Introgression of a synthetic sex ratio distortion system from Anopheles gambiae into Anopheles arabiensis

Abstract

I-PpoI is a homing endonuclease that has a high cleavage activity and specificity for a conserved sequence within the ribosomal rDNA repeats, located in a single cluster on the Anopheles gambiae X chromosome. This property has been exploited to develop a synthetic sex ratio distortion system in this mosquito species. When I-PpoI is expressed from a transgene during spermatogenesis in mosquitoes, the paternal X chromosome is shredded and only Y chromosome-bearing sperm are viable, resulting in a male-biased sex ratio of >95% in the progeny. These distorter male mosquitoes can efficiently suppress caged wild-type populations, providing a powerful tool for vector control strategies. Given that malaria mosquito vectors belong to a species complex comprising at least two major vectors, we investigated whether the sex distorter I-PpoI, originally integrated in the A. gambiae genome, could be transferred via introgression to the sibling vector species Anopheles arabiensis. In compliance with Haldane's rule, F1 hybrid male sterility is known to occur in all intercrosses among members of the Anopheles gambiae complex. A scheme based on genetic crosses and transgene selection was used to bypass F1 hybrid male sterility and introgress the sex distorter I-PpoI into the A. arabiensis genetic background. Our data suggest that this sex distortion technique can be successfully applied to target A. arabiensis mosquitoes.

Figure 1

Generation of F1 hybrids in reciprocal crosses between A. arabiensis and A. gambiae PMB1 mosquitoes. (a) A. arabiensis females (blue) are crossed to PBM1 males (red) to generate F1 hybrids. The transgene in PMB1 strain, containing the sex distorter I-PpoI and GFP, is highlighted (green dot) in one of the autosomes. (b) Reciprocal cross. The parental genetic contribution in the progeny follows the colour code indicated in the legend. Sex ratio values for F1 progeny are indicated as well as the number of females and males counted (n). Significance (p < 0.0001, two-tailed Fisher’s exact test) was tested comparing number of males and females obtained from crosses of A. arabiensis females and PMB1 males versus number of males and females obtained from crosses of PMB1 females and A. arabiensis males.

Figure 2

Transmission (TM) and fluorescence (FM) microscopy analysis of testes dissected from PMB1 and F1 hybrid males generated in reciprocal crosses of A. arabiensis and A. gambiae PMB1 mosquitoes. (a) Testes from PMB1 males looked healthy and expressed the typical β2:GFP decoration pattern along their axis. (b) F1 hybrid males generated from PMB1 fathers showed complete organ atrophy and lack of GFP expression. (c) F1 hybrid males generated from A. arabiensis fathers showed normal-shaped testes but lacked mature sperm. A patchy distribution of GFP is observed in testes of transgenic males.

Figure 3

Crossing scheme implemented to generate back cross generation 1 (BC₁) males. Transgenic F1 hybrid females (blue/red), generated from A. arabiensis mothers and PMB1 fathers, were crossed to A. arabiensis males (blue). The autosomal transgene is highlighted (green dot). BC₁ males inherited A. arabiensis DNA from their fathers whereas the maternal genetic contribution is the result of meiotic recombination between heterospecific chromosomes (grey). Sex ratio values for BC₁ progeny are indicated as well as the number of females and males counted (n). Significance (p = 0.0002, two-tailed Fisher’s exact test) was tested comparing number of males and females obtained from crosses of F1⁺ females and A. arabiensis males versus number of males and females expected according to Mendelian rules of inheritance.

Figure 4

Transmission (TM) and fluorescence (FM) microscopy analysis of testes dissected from BC₁ males. Organ development and GFP expression were analysed in a total of 50 males. (a) The majority (38/50) of testes analysed showed no obvious morphological anomalies and the testes of transgenic males showed a GFP fluorescent signal in line with the transcription pattern of the β2 tubulin promoter. (b) Testes dissected from 12 males showed undeveloped cells and lack of mature sperm.

Figure 5

Crossing scheme implemented to generate BC₂ males. BC₁ males (blue/grey) were crossed to A. arabiensis females (blue) to generate BC₂ males. The autosomal transgene is highlighted (green dot).

Figure 6

Crossing scheme implemented to generate BC₃ males. Transgenic (a) and non-transgenic (b) BC₂ males (blue/grey) were crossed in single copula mating to A. arabiensis females (blue) to generate BC₃ males. The autosomal transgene is highlighted (green dot). (c) To assess the fertility of BC₂ males and to investigate the activity of the sex distorter in the A. arabiensis genetic background, the oviposition (left panel) and hatching rate (middle panel), as well as the sex ratio in the progeny (right panel), were analysed for each cross. For oviposition, significance (p = 0.7549, two-tailed unpaired t test with Welch’s correction) was tested comparing number of eggs laid by females after mating with ${\mathrm{BC}}_2^{+}$ males versus number of eggs laid by females after mating with ${\mathrm{BC}}_2^{-}$. For hatching rate, significance (p = 0.4625, two-tailed unpaired t test with Welch’s correction) was tested comparing hatching rate values from progeny of females mated with ${\mathrm{BC}}_2^{+}$ males versus hatching rate values from progeny of females mated with ${\mathrm{BC}}_2^{-}$. For sex ratio, significance (p < 0.0005, two-tailed unpaired t test with Welch’s correction) was tested comparing number of males from progeny of females mated with ${\mathrm{BC}}_2^{+}$ males versus number of males from progeny of females mated with ${\mathrm{BC}}_2^{-}$.

Figure 7

Graph showing the percentage of sex distortion recovered from I-PpoI introgressed males at different backcross generations (from generation BC₅–BC₁₀ and BC₄₀).

Figure 8

Fertility assay of transgenic and non-transgenic I-PpoI introgressed males (BC₃₈). Fertility was assessed in a single copula mating experiment with A. arabiensis females. Number of eggs laid by individual females and hatching rate are shown on the left and right panel respectively. For oviposition, significance (p = 0.0302 and p = 0.0007, two-tailed unpaired t test with Welch’s correction) was tested comparing number of eggs laid by females after mating with A. arabiensis males versus number of eggs laid by females after mating with ${\mathrm{BC}}_{38}^{+}$ or ${\mathrm{BC}}_{38}^{-}$ males. For hatching rate, significance (p = 0.9272 and p = 0.8531, two-tailed unpaired t test with Welch’s correction) was tested comparing hatching rate values from progeny of females mated with A. arabiensis males versus hatching rate values from progeny of females mated with ${\mathrm{BC}}_{38}^{+}$ or ${\mathrm{BC}}_{38}^{-}$ males.