Differential gene expression, including Sjfs800, in Schistosoma japonicum femalesbefore, during, and after male-female pairing

Schistosomiasis is a prevalent but neglected tropical disease caused by parasitic trematodes of the genus Schistosoma, with the primary disease-causing species being S. haematobium, S. mansoni, and S. japonicum. Male-female pairing of schistosomes is necessary for sexual maturity and the production of a large number of eggs, which are primarily responsible for schistosomiasis dissemination and pathology. Here, we used microarray hybridization, bioinformatics, quantitative PCR, in situ hybridization, and gene silencing assays to identify genes that play critical roles in S. japonicum reproduction biology, particularly in vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. Microarray hybridization analyses generated a comprehensive set of genes differentially transcribed before and after male-female pairing. Although the transcript profiles of females were similar 16 and 18 days after host infection, marked gene expression changes were observed at 24 days. The 30 most abundantly transcribed genes on day 24 included those associated with vitellarium development. Among these, genes for female-specific 800 (fs800), eggshell precursor protein, and superoxide dismutase (cu-zn-SOD) were substantially upregulated. Our in situ hybridization results in female S. japonicum indicated that cu-zn-SOD mRNA was highest in the ovary and vitellarium, eggshell precursor protein mRNA was expressed in the ovary, ootype, and vitellarium, and Sjfs800 mRNA was observed only in the vitellarium, localized in mature vitelline cells. Knocking down the Sjfs800 gene in female S. japonicum by approximately 60% reduced the number of mature vitelline cells, decreased rates of pairing and oviposition, and decreased the number of eggs produced in each male-female pairing by about 50%. These results indicate that Sjfs800 is essential for vitellarium development and egg production in S. japonicum and suggest that Sjfs800 regulation may provide a novel approach for the prevention or treatment of schistosomiasis. Author Summary Schistosomiasis is a common but largely unstudied tropical disease caused by parasitic trematodes of the genus Schistosoma. The eggs of schistosomes are responsible for schistosomiasis transmission and pathology, and the production of these eggs is dependent on the pairing of females and males. In this study, we determined which genes in Schistosoma japonicum females were differentially expressed before and after pairing with males, identifying the 30 most abundantly expressed of these genes. Among these 30 genes, we further characterized those in female S. japonicum that were upregulated after pairing and that were related to reproduction and vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. We identified three such genes, S. japonicum female-specific 800 (Sjfs800), eggshell precursor protein, and superoxide dismutase, and confirmed that the mRNAs for these genes were primarily localized in reproductive structures. By using gene silencing techniques to reduce the amount of Sjfs800 mRNA in females by about 60%, we determined that Sjfs800 plays a key role in development of the vitellarium and egg production. This finding suggests that regulation of Sjfs800 may provide a novel approach to reduce egg counts and thus aid in the prevention or treatment of schistosomiasis.


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Schistosomiasis, also known as bilharzia, is a tropical disease caused by parasitic  Ongoing work in our laboratory has indicated that during the development of S. 102 japonicum, no male-female pairing occurs up to 16 days after the host is infected. 103 Some pairing occurs 17 days post infection (dpi), and pairing is common 18 dpi. 104 Paired females begin laying eggs approximately 24 dpi. Therefore, in the present 105 study, to identify genes that likely contribute to pairing and reproduction, we used 106 microarray technology to determine differential gene expression in females 16, 18, 107 and 24 dpi. We identified genes that play critical roles in the development of the 108 vitellarium and in the production of eggs, providing a clearer understanding of gene 109 regulation before and after male-female pairing in the S. japonicum female and 110 insights on schistosome reproduction biology.    Quantitative PCR (qPCR) 172 Thirteen genes whose expression levels were increased and two genes whose  Total RNAsfrom eggs, cercariae, schistosomula, and females 24 and 42 dpi were 193 extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions.

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The total RNA concentration and purity were measured using a NanoDrop 2000 195 (Thermo Fisher). Quantitative PCR was performed as described above using primer 196 (Table 1)

Bioinformatics analysis of differentially expressed genes 284
To determine the potential function of these upregulated genes, the Gene

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Fifteen differentially expressed genes were selected for confirmation using qPCR.

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The analysis for each gene was repeated three times, and PSMD4 was used as the The gene expression levels for Sjfs800, cu-zn-SOD, and eggshell precursor 311 protein were determined using qPCR of the eggs, cercariae, schistosomula (at 16 dpi), 312 and female worms 24 and 42 dpi. As above, the results were normalized to the 313 housekeeping gene PSMD4, and the relative expression was then determined using 314 the 2 -ΔΔct method. We found three genes that were highly expressed in females 42 dpi, 315 and eggshell precursor protein and superoxide dismutase were also highly expressed 22 316 in females 24 dpi. The expression levels of Sjfs800 were low in the eggs, cercariae, 317 and schistosomula (at 16 dpi) and modestly increased in female worms at 24 dpi, with 318 a further increase at 42 dpi (Fig 2). These results suggested that these genes may be eggshell precursor protein, and Sjfs800 in female S. japonicum 24 dpi (Fig 3).

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Although cu-zn-SOD mRNA was located throughout much of the whole body, the 23 331 signal intensity in the ovary and vitellarium was markedly stronger than that in other 332 parts. Eggshell precursor protein was substantially expressed in the ovary and ootype 333 and was also found in the vitellarium. The expression of Sjfs800 mRNA was observed 334 only in the vitellarium.  an approximately 60% reduction in Sjfs800 mRNA levels on the tenth day later, and 360 this experiment was repeated three times (Fig 4).  . We also observed eggshell precursor protein signals in the ootype.

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Thus, the findings of the present study suggest that eggshell precursor protein maybe 457 play an important role in helping the vitellarium promote eggshell formation. the pairing rate and oviposition rate were also significantly decreased. Therefore, we 466 concluded that fs800 is vital for vitelline cell development and maturation and that 467 maturation of the vitellarium is required for S. japonicum females to produce eggs.

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The number male-female pairings was reduced by approximately 70% after Sjfs800