Fig. 2.

Suppression of pantothenate pathway contributes to female-to-male sex reversal. (A) The amount of pantothenate between normally feeding and starvation conditions. Whole XX larvae were analyzed based on CE-TOF-MS (an average of three experiments, each using 20 larvae) or IC-FTMS (an average of ten single larvae) at 5 dph. Pantothenate levels increase under starvation conditions. The values indicate the average and the bars indicate s.e.m. Student's t-test was used as statistical analysis. *P-value<0.05. (B) The amount of metabolites in the pantothenate pathway of whole XX larvae based on CE-TOF-MS analysis at 5 dph (an average of three experiments, each using 20 larvae). The vertical axis indicates the peak area of each metabolite in the CE-TOF-MS analysis. The values indicate the average and the bars indicate s.e.m. (C) pank1a, pank2 and pank4 transcript levels extracted from one whole XX larva at 5 dph (fed n=16, starved n=13) were determined by RT-qPCR. The expression level of pank1a is suppressed by starvation treatment. Transcript levels in starved samples were normalized to the average of those in fed samples for each gene. The values indicate the average and the bars indicate s.e.m. Student's t-test was used as statistical analysis. *P-value<0.03. (D) The ratio of pantothenate and 4-phosphopantothenate under several conditions. Larvae treated with Pank inhibitor accumulate pantothenate as seen under starvation conditions (XX fed n=4, XX starved n=5, XX Pank inhibitor n=5, XY fed n=5, XY starved n=5, XY Pank inhibitor n=5). The average pantothenate/4-phosphopantothenate ratio in fed XX was used to normalize each sample. PI, Pank inhibitor. The values indicate the average and the bars indicate s.e.m. A two-way ANOVA followed by Dunnett’s test was used as statistical analysis. a*: P-value<0.05 in a two-way ANOVA. b*: P-value<0.05 in Dunnett’s test. (E) The relative amount of lipids in 5 dph whole larvae (n=3). The total lipids were extracted from four larvae. The amount of TG is lowered by Pank inhibitor treatment, as seen under starvation conditions. The values indicate the average and the bars indicate s.e.m. A one-way ANOVA followed by Dunnett’s test was used used as statistical analysis. a*: P-value<0.05 in a two-way ANOVA. b*: P-value<0.05 in Dunnett’s test. See the legend of Fig. 1D for abbreviations. (F) Pank inhibitor treatment causes female-to-male sex reversal. Sex-reversed individual (XX male) indicates a male type of dorsal and anal fins with normal testis. The result of genotyping is shown in Table 1. Scale bars: 1 mm.