trematode parasites of the genus Schistosoma is a serious parasitic disease that infects more than 258 million people worldwide.As no commercial vaccine available,praziquantel(PZQ)is the only effective drug applied to against adult worms,but its extensive use raises the potential of emerging resistance strains,which aggravates the needs for alternative drug or vaccine.A portion of eggs produced by female accumulates in tissues which induce inflammatory granuloma is mainly responsible for the pathogenic consequences of this disease.Elucidating the function of reproductive process relevant genes may provide theoretical basis for developing effectivenovel vaccines or drugs.Serine/threonine protein phosphatase 1(PP1)has been proved to be involved in cell proliferation,spermiogenesis and parasite growth.The specific function of PP1 was achieved by the PP1 catalytic subunit binding with different regulatory subunits.Our study aims to illustrate the role of PP1 in the reproductive process of Schistosoma japonicum.Molecular and bioinformatic methods were employed to isolate and characterize PP1 catalytic subunit(PP1c)encoding genes.In the present study,three genes were isolated and characterized,namely Sj-ppl-1,Sj-ppl-2 and Sj-ppl-3.Transcriptional patterns of Sj-ppl-1/2/3 were investigated in different developmental stages by real-time PCR and different tissue sections of adult worms by in-situ hybridization;RNAi technology was used to study the potential roles of Sj-pplc in the reproductive development;RNA-seq technology was employed to analyze the transcriptional profile changes in worms after silencing Sj-pplc,which may reveal the potential mechanism of PPlc to regulate development processes in schistosomes.In the present study,we obtained the following findings:(1)The gene characterization of Sj-pplcThree PPlc encoding genes were identified in S.japonicum.The amino acid sequence alignment showed high homology among Sj-pplc genes and protein phosphatase signature motif,metal ion binding site,regulatory subunit and specific inhibitors binding sites are conserved in the sequences.Phylogenetic analyses revealed that-Sj-PP1-1,Sj-PP1-2 and-Sj-PP1-3 belong to PP1beta,PP1 gammal and PP1 alpha subfamily,respectively.(2)Transcriptional patterns of Sj-pplcSj-ppl-1/2/3 genes were transcribed in different developmental stages of S.japonicum with enriched transcription in eggs.Sj-ppl-1/2/3 genes were mainly transcribed in reproductive organs and tissues.(3)RNAi effect on Sj-pplcThe post-infected 21 d paired worms were treated with Sj-ppl-1,Sj-ppl-2,Sj-ppl-3 dsRNA respectively by soaking method,but no obvious phenotype was observed,which may be caused off target effect due to high similarity between sequences of three Sj-pplc and also maybe due to the potential functional overlap among the three molecules.Therefore,the combined Sj-ppl-1/2/3 dsRNA were used together to knock down three genes at the same time that sufficiently decreased the transcription level by 60%-96%(P<0.01).(4)Knockdown of Sj-pplc interfered the development of S.japonicum and caused morphology changeThe worm length of Sj-pplc dsRNAs treated worms were significantly shorter than that of non-treated worms(P<0.001)after RNAi for 7 d,which indicated the suppression of Sj-pplc induced stunted growth of worms.Continuing RNAi to 12 d caused morphological changes in Sj-pplc dsRNAs treated males including bulges of varying size and swelling in posterior part of their body.(5)Knockdown of Sj-pplc reduced pairing stability and motility.Knockdown of Sj-pplc reduced pairing stability between female and male.After RNAi for 12 d,there is no pairing couple in Sj-pplc dsRNAs treated group.Worms from Sj-pplc dsRNAs treated group have lower motility compared with worms from control group.(6)Knockdown of Sj-pplc inhibited the maturation of reproductive systemKnockdown of Sj-pplc significantly reduced cell proliferation activity in ovary,vitellarium,testis and parenchyma.Meanwhile,it also resulted in delayed and defective maturation of ovary,vitellarium and testis as well as density reduction of mature sperms in sperm vesicle.In addition,the number of eggs produced by females that treated with Sj-pplc dsRNAs was significantly less than that from non-treated female after RNAi for 10 d in vitro.(7)Knockdown of Sj-pplc changes transcriptional profile in both male and female S.japonicumThe RNA-seq analysis between non-treated group and Sj-pplc dsRNAs treated group showed that a large number of genes were differentially expressed in male more than that in female(2291:229).These differentially expressed genes mainly involved in cell catabolism,lipid metabolism,cytoskeleton organization,DNA replication and egg synthesis.It indicated Sj-pplc may function in worm growth,reproductive system maturation and female-male interaction processes through regulating nutrition uptake and metabolism,cell proliferation and differentiation,morphological maintenance as well as other biological processes.Taken together,our research characterized three PP1c in S.japonicum and investigated their roles in worm growth and reproductive process,which suggested the function of PPlc in worm growth,germ cell development and pairing behavior.Our present study would provide experimental data for the research of the molecular mechanism of female-male interaction,it could also provide information and laid foundation for the development of novel drugs or vaccines which target the reproductive processes of S.japonicum. |
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