Figure 2.
Stable fle Knockdown in A. gambiae Causes Female Masculinization and Death through Abnormal Upregulation of Transcription from the X Chromosome
(A) Schematic representation of the transgenic fle knockdown construct.
(B) Pupal abdominal termini of the wild-type and fle knockdown transgenic mosquito lines. Scale bars, 200 μm.
(C) Adults. From top: wild-type male, 4M4B line female, wild-type female. Note male-like antennae (short arrows) and palps (long arrows), and a slender abdomen with a male-like uninverted copulatory apparatus.
(D) Reproductive system: ejaculatory pump (long white arrows) and accessory glands (short white arrows) in a wild-type male and 4M4B female. Scale bar, 100 μm.
(E) RT-PCR analysis of dsx and fru splicing patterns in females from selected transgenic lines.
(F) A sexually dimorphic pair of dorsal muscles (indicated by white arrows) in the fifth segment of the abdomen (A5), likely homologous to the Drosophila muscle of Lawrence, is of similar thickness in wild-type males and transgenic females, and considerably thicker than in wild-type females. Scale bars, 200 μm.
(G) Proportions of individuals reaching adulthood in selected transgenic lines. The lines have been maintained by backcrossing of transgenic males with the wild-type line females at every generation, which should yield 50% of wild-type progeny and equal proportions of the sexes in the transgenic and wild-type groups. However, apart from line 4M6, a highly significant deficiency of transgenic females was observed (∗p < 0.0001; chi-square test). In line 4M2B, no transgenic females developed beyond the embryonic stage.
(H) Pupal abdominal terminus with a characteristic black tumor (arrow) in a 4M4B female.
(I) Fluorescent marker expression in selected transgenic lines.
(J) Levels of fle transcripts in the pupae of the wild-type line G3 and in selected transgenic lines quantified by qRT-PCR and normalized relative to the fle/rpS7 transcription level in wild-type females. The error bars represent standard deviations. ∗p < 0.01, ∗∗p < 0.001; Student’s t test.
(K) Comparison of transcription levels from the autosomes and the X chromosomes in transgenic line 4M4B and wild-type line G3 females. Shown are transcripts per kilobase million (TPM) ratios. The median expression ratio between the 4M4B and G3 lines for the autosomes (0.92) and for the X chromosomes (1.34) indicates that the X chromosome genes in the transgenic line females are significantly overexpressed; p < 10−15; Mann-Whitney U test. A similar trend for the median shrunken fold change for the autosomal (0.985) and for the X linked (1.22) transcripts confirms that the effect is statistically robust.
See also Table S1.