Fig 1.
(A) A schematic of the pTRE-Nix/NixPro-tTA transformation construct and its landing site on the genome of transgenic lines. PiggyBac right arm (pBac R. A.) and left arms (pBac L. A.) are indicated, along with the 3XP3-DsRED marker cassette. The dotted lines represent sequence from the plasmid backbone. The pTRE-Nix/NixPro-tTA construct introduced another two genes: 1) The P element week promoter (pEw) driving the expression of the Nix protein. Upstream the promoter is the tetracycline responsive element (TRE) and downstream is the leader sequence of the heat shock protein 70 (HSP70). 2) The Nix promoter sequence driving the expression of the tetracycline Trans activator (tTA). All three genes on this construct have the 3’UTR from the simian virus 40 (SV40). (B) Schematic representation of the Tet-off system. Nix promoter (Nix Prom.). Illustrations were created using Biorender.com through a license to Texas A&M University.
Table 1.
Microinjection results of Nix construct.
Fig 2.
Intersex phenotypes in transgenic Ae. aegypti with Nix under the control of the TRE.
(A) Sex ratio of each transgenic line. Binomial test was used to access statistical significance (B) Representative images of the head of the seven transgenic lines, male and female Liverpool strain depicting different maxillary palp lengths, red arrow indicating the maxillary palps from male and female wild type. (C) Maxillary palp size in millimeters from female mosquitoes of the seven transgenic lines, male and female from Liverpool in the absence of Dox. Bars represent the median. (D) Body size in millimeters from female mosquitoes of the seven transgenic lines, male and female from Liverpool strain in the absence of Dox. Bars represent the median. (E) Number of females from each transgenic line in absence of Dox that developed the male genitalia with the presence of one gonostilus (F) Pictures of F3 transgenic line, male and female Liverpool strain abdomen, the arrows are pointing to the genitalia depicting the presence (male), absence (female) or the presence of only one gonostilus (F3 line). (C) and (D) Numbers followed by different letters are statistically different P≤ 0.05 (Tukey’s test).
Fig 3.
Transgenic females displaying higher levels of masculinization are not able to blood feed in the presence of a physical barrier.
(A) Schematic representation of the two artificial blood meals offered, one with a physical barrier (artificial system) and one without the physical barrier (blood-soaked cotton ball) (B) Number of blood fed (BF) and non-blood fed (NBF) females from transgenic lines and Liverpool strain in the absence of Dox in the artificial blood meal system and (C) blood-soaked cotton ball. Percentages represent blood fed females. Illustrations were created using Biorender.com through a license to Texas A&M University.
Fig 4.
Reduced fecundity and fertility in masculinized transgenic females.
Transgenic females were offered a blood meal in the artificial apparatus or through a warm blood-soaked cotton. Blood fed females were put into an Eagal plate and number of eggs per female (fecundity) and number of larvae per eggs (fertility) was scored. (A) Fecundity from P5, (B) fertility from P5 (C) fecundity from F6b, and (B) fertility from F6b transgenic lines. A Mann–Whitney U test was used to evaluate statistical significance. ns: P>0.05; ***: P<0.0001.
Fig 5.
Transgenic females displaying higher masculinization have higher mortality.
Survival curves of transgenic (A) transgenic females (at least 100 individuals per transgenic line) and (B) males (at least 75 individuals per transgenic line) and Liverpool strain (150 females and 75 males) in a total of 38 days of observation. Log-rank test was used to determine P values and statistical significance was determined after the Bonferroni method to correct for multiple testing. *: P < .07.
Fig 6.
Doxycycline treatment partially restores maxillary palps size in transgenic females.
Mosquitoes were reared in the absence or presence of 50ug/mL doxycycline and supplemented with SURE cells during larval and pupal stages and after emergence, adult pictures were taken and maxillary palps were measured from F3 (A), F6b (B) and P5 (C) transgenic lines and Liverpool strain. The bars from the maxillary palps measurements represent the median. A Mann–Whitney U test was used to evaluate statistical significance. ns: P>0.05; ***: P<0.0001.
Fig 7.
Transgenic Nix expression profile in transgenic mosquitoes.
(A) Schematic representation of the pTRE-Nix/NixPro-tTA transgene (top) and endogenous Nix gene (bottom) and Nix T (specific for the transgene) and Nix End (binds on the Nix open reading frame on both the transgene and endogenous Nix) primer binding localization. (B) Quantification of endogenous or transgenic Nix transcripts in F3, F6b and P5 transgenic adult female mosquitoes and Liverpool males of at least 3 experiments containing 5 mosquitoes each. Quantification of accumulated transcript in the maxillary palps (C, E) and antennae (D, F) from 25–30 male Liverpool or female transgenic of transgenic Nix and Nix endogenous (C-D) and tTA and transgenic Nix (E-F). Columns represent the mean values and bars the standard. One-way ANOVA test was used to determine the statistical significance; ns: P>0.05; ****: P<0.0001. T Nix, transgenic Nix transcript; Nix End, endogenous Nix transcript, tTA, tetracycline transactivator.
Fig 8.
Transgenic and endogenous Nix expression profile in transgenic mosquitoes.
Comparison of accumulated transgenic Nix transcript (A) and quantification of accumulated transgenic Nix transcript and Nix endogenous (B) from embryos collected 0-2h, 2-4h, 4-6h, 6-8hs after being laid from F3, F6b, and P5 transgenic lines. Columns represent the mean values and bars show the standard deviation of at least two independent experiments containing at least 50 embryos each. One-way ANOVA test was used to determine the statistical significance (B); ns: P>0.05; ***: P<0.0001.
Fig 9.
Impact of doxycycline on transgenic Nix expression.
Quantification of accumulated transgenic Nix transcript treated and non-treated with 50μg/ml of Doxycycline from embryos collected 0-2h, 2-4h, 4-6h, 6-8hs after being laid from F3, F6b, and P5. (A) Transgenic lines and (B) Adult female mosquitoes. Columns represent the mean values and bars the standard deviation (A-B) of at least two independent experiments containing at least 50 embryos each (A) and 3 independent experiments (B). Multiple t test was used to determine the statistical significance (B); ns: P>0.05; ***: P<0.0001.