GAL4-UAS mediated expression of <i>Masc-R</i> caused severe lethality in females.

<p>(A) Forced expression of <i>Masc-R</i> utilizing the GAL4-UAS system in the transgenic strains. The expression of <i>Masc-R</i> mRNA in first instar larvae just after hatching was detected by RT-PCR. Individual sex was determined by the expression of <i>Fem</i> RNA. The universally expressed <i>ribosomal protein S3</i> (<i>rpS3</i>) was used as an internal control. Fluorescent-positive animals expressing either DsRed (R / +), EGFP (G / +), or both (R / G) were examined. R / + and G / + animals carried either BmA3-GAL4 or UAS-<i>Masc-R</i> transgenes, respectively. R / G animals possessed both BmA3-GAL4 and UAS-<i>Masc-R</i>. (B) Kaplan—Meier survival curve was plotted with survival rate (%) on <i>Y</i>-axis and the number of days after hatching on the <i>X</i>-axis. BmA3-GAL4 (R/+) n = 68, BmA3-GAL4; Sumi13-1 (R/G) n = 48, BmA3-GAL4; Sumi13-3 (R/G) n = 27, P = 0.00013 [BmA3-GAL4 (R/+) versus BmA3-GAL4; Sumi13-1 (R/G)], p = 0.00020 [BmA3-GAL4 (R/+) versus BmA3-GAL4; Sumi13-3(R/G)], log rank test. (C)(D) The sex ratio of F1 adult moths obtained by crossing BmA3-GAL4 females either with UAS-<i>Masc-R</i> (Sumi13-1 and Sumi13-3) males (C and D). Blue and red bars indicate the number of males and the number of females, respectively. (E) The sexing of F1 individuals obtained by crossing BmA3-GAL4 females with UAS-<i>Masc-R</i> males (Sumi13-1). PCR-amplification of <i>Musashi</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006203#pgen.1006203.ref028" target="_blank">28</a>], a marker gene on the W chromosome, using genomic DNA isolated from F1 dead larvae (upper panel) or animals that reached the adult stage (lower panel). Two different sizes of DNA fragments were amplified in females, whereas the same PCR reactions detected only a single DNA fragment in males. Based on the size of the amplified products, upper band corresponded to the W-chromosome specific marker <i>Musashi</i>, whose size is 574 bp [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006203#pgen.1006203.ref028" target="_blank">28</a>]. The smaller DNA fragment amplified in both males and females was assumed to be a non-specifically amplified DNA derived from autosome.