Fig. 4
Dr-SLC38A9 knockdown phenotypes under sex-inducing conditions. a Scheme of an experimental schedule of RNA interference (RNAi) under sexual induction. b-d qRT-PCR data are shown relative to the expression level (normalized to Dr-ef1a mRNA level) in the EGFP KD worm; log2 (relative expression) on the vertical axis indicates −∆∆Ct. Each circle indicates an individual EGFP KD worm or Dr-SLC38A9 KD worm sample. The bars in the plots indicate the averages of −∆∆Ct. b Dr-SLC38A9 transcript abundance after 4 weeks of treatment. Six replicates were performed. Significance was calculated using Welch’s t-test (*P < 0.05). c DrY1 transcript abundance after 4 weeks of treatment. Significance was calculated using Student’s t-test (***P < 0.001). d Dryg transcript abundance after 6 weeks of treatment. Significance was calculated using Student’s t-test (**P < 0.01). e, f Development of ovaries (white arrowheads) and copulatory apparatus (black arrowhead). A ventral view is shown, with the anterior of the worm at the top. g, h Development of the genital pore (black arrow). A ventral view is shown, with the anterior of the worm at the top. Scale bar, e-h 1 mm. i-l Transverse sections from asexual worms fed daily with minced B. brunnea worms supplemented with EGFP (control; i, k, m) or Dr-SLC38A9 (j, l, n) dsRNA for 6 weeks. The anterior part of the worm is to the left, and the dorsal part is at the top. i, j HE staining of the copulatory apparatus (red circles) after 4 weeks of treatment. k, l HE staining of the testes (blue circles) after 4 weeks of treatment. m, n HE staining of the yolk glands (orange circles) after 6 weeks of treatment. Yolk glands did not develop in the Dr-SLC38A9-knockdown worms. Scale bars, i, j 200 μm, k, l 50 μm, m, n 100 μm