Estimation of the onset of the <i>Tdic</i>-<i>dsx</i> dependent horn developmental program by <i>Tdic-tra</i> RNAi.

(A) Relationship between the timepoints of Tdic-tra dsRNA injection and the extent of sex transformation. The extent of the sex transformation (masculinization) phenotypes of Tdic-tra RNAi females were categorized into three classes: (i) prominent HH and TH were formed (Tdic-tra dsRNA injection before pupation in each class. The green dot, the magenta dot, and the blue dot indicate -3 h APF, -7 h APF and 73 h APF, respectively. Scale bars are 5 mm. (B) Time course of relative Tdic-tra and Tdic-dsxF mRNA expression level after Tdic-tra dsRNA injection at 24 h APF. mRNA expression levels at each timepoint were quantified by qRT-PCR. The expression levels of Tdic-tra were significantly decreased at 36 hours after injection (pTdic-dsxF tended to be decreasing as time went on, but there were no statistically significant difference (pTdic-dsx dependent horn developmental program. The magenta line is the boundary timepoints of phenotype (i). Red arrowheads indicate the timepoints corrected by the mRNA degradation delay (36 hours), which estimated by qRT-PCR. APF, after puapal-chamber formation; Red arrowhead, the estimated onset of the developmental program for sexually dimorphic horn formation driven by Tdic-dsx.