Transcriptomic analysis reveals differences in the regulation of amino acid metabolism in asexual and sexual planarians

Many flatworms can alternate between asexual and sexual reproduction. This is a powerful reproductive strategy enabling them to benefit from the features of the two reproductive modes, namely, rapid multiplication and genetic shuffling. The two reproductive modes are enabled by the presence of pluripotent adult stem cells (neoblasts), by generating any type of tissue in the asexual mode, and producing and maintaining germ cells in the sexual mode. In the current study, RNA sequencing (RNA-seq) was used to compare the transcriptomes of two phenotypes of the planarian Dugesia ryukyuensis: an asexual OH strain and an experimentally sexualized OH strain. Pathway enrichment analysis revealed striking differences in amino acid metabolism in the two worm types. Further, the analysis identified serotonin as a new bioactive substance that induced the planarian ovary de novo in a postembryonic manner. These findings suggest that different metabolic states and physiological conditions evoked by sex-inducing substances likely modulate stem cell behavior, depending on their different function in the asexual and sexual reproductive modes. The combination of RNA-seq and a feeding assay in D. ryukyuensis is a powerful tool for studying the alternation of reproductive modes, disentangling the relationship between gene expression and chemical signaling molecules.

S1: Summary of RNA-seq, de novo assembly, and mapping statistics Table. S2: GO enrichment analysis of asexual and sexual DEGs  Table. S4: Detail information of the DEGs in three amino acid metabolic pathways enriched both in asexual and sexual worms; Tryptophan metabolism; Glycine, serine, and threonine metabolism; and Arginine and proline metabolism Table. S5: Primer sets used for the synthesis of whole-mount in situ hybridization probes  Figure S1. Differences in gene expression between the asexual and sexual modes of reproduction. The sexual to asexual expression ratios (log 2 FC sexual/ asexual) are plotted against the average expression intensity (log 2 CPM; CPM counts per million), using the edgeR v3.12.0 63,64 in R v3.2.2 70 . Asexual (log 2 FC < 0) and sexual (log 2 FC > 0) DEGs are indicated in red. The 7 asexual DEGs and 21 sexual DEGs examined for whole-mount in situ hybridization and qRT-PCR are indicated in pink. The DEGs from the enriched KEGG pathways are indicated in green. Especially, the DEGs with a KEGG gene name in green are from three of the amino acid metabolic pathways enriched both in asexual and sexual worms: tryptophan metabolism; glycine, serine, and threonine metabolism; and arginine and proline metabolism. The plot was generated based on four biological replicates for each worm type (see RNA-seq section in the Materials and Methods).

Supplementary Figure S2. Qualitative and quantitative validation of asexual DEGs.
Representative whole-mount in situ hybridization patterns for the ventral and dorsal sides of worm are shown. The expression pattern was judged based on three replicates.
Signals were seen as blue/purple staining. The white arrowheads indicate ovarian primordia. The qRT-PCR data are shown relative to the expression level in the sexual worm, and log 2 (relative expression) on the vertical axis indicates -ΔΔCt. Each circle indicates an individual asexual or sexual worm. Five replicates were used, but data are not shown if the expression was too low to be detected (handled as NA). The bars in the plots indicate the averages of -ΔΔCt. Asterisks indicate significant differences between the asexual and sexual worms (Student's or Welch's t-test: * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant).  Figure S3. KEGG pathway mapping of the glycine, serine, and threonine metabolism. Annotated genes in the RNA-seq analysis were mapped against KEGG pathway maps (www.kegg.jp/kegg/kegg1.html) 26-28 using a KEGG mapper tool (http://www.kegg.jp/kegg/tool/map_pathway2.html). Cyan indicates genes identified as asexual DEGs, and pink indicates genes identified as sexual DEGs. Gray indicates genes not identified as DEGs but expressed in the planarian transcriptome in the present study. The qRT-PCR data for each DEG are shown relative to the expression level in the sexual worm, and log 2 (relative expression) on the vertical axis indicates -ΔΔCt.
Each circle indicates an individual asexual or sexual worm. Eight to ten replicates were used, but data are not shown if the expression was too low to be detected or in the case of outliers (handled as NA). The bars in the plots indicate the averages of -ΔΔCt.
Asterisks indicate significant differences between the asexual and sexual worms (Student's or Welch's t-test: * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant).  Figure S4. KEGG pathway mapping of arginine and proline metabolism. Annotated genes in the RNA-seq analysis were mapped against KEGG pathway maps (www.kegg.jp/kegg/kegg1.html) 26-28 using a KEGG mapper tool (http://www.kegg.jp/kegg/tool/map_pathway2.html). Cyan indicates genes identified as asexual DEGs, and pink indicates genes identified as sexual DEGs. Gray indicates genes not identified as DEGs but expressed in the planarian transcriptome in the present study. The qRT-PCR data for each DEG are shown relative to the expression level in the sexual worm, and log 2 (relative expression) on the vertical axis indicates -ΔΔCt.
Each circle indicates an individual asexual or sexual worm. Eight to ten replicates were used, but data are not shown if the expression was too low to be detected or in the case of outliers (handled as NA). The bars in the plots indicate the averages of -ΔΔCt.