The RIO protein kinase-encoding gene Sj-riok-2 is involved in key reproductive processes in Schistosoma japonicum

Abstract

Background: Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is caused by parasitic trematodes of the genus Schistosoma. The pathogenesis of schistosomiasis is caused by eggs whose production is the consequence of the pairing of schistosomes and the subsequent sexual maturation of the female. Previous studies have demonstrated that protein kinases are involved in processes leading to the male-induced differentiation of the female gonads, ovary and vitellarium. Right open reading frame protein kinase 2 (RIOK-2) is a member of the atypical kinase family and shown in other organisms to be responsible for ribosomal RNA biogenesis and cell-cycle progression, as well as involves in nematode development. However, nothing is known about its functions in any trematode including schistosome.

Methods: We isolated and characterized the riok-2 gene from S. japonicum, and detected the transcriptional profiles of Sj-riok-2 by using real-time PCR and in situ hybridization. RNAi-mediated knockdown of Sj-riok-2 was performed, mitotic activities were detected by EdU incorporation assay and morphological changes on organs were observed by confocal laser scanning microscope (CLSM).

Results: In silico analyses of the amino acid sequence of Sj-RIOK-2 revealed typical features of this class of kinases including a winged helix (wHTH) domain and a RIO kinase domain. Sj-riok-2 is transcribed in different developmental stages of S. japonicum, with a higher abundance in adult females and eggs. Localization studies showed that Sj-riok-2 was mainly transcribed in female reproductive organs. Experiments with adult schistosomes in vitro demonstrated that the transcriptional level of Sj-riok-2 was affected by pairing. Knocking down Sj-riok-2 by RNAi reduced cell proliferation in the vitellarium and caused the increased amount of mature oocytes in ovary and an accumulation of eggs within the uterus.

Conclusions: Sj-riok-2 is involved in the reproductive development and maturation of female S. japonicum. Our findings provide first evidence for a pairing-dependent role of Sj-riok-2 in the reproductive development and maturation of female S. japonicum. Thus this study contributes to the understanding of molecular processes controlling reproduction in schistosomes.

Keywords: Gonad; RIO2 kinase; RNA interference; Reproductive development; Schistosoma japonicum.

Fig. 1

Schematic diagram showing the genomic organization of riok-2 of Schistosoma japonicum. Black boxes indicate exons, with the numbers above indicating the length (bp) of an exon. Introns are indicated by slanted lines between the exons, with the numbers indicating intron length (bp)

Fig. 2

Alignment of the inferred amino acid sequences of Schistosoma japonicum Sj-RIOK-2 with RIOK-2 orthologs of eight other species. The eight selected species are Schistosoma mansoni (CCD79377.1, Sm-RIOK-2), Caenorhabditis elegans (CAC70109.2, Ce-RIOK-2), Homo sapiens (NP_060813.2, Hs-RIOK-2), Xenopus tropicalis (NP_001016682.1, Xr-RIOK-2), Danio rerio (NP_998719.2, Dr.-RIOK-2), Drosophila melanogaster (NP_651365.1, Dm-RIOK-2), Saccharomyces cerevisiae (CAC70109.2, Sc-RIOK-2), Arabidopsis thaliana (AEE78772.1, At-RIOK-2). Functional domains including the wHTH region (grey), ATP binding motif (red), flexible loop (yellow), hinge region (blue), active site (green), metal binding motif (orange) are highlighted and labeled below the alignment. Red arrows indicate conserved residues within the wHTH domain and “Asp” and “Asn” residues responsible for phosphoryl transfer exits within the active site, and “Asp” required for the positioning of metal ions exits within the metal binding motif. Asterisks under the alignment indicate identical residues. “:” represents strong similarity, “.” represents weak similarity, and a lack of a symbol indicates no similarity among residues

Fig. 3

The neighbor-joining (NJ) tree of Schistosoma japonicum RIOK-2 with 13 homologues from 13 selected species. The species selected were nine invertebrates including Brugia malayi (CRZ23003.1), Clonorchis sinensis (GAA29846.2), Caenorhabditis elegans (CAC70109.2), Drosophila melanogaster (NP_651365.1), Haemonchus contorts (ADW27445.1), Loa loa (EFO24525.1), Schistosoma haematobium (XP_012792720.1), Schistosoma mansoni (CCD79377.1), Strongyloides ratti (CEF64700.1) and four vertebrates including Danio rerio (NP_998719.2), Homo sapiens (NP_060813.2), Mus musculus (NP_080210.1), Xenopus tropicalis (NP_001016682.1). The RIOK-2 from Saccharomyces cerevisiae (KZV08416.1) was used as the outgroup. Bootstrap values are displayed above or below the branches

