Sex Change in Clownfish: Molecular Insights from Transcriptome Analysis

Abstract

Sequential hermaphroditism is a unique reproductive strategy among teleosts that is displayed mainly in fish species living in the coral reef environment. The reproductive biology of hermaphrodites has long been intriguing; however, very little is known about the molecular pathways underlying their sex change. Here, we provide the first de novo transcriptome analyses of a hermaphrodite teleost´s undergoing sex change in its natural environment. Our study has examined relative gene expression across multiple groups-rather than just two contrasting conditions- and has allowed us to explore the differential expression patterns throughout the whole process. Our analysis has highlighted the rapid and complex genomic response of the brain associated with sex change, which is subsequently transmitted to the gonads, identifying a large number of candidate genes, some well-known and some novel, involved in the process. The present study provides strong evidence of the importance of the sex steroidogenic machinery during sex change in clownfish, with the aromatase gene playing a central role, both in the brain and the gonad. This work constitutes the first genome-wide study in a social sex-changing species and provides insights into the genetic mechanism governing social sex change and gonadal restructuring in protandrous hermaphrodites.

Figure 1. Changes in the gene expression profile of the brain during the sex change of clownfish (Amphiphrion bicinctus).

Contigs are plotted by their correlation with the index of sex-change (x-axis) and the dispersion in expression among individuals (y-axis, only values above 1 are plotted). Significant correlations are highlighted in blue for negative and in red for positive coefficients, indicating up-regulation in males and females, respectively. Examples from selected contigs (marked with a circle) regarding its position in the plot are highlighted. Labels: M - males; TM – transitional males; TF – transitional females; FI – immature females; and FM – mature females.

Figure 2. Changes in gonadal expression during the male-to-female sex change of clownfish (Amphiphrion bicinctus).

Contigs are plotted by their correlation with the index of sex change (x-axis) and the dispersion in expression among individuals (y-axis, only values above 1 are plotted). Significant correlations are highlighted in blue for negative and in red for positive coefficients, indicating up-regulation in males and females, respectively. Examples from selected contigs (marked with a circle) regarding its position in the plot are highlighted. Labels: M - males; TM – transitional males; TF – transitional females; FI – immature females; and FM – mature females.

Figure 3. Multivariate analyses of RNA-Seq data in clownfish (Amphiphrion bicinctus).

Gene expression changes were investigated by (A) Principal Component Analyses performed on normalized RNA-Seq data of a selected set of 173 transcripts from the brain (left) and 768 transcripts from the gonad (right). (B) Hierarchical dendrogram clustering on the same data. The clustering of transcripts results in three (brain: BG1 to BG3) and five (gonad: GG1 to GG5) clearly differentiated expression profiles. Labels: M – males (dark blue); TM – transitional males (light blue); TF – transitional females; FI (pink) – immature females (purple); and FM – mature females (brown).

Figure 4. Expression of selected key genes during sex-change stages in brain and gonad in clownfish (Amphiphrion bicinctus).

The vertical axis shows the normalized gene expression levels, bars represent the mean for each sex category ± SE. The description of each gene is found in Table 3, and the associated statistics in Supplementary Tables S2 and S3. Labels: M – males; TM – transitional males; TF – transitional females; FI– immature females; and FM – mature females.

Figure 5. Schematic representation of the proposed genetic mechanism underlying sex change in A. bicinctus at the brain level (upper panel) and the gonad (lower panel).

Solid arrows represent the known regulations of genes involved in sexual differentiation in A. bicinctus, while dashed arrows correspond to the suggested ones in females (upper part of the figure, both panels, ♀) and males (lower part of the figure, both panels, ♂). +indicates up-regulation/increase, − indicates down-regulation/decrease. Abbreviations: HPG, hypothalamic-pituitary-gonadal; cyp19a1b, cytochrome P450, family 19, subfamily A, polypeptide 1b; sox6, SRY (sex determining region Y)-box 6; foxp4, forkhead box P4; foxl2, forkhead box L2; Foxl2, forkhead box L2 protein; dmrt1/Dmrt1, doublesex and mab-3 related transcription factor 1; cyp19a1a, cytochrome P450, family 19, subfamily A, polypeptide 1a; amh, anti-Mullerian hormone; sox8, SRY (sex determining region Y)-box 8.

Figure 6. Diagram showing the experimental design used to profile molecular events related to sex-change in A. bicinctus.

Top: sixteen clownfish families were involved in the study, two functional males and all females were removed at time point 0 to trigger the sex-change; individual codes and the planned time point for collection (days after female removal) of the 14 sex-changing individuals along the 50-day experimental period is illustrated; bottom: flowchart depicting the transcriptome analysis pipeline.