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. 2021 Feb 15;376(1818):20190808.
doi: 10.1098/rstb.2019.0808. Epub 2020 Dec 28.

Genetic sexing strains for the population suppression of the mosquito vector Aedes aegypti

Panagiota Koskinioti  1   2 , Antonios A Augustinos  1 , Danilo O Carvalho  1 , Muhammad Misbah-Ul-Haq  1   3 , Gulizar Pillwax  1 , Lucia Duran de la Fuente  1 , Gustavo Salvador-Herranz  4 , Rafael Argilés Herrero  5 , Kostas Bourtzis  1
Affiliations

Genetic sexing strains for the population suppression of the mosquito vector Aedes aegypti

Panagiota Koskinioti et al. Philos Trans R Soc Lond B Biol Sci. .

Abstract

Aedes aegypti is the primary vector of arthropod-borne viruses including dengue, chikungunya and Zika. Vector population control methods are reviving to impede disease transmission. An efficient sex separation for male-only releases is crucial for area-wide mosquito population suppression strategies. Here, we report on the construction of two genetic sexing strains using red- and white-eye colour mutations as selectable markers. Quality control analysis showed that the Red-eye genetic sexing strains (GSS) is better and more genetically stable than the White-eye GSS. The introduction of an irradiation-induced inversion (Inv35) increases genetic stability and reduces the probability of female contamination of the male release batches. Bi-weekly releases of irradiated males of both the Red-eye GSS and the Red-eye GSS/Inv35 fully suppressed target laboratory cage populations within six and nine weeks, respectively. An image analysis algorithm allowing sex determination based on eye colour identification at the pupal stage was developed. The next step is to automate the Red-eye-based genetic sexing and validate it in pilot trials prior to its integration in large-scale population suppression programmes. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Keywords: Zika; dengue; sterile insect technique; vector control.

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Conflict of interest statement

We declare we have no competing interests.

Figures

Figure 1.
Figure 1.
The eye colour of wild-type, Red-eyes and Higgs is dark brown (a–c), red (d–f) and white (g–i), respectively, consistent in larva L3 (a,d,g), pupa (b,e,h), and adult (c,f,i).
Figure 2.
Figure 2.
Backcrossing scheme for the assessment of recombination rates between re or w and M locus and the construction of Red-eye GSS and White-eye GSS. (a) Wild-type BRA males + M/+ m were crossed to virgin Red-eye females re m/re m. F1 males + M/re m were backcrossed to virgin Red-eye females and backcross progeny were screened for sex and eye colour. Red-eye females and wild-type males are the parental genotypes (circled with green), while Red-eye males and wild-type females are the recombinant genotypes (circled with red). (b) Wild-type males + M/+ m were crossed to virgin Higgs females w m/w m. F1 males + M/w m were backcrossed to virgin Higgs females and backcross progeny were screened for sex and eye colour. White-eye females and wild-type males are the parental genotypes (circled with green), while white-eye males and wild-type females are the recombinant genotypes (circled with red). (Online version in colour.)
Figure 3.
Figure 3.
The cage suppression experiment. The Red-eye GSS (a) and the Red-eye GSS/Inv35 (b) were tested for their ability to suppress a target population in laboratory cage experiments by setting up three types of cages with three replicates each: the ‘1 : 1 : 0' fertile control cage (1 wild-type female: 1 wild-type male: 0 irradiated Red-eye GSS males), the ‘1 : 1 : 1' suppression cage and the ‘1 : 1 : 10' suppression cage. Only the two later are presented (‘1 : 1 : 1' and ‘1 : 1 : 10’) since the data from the fertile control cage have been used to assess the baseline fertility used for the calculations. Releases of irradiated Red-eye GSS were performed twice a week for six weeks, and releases of Red-eye GSS/Inv35 males were performed twice a week for a period of nine weeks. The thicker straight line represents the generalized model with the grey-shaded area as the standard error, while the jagged thinner line represents the mean of each week; the points indicate the observed data. (Online version in colour.)
Figure 4.
Figure 4.
Identification of the eye colour. (a) Automatic pupal sex determination is shown in the top right of each box. The position of the eyes in males is also shown. (b) Detail of the contour, dorsal parts of the cephalothorax, centroids of the entire contours (black dot) and cephalothorax (dark grey dot), computed orientation lines and BLOBS that have overcome the filtering process are shown, and these features are used to determine the gender of the pupa. The value in the lower right corner of each box represents the circularity index of the convex hull of the dorsal part of the cephalothorax (‘perfect circularity' being 1). (Online version in colour.)
Figure 5.
Figure 5.
A hypothetical pipeline combining different sex separation strategies to eliminate female contamination in male release batches. The pipeline incorporates male protandry, pupal size dimorphism and pupal eye colour dimorphism. Male protandry is already exploited in mass-rearing facilities and results in batches that are enriched in males in the range of 70–80%. Pupal size sorting performed by skilled technicians using the Fay and Morlan adjustable glass plates system results in contamination rates of 0.1–0.01% with a speed of 15 000–20 000 males per hour. Incorporation of the red eye in the sexing strategy can reduce female contamination by a factor of 1–2.5% (independent markers) while the introduction of Inv35 can reduce this factor to 0.22%. Such a strategy, supported by the appropriate technical advances, can result in female contamination ranging between 2.2 females per million males and 2.2 females per ten million males. (Online version in colour.)
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