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. 2019 Apr 15;12:170. doi: 10.1186/s13071-019-3437-4

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© The Author(s) 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 1 — Doublesex sequence and profile expression in An. gambiae G3. a Sequence alignment between the two previously annotated sequences for the dsx gene in An. gambiae with the sequence of PCR products amplified from the MR4 An. gambiae G3 and An. stephensi STE2. The An. gambiae G3 sequence matched the KM978939 sequence and the An. stephensi STE2 sequence matched the DQ137802 sequence. Marked in blue are the sequences that match in at least four consecutive bases the KM978939 sequence for AgdsxF exon. Three other regions from our An. stephensi strain resulted in 100% match to DQ137802 (Additional file 3). Thus, all subsequent primer designs were based on the KM978939 sequence. b Sequence conservation among Anopheles for the AgdsxF region of interest (1823–2066) was evaluated. PCR products were amplified from An. gambiae, An. minimus, An. stephensi and An. albimanus cDNAs (Additional file 3) and the sequences analyzed. A MAFFT alignment is shown, indicating regions with significant similarity in green. c PCR amplification of a AgdsxF 260 bp fragment, in the fifth exon, showed that the expression was limited to females (using cDNAs of whole body extract) of each sex, consisting of three 1-day-old pupae or five 5-day-old adults. d Real-time PCR (RT-PCR) analyses of relative gene expression reveal low levels of AgdsxF expression between the L1 and L3 stages, and significantly higher from L4 forward. Two-way ANOVA with Tukey’s multiple comparisons test was performed. e Gene expression analysis for AgdsxF by qPCR of different tissues. In adult female mosquitoes less than 24 h post-emergence (virgin females), dsxF expression was detected only in ovarian tissue (Ov) and salivary glands (SG). In mature adult females (more than 5-days-old and mated) detectable levels of AgdsxF expression were observed in the midgut (Mg) and ovaries (Ov), and especially high levels in the salivary glands (SG). Black points indicate the samples that had no detectable expression. AgdsxF expression was not detected in either time-point in the head, including antennas and proboscis (Hd) or fat body (FB). Pools of tissue from 12–15 individuals were analyzed