Fig. 4

Transcriptional profiles of Sj-riok-2 detected in different developmental stages of Schistosoma japoncium by real-time PCR. Relative expression levels of transcripts were analyzed by the 2^-△△Ct method, and Sj-β-tubulin was used as internal standard. Data are representative of the mean ± SD of three separate experiments. All stages were compared with the cercariae stage, and statistically significant differences are shown as ** (P = 0.001) and *** (P < 0.0001); ### represents the significant difference (P < 0.0001) in transcript abundances between female and male worms

Fig. 5

Transcription localization of Sj-riok-2 detected by in situ hybridization on sections of adult worms of Schistosoma japonicum. a Semi-quantification of the Sj-eggshell (Sj-es) gene in 42 d adult worms. b Histological localization of Sj-es, with antisense probe (left) and sense probe (right) of Sj-es. c Histological localization of Sj-riok-2, with antisense probe (c1-4) and sense probe (c5-8). Transcripts of Sj-es were detected in the vitellarium (v), and Sj-riok-2 was localized mainly in all reproductive organs: ovary (o), vitellarium (v), mature oocyte (mo), immature oocyte (imo), vitelloduct (vtd), ootype-surrounding area (ot), testis (t) and gastrodermis (ga). Scale-bars: 50 μm

Fig. 6

Effects of pairing on the transcript level of Sj-riok-2 in the female and male Schistosoma japonicum. Worm couples, single females, and single males were cultured for 3 d (a) and for 9 d (b) in vitro. After 3 d, the same number of single females and males were combined for re-pairing for another 6 d (b). Singly cultured and re-paired worms were compared with paired worms, and statistically significant differences were shown as ** (P < 0.01); #/### represent the significant differences (P < 0.05 and P < 0.001) in transcript abundances between re-paired worms and single worms

Fig. 7

Transcriptional levels of Sj-riok-2 and Sj-plk-1 in worms treated with Sj-riok-2 dsRNA or/and Sj-plk-1 dsRNA. Relative Sj-riok-2 (a) and Sj-plk-1 (b) transcript levels of females and males, respectively, after treatment with Sj-riok-2 dsRNA, Sj-plk-1 dsRNA, or both Sj-riok-2 and Sj-plk-1 dsRNAs; as controls Sj-stk-6 dsRNA or no dsRNA were used. All dsRNA-treated groups were compared with control (non-treated) groups. Data are representative of the mean ± SD of three separate experiments. **, *** indicate P < 0.01, P < 0.001

Fig. 8

Cell proliferation activity of Schistosoma japonicium couples treated with dsRNAs of Sj-riok-2 and/or Sj-plk-1. The EdU-incorporation cells of non-treated worms were detected in the vitellarium and ovary of females (a, e), testes and parenchyma of males (i, m). EdU+ cells were detected in Sj-riok-2 dsRNA (b, f, j, n)-, Sj-plk-1 dsRNA (c, g, k, o)-, Sj-riok-2- and Sj-plk-1 dsRNA-group (d, h, l, p). Abbreviations: v, vitellarium; o, ovary; imo, immature oocytes; t, testes; p, parenchyma. Scale-bars: 50 μm

Fig. 9

Morphology of the reproductive organs from Schistosoma japonicium couples treated with dsRNAs of Sj-riok-2 and/or Sj-plk-1. Whole mount preparations of non-treated worms (a, e, i, m), Sj-riok-2 dsRNA-treated worms (b, f, j, n), Sj-plk-1 dsRNA- treated worms (c, g, k, o) and Sj-riok-2 plus Sj-plk-1 dsRNA doubly-treated worms (d, h, l, p) were observed by confocal scanning laser microscope. Abbreviations: o, ovary; imo, immature oocytes; mo, mature oocytes; v, vitellarium; vtd, vitelloduct; ut, uterus; t, testes; sv, sperm vesicle. Scale-bars: 100 μm