Schistosome sex matters: a deep view into gonad-specific and pairing-dependent transcriptomes reveals a complex gender interplay

Abstract

As a key event for maintaining life cycles, reproduction is a central part of platyhelminth biology. In case of parasitic platyhelminths, reproductive processes can also contribute to pathology. One representative example is the trematode Schistosoma, which causes schistosomiasis, an infectious disease, whose pathology is associated with egg production. Among the outstanding features of schistosomes is their dioecious lifestyle and the pairing-dependent differentiation of the female gonads which finally leads to egg synthesis. To analyze the reproductive biology of Schistosoma mansoni in-depth we isolated complete ovaries and testes from paired and unpaired schistosomes for comparative RNA-seq analyses. Of >7,000 transcripts found in the gonads, 243 (testes) and 3,600 (ovaries) occurred pairing-dependently. Besides the detection of genes transcribed preferentially or specifically in the gonads of both genders, we uncovered pairing-induced processes within the gonads including stem cell-associated and neural functions. Comparisons to work on neuropeptidergic signaling in planarian showed interesting parallels but also remarkable differences and highlights the importance of the nervous system for flatworm gonad differentiation. Finally, we postulated first functional hints for 235 hypothetical genes. Together, these results elucidate key aspects of flatworm reproductive biology and will be relevant for basic as well as applied, exploitable research aspects.

Figure 1. Sample relationships revealed by three different approaches.

(a) Multidimensional scaling (replicates for each sample are indicated by the same color). (b) Sample distance matrix based on a black/white scale. The samples were colored the same as in (a). (c) Hierarchical clustering based on RPKM values of all genes and the scaling was done across all samples for each gene. bM, bisex (paired) males; sM, single-sex (unpaired) males; bT, testes from bisex males; sT, testes from single-sex males; bF, bisex (paired) females; sF, single-sex (unpaired) females; bO, ovaries from bisex females; sO, ovaries from single-sex females.

Figure 2. Overview of the divisions of DEGs of testes and ovaries into 8 categories (Cat) based on their tissue-preferential/specific and pairing-dependent transcript occurrence.

Cat 1.1: Testis-preferential/specific and pairing-affected genes. Cat 1.2: Testis-preferential/specific and pairing-unaffected genes. Cat 2.1: Ovary-preferential/specific and pairing-affected genes. Cat 2.2: Ovary-preferential and pairing-unaffected genes. Cat 3.1: Pairing-affected genes in both gonads. Cat 3.2: Pairing-unaffected genes in both gonads. Cat 4.1: Transcripts of genes affected by pairing in testes but not in ovaries. Cat 4.2: Transcripts of genes affected by pairing in ovaries but not in testes. (a) Graphical illustration of the categories. The left and right parts indicate DEGs in testes (bT/sT) and ovaries (bO/sO), respectively. Grey color indicates genes that were not found to be significantly differentially transcribed. Blue and yellow areas represent genes whose transcript numbers were significantly lower or higher after pairing, respectively. (b) Transcript profiles of one representative gene of each category on a relative basis of transcript amounts. To provide more information about the transcript profiles of categorized genes, we added their relative expression also for the adult samples. The selected genes represent a cell-division cycle phosphatase cdc25 (Smp_152200; Cat 1.1), elav (embryonic lethal, abnormal visual system) (Smp_194950; Cat 1.2), cpeb1 (cytoplasmic polyadenylation element binding) (Smp_070360; Cat 2.1), a sox transcription factor (Smp_076600; Cat 2.2), a potassium channel of the twik (tandem of P domains in a weakly inward rectifying K⁺ channel) family (Smp_147550; Cat 3.1), lin-9 (Smp_133660; Cat 3.2), a von-Willebrand factor A (vWA) domain-containing protein gene (Smp_127480; Cat 4.1), and melk (maternal embryonic leucine zipper kinase) (Smp_166150; Cat 4.2).

Figure 3. Heatmap presentation of the hierarchical clustering of 3,748 female DEGs based on their higher (upper panel, 1,591 genes) or lower (lower panel, 2,157 genes) transcript numbers.

Transcript abundance across all worm samples is shown as follows: sM (1. column), sF (2. column), bM (3. column), and bF (4. column). Yellow and blue colors indicate high and low abundance, respectively. Exemplary genes (comprising largest fold-changes) are indicated by name showing that e.g. egg synthesis-associated genes such as tyrosinase, p14, and p48 are transcribed at the highest levels in bF (upper panel). In contrast, transcript levels of genes associated with motor function such as kinesin or troponin, or cell adhesion such as cell adhesion molecular or protocadherin decreased in bF (lower panel).

Figure 4. Transcript plots of exemplary genes involved in stem-cell proliferation and differentiation.

Gene identifiers and Smp numbers are given. bM, bisex males; sM, single-sex males; bT, testes from bisex males; sT, testes from single-sex males; bF, bisex females; sF, single-sex females; bO, ovaries from bisex females; sO, ovaries from single-sex females. SmPlk1, polo-like kinase 1, Smvlg1-2, vasa-like genes 1-3, SmFGFRa/b, fibroblast growth factor receptor, SmAgo, argonaute, and SmPCNA, proliferating cell nuclear antigen.

Figure 5. Hierarchical clustering of genes involved in neural processes.

Blue and yellow colors indicate low and high transcript levels, respectively (see color key). Right labeling: Smp numbers of the 61 genes, whose function has been associated with neural processes. Exemplary genes are indicated by name according to their annotation. Bottom line: all compared samples (bM, bisex males; sM, single-sex males; bT, testes from bisex males; sT, testes from single-sex males; bF, bisex females; sF, single-sex females; bO, ovaries from bisex females; sO, ovaries from single-sex females).

Figure 6. Expression of neuropeptide precursor and convertase genes.

(a) Hierarchical clustering of neuropeptide precursor genes. Blue and yellow colors indicate low and high transcript levels, respectively (see color key). Right labeling: Smp numbers of the 13 neuropeptide precursor genes. (b) Transcript profile of the neuropeptide convertase gene.