Figure 5
Developmental expression analyses of the Aeadsx gene. The analyses were performed on the following samples: E1 = 0-1.5 h embryos; E2 = 1.5-2 h embryos; E3 = 2-5 h embryos; E4 = 8-12 h embryos; E: 0-36 h embryos; O = dissected ovaries; FC = female carcasses depleted of ovaries; L12= early larvae; L34= late larvae; P = pupae; M = adult males; F = adult female. Except for M, F, O and FC all samples are composed of mixed sexes. Negative controls are not shown. (A) Amplification of Ae. aegypti rp49 transcripts with the Aearp49+/Aearp49- primer pair. The Aedes aegypti ribosomal gene rp49 is constitutively expressed throughout development. (B) Aeadsx developmental expression pattern. (B.1 and B.4) The dsx3/dsx5 primer combination amplified at adult stages a 0.5-kb male-specific cDNA fragment and two female-specific cDNA fragments (1.0 kb and 1.5 kb). These three bands were detected in pupae and late larvae, while the 1.5-kb band was absent in embryos and mid-larvae but present in ovaries and female carcasses. (B.2 and B.5) The dsx3-dsx4 primer combination amplified at adult stages a female-specific cDNA fragment. A cDNA product of identical size was amplified at all developmental stages, including embryos, suggesting an early Aeadsx female-specific regulation. (B.3 and B.6) The dsx1/dsx2 primer combination amplified in all samples two slightly different cDNA fragments (0.37 kb and 0.31 kb), corresponding to the alternatively spliced isoforms of exon 2 either containing (0.37 kb) or not containing (0.31 kb) the 63-bp intronic sequence. In contrast to the data reported in Figure B.1-3, the RT-PCR results in Figure B.4, B.5 and B.6 lack of a positive semiquantitative control and the apparent changes in expression levels of Aeadsx isoforms during embryonic stages have to be further investigated. (C) A northern blot analysis was performed on total RNA (20 μg) extracted from male and female Ae. aegypti adults. The genomic position of the utilized probe is indicated in Figure 5B. The observed molecular size of Aeadsx transcripts confirms that isolated Aeadsx cDNA clones were not full-length at the 3' and 5' ends.