De novo transcriptome assembly from the gonads of a scleractinian coral, Euphyllia ancora: molecular mechanisms underlying scleractinian gametogenesis

Abstract

Background: Sexual reproduction of scleractinians has captured the attention of researchers and the general public for decades. Although extensive ecological data has been acquired, underlying molecular and cellular mechanisms remain largely unknown. In this study, to better understand mechanisms underlying gametogenesis, we isolated ovaries and testes at different developmental phases from a gonochoric coral, Euphyllia ancora, and adopted a transcriptomic approach to reveal sex- and phase-specific gene expression profiles. In particular, we explored genes associated with oocyte development and maturation, spermiogenesis, sperm motility / capacitation, and fertilization.

Results: 1.6 billion raw reads were obtained from 24 gonadal samples. De novo assembly of trimmed reads, and elimination of contigs derived from symbiotic dinoflagellates (Symbiodiniaceae) and other organisms yielded a reference E. ancora gonadal transcriptome of 35,802 contigs. Analysis of 4 developmental phases identified 2023 genes that were differentially expressed during oogenesis and 678 during spermatogenesis. In premature/mature ovaries, 631 genes were specifically upregulated, with 538 in mature testes. Upregulated genes included those involved in gametogenesis, gamete maturation, sperm motility / capacitation, and fertilization in other metazoans, including humans. Meanwhile, a large number of genes without homology to sequences in the SWISS-PROT database were also observed among upregulated genes in premature / mature ovaries and mature testes.

Conclusions: Our findings show that scleractinian gametogenesis shares many molecular characteristics with that of other metazoans, but it also possesses unique characteristics developed during cnidarian and/or scleractinian evolution. To the best of our knowledge, this study is the first to create a gonadal transcriptome assembly from any scleractinian. This study and associated datasets provide a foundation for future studies regarding gametogenesis and differences between male and female colonies from molecular and cellular perspectives. Furthermore, our transcriptome assembly will be a useful reference for future development of sex-specific and/or stage-specific germ cell markers that can be used in coral aquaculture and ecological studies.

Keywords: Euphyllia ancora; Gonads; Oogenesis; Ovary; Phase-specific; RNA-seq; Scleractinian corals; Sex-specific; Spermatogenesis; Testis; Transcriptome assembly.

Fig. 1

Euphyllia ancora and its germ cells observed histologically in isolated gonads at different sampling times. a External appearance of an E. ancora colony. b External appearance of tentacles of an E. ancora colony. Anchor-like tentacles and the flabello-meandroid skeleton typify E. ancora. The pictures were taken at Nanwan Bay, Kenting National Park, in southern Taiwan in October 2016. c A top view of an E. ancora skeleton. The picture was taken after removal of polyp tissue in the laboratory. d Periods of oogenesis (pink arrow) and spermatogenesis (blue arrow) and predicated spawning timing (*). Letters (e-l) on the arrows correspond to Figure 1 (e-l) below, and indicate the timing (month) of sampling for ovaries and testes. e-h The external appearance of isolated ovaries in October and December 2016 and February and April 2017. e’-h’ Histological observation of the isolated ovaries. e, e’ The early phase of ovaries. f, f’ The middle phase of an ovary. g, g’ The late phase of an ovary. h, h’ The premature/mature phase of an ovary. i-l The external appearance of isolated testes in February, March, April, and June 2017. i’-l’ Histological observation of isolated testes. i, i’ The early phase of a testis with spermatogonia. j, j’ The middle phase of a testis having spermatogonia and primary spermatocytes. k, k’ The late phase of a testis with secondary spermatocytes and spermatids. l, l’ The mature phase of a testis with mature sperm. Sections were stained with hematoxylin and eosin. Scale bars = 1 cm (c); 500 μm (e-l); 50 μm (e’-h’); 10 μm (i’-l’)

Fig. 2

Identification of E. ancora contigs from the transcriptome assembly of an E. ancora holobiont. a A flow chart for identification of E. ancora contigs from the transcriptome assembly (all contigs) that contains contigs from the host coral, symbiotic (dinoflagellates), and other organisms (bacteria). b Distribution of GC percentages of assembled contigs. Red line: all contigs, green line: E. ancora contigs, blue line: extracted Symbiodiniaceae contigs. c Proportions of contigs from E. ancora, Symbiodiniaceae, and other symbiotic organisms (other contigs) in the initial whole holobiont transcriptome assembly. Only extracted E. ancora contigs (56.7%) were used for further analysis

Fig. 3

Contig numbers in the reference E. ancora gonadal transcriptome that matched SWISS-PROT and Pfam databases. a Results of BLAST searches against the SWISS-PROT database (cut-off -evalue le-5). Note that 21,569 out of 35,802 contigs (60.2%) had significant similarities with database sequences. bIdentification of protein domains using the Pfam database (cut-off -evalue le-5) for contigs from the reference E. ancora gonadal transcriptome. Note that 23,686 out of 35,802contigs (66.2%) had conserved protein domains

Fig. 4

Differentially expressed genes in oogenesis and spermatogenesis of E. ancora. a 2023 and 678 genes were differentially expressed during oogenesis and spermatogenesis, respectively, and 67 of those genes were differentially expressed in both oogenesis and spermatogenesis (q-value< 0.05, ANOVA). b Relative gene expression levels of differentially expressed genes (2023 genes) at different phases of ovaries. CPM values were scaled to row Z-scores for each of the genes. In premature/mature ovaries, 631 genes were expressed at higher levels than in the other 3 phases. Among the 631 genes, 446 genes (71%) matched the SWISS-PROT human database, as shown in the pie chart. c Relative gene expression levels of differentially expressed genes (678 genes) at different phase of testes. CPM values were scaled to row Z-scores for each of the genes. In mature testes, 538 genes were expressed more highly than in the other 3 phases. Among the 538 genes, 305 (57%) matched the SWISS-PROT human database, as shown in the pie chart. In the heatmaps, each row represents a differentially expressed gene and the columns represent time points. The color bar on the left indicates expression levels

Fig. 5

Genes potentially involved in oocyte development and maturation, and in sperm motility/capacitation, and fertilization in E. ancora. Genes indicated in red have been reported in our previous studies