New self-sexing Aedes aegypti strain eliminates barriers to scalable and sustainable vector control for governments and communities in dengue-prone environments

Abstract

For more than 60 years, efforts to develop mating-based mosquito control technologies have largely failed to produce solutions that are both effective and scalable, keeping them out of reach of most governments and communities in disease-impacted regions globally. High pest suppression levels in trials have yet to fully translate into broad and effective Aedes aegypti control solutions. Two primary challenges to date-the need for complex sex-sorting to prevent female releases, and cumbersome processes for rearing and releasing male adult mosquitoes-present significant barriers for existing methods. As the host range of Aedes aegypti continues to advance into new geographies due to increasing globalisation and climate change, traditional chemical-based approaches are under mounting pressure from both more stringent regulatory processes and the ongoing development of insecticide resistance. It is no exaggeration to state that new tools, which are equal parts effective and scalable, are needed now more than ever. This paper describes the development and field evaluation of a new self-sexing strain of Aedes aegypti that has been designed to combine targeted vector suppression, operational simplicity, and cost-effectiveness for use in disease-prone regions. This conditional, self-limiting trait uses the sex-determination gene doublesex linked to the tetracycline-off genetic switch to cause complete female lethality in early larval development. With no female progeny survival, sex sorting is no longer required, eliminating the need for large-scale mosquito production facilities or physical sex-separation. In deployment operations, this translates to the ability to generate multiple generations of suppression for each mosquito released, while being entirely self-limiting. To evaluate these potential benefits, a field trial was carried out in densely-populated urban, dengue-prone neighbourhoods in Brazil, wherein the strain was able to suppress wild mosquito populations by up to 96%, demonstrating the utility of this self-sexing approach for biological vector control. In doing so, it has shown that such strains offer the critical components necessary to make these tools highly accessible, and thus they harbour the potential to transition mating-based approaches to effective and sustainable vector control tools that are within reach of governments and at-risk communities who may have only limited resources.

Keywords: Aedes aegypti; genetic sexing; self-limiting insects; sustainability; vector control.

FIGURE 1

(A) Linear plasmid map showing the two genes (DsRed2 and tTAV) between the non-autonomous vector piggyBac sequences, inserted in the OX5034 Aedes aegypti strain. Due to the splicing module tTAV protein is only expressed in females in the absence of tetracycline family antibiotics. (B) Sex-specific splicing of endogenous Aeadsx and of the Aeadsx-ubiquitin-tTAV gene was confirmed in Aedes aegypti mosquitoes (pupal life stage, mosquitoes reared with doxycycline in the rearing medium) by RT-PCR reactions using either primers SS1997 and TD3349 (endogenous dsx), or TD226 and SJ124 (OX5034 dsx). All splicing isoforms of the endogenous Aeadsx gene (M, F1 and F2) were detected in WT as well as OX5034 mosquitoes in a sex-specific manner. The Aeadsx-ubiquitin-tTAV gene was also splicing in a sex-specific manner and was only detectable in mRNA extracted from OX5034 Aedes aegypti mosquitoes. In the absence of tetracycline antibiotics, the F2 isoform mRNA results in female-specific tTAV protein expression, and dsx and ubiquitin amino acids are cleaved from tTAV protein by endogenous cellular processes. Red octagons indicate stop codons.

FIGURE 2

Trait decline of OX5034 Aedes aegypti. Boxplots showing the results from 500 iterations of a stochastic model simulating the extinction of a male-selecting genetic trait under restrictive conditions (off-doxycycline rearing). Horizontal bold lines represent generational medians; upper and lower box lines represent first and third quartiles, respectively; outer horizontal lines represent 1.5× the interquartile range; and open circles represent data points over 1.5× above or below the first and third quartiles. Overlaid onto the box plots are lines (red, blue and green) showing male-selecting trait frequency changes from three replicates of caged experiments. Generation 1 represents a post-field release population with a trait frequency of 0.25.

FIGURE 3

OX5034 Aedes aegypti release neighbourhoods in the city of Indaiatuba, São Paulo State. Four treatment areas are shown, with blue shading indicating low-dose treatment areas (Jardim Itamaracá and Jardim Moacyr Arruda), and orange shading indicating high-dose treatment areas (CECAP, Morada do Sol). The control area (Jardim Oliveira Camargo) is shown with a dark blue outline. The geographic coordinates of the city are 23°05′24″ S and 47°13′04″ W. Release points (triangles) and trap locations (small circles) are shown in each of the four treatment areas. Buffer zones (deployed from February 2019) are shown for Jardim Moacyr Arruda (pale blue shading) and Morada do Sol (pale orange shading).

FIGURE 4

(A) Absolute suppression chart in all areas in Indaiatuba treated with OX5034 Aedes aegypti adult males. Solid lines indicate suppression calculated for each release area, relative to the untreated control area. Shaded areas indicate 95% confidence intervals. (B) OX5034 Aedes aegypti male mosquito releases during the Indaiatuba field trial. Releases began in May 2018 (Week 22) and ended in April 2019 (Week 14). Additional buffer zone releases were added to two neighbourhoods, Morada do Sol (high dose) and Jardim Moacyr Arruda (low dose) in February 2019. (C) Post-release monitoring of the four treatment neighbourhoods demonstrated that the transgenic mosquitoes were no longer detected in the environment after 13 weeks (CECAP, Jardim Itamaracá and Jardim Moacyr Arruda), and after 24 weeks in Morada do Sol, a high-dose area with added buffer zone. Monitoring continued until 48 weeks after the end of OX5034 Aedes aegypti male releases.

FIGURE 5

Number of fluorescent adults hatched from ovitraps-collected eggs from OX5034-treated areas, Indaiatuba, São Paulo State, Brazil. Each bar represents fluorescent adults from one of the four field sites (CECAP, Jardim Itamaracá, Jardim Moacyr Arruda, Morada do Sol). Low mosquito seasons (May-October) are shown with grey boxes. No fluorescent females were obtained from any ovitrap-collected eggs at any time throughout the 2018–2019 field trial of OX5034 Aedes aegypti. Data are reported as totals for each calendar month throughout the OX5034 Aedes aegypti release period.