Fluorescent sexing strains to facilitate the Sterile Insect Technique in the dengue and tiger mosquitoes

Fluorescent sexing strains to facilitate the Sterile Insect Technique in the dengue and tiger mosquitoes
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Mosquitoes of the Aedes genus, in particular Aedes aegypti and Aedes albopictus, are not only a nuisance, they also transmit pathogenic viruses such as dengue, Zika, chikungunya and yellow fever, which have a major impact on public health. Both species thrive in urban environments, where they use people as food and various types of human-made water containers as breeding sites for their larvae. They have a strong tendency to be invasive species, taking advantage of human mobility and the transport of goods across the world to spread and settle in new territories favored by climate change. The tiger mosquito Aedes albopictus invaded the world from South-East Asia and continues to spread as far north as Europe and North America, while its cousin the yellow fever mosquito Aedes aegypti long ago completed its invasion of all tropical regions, initially moving from Africa to the South American continent with the slave trade. More recently, it has been rediscovered in Europe, where it is in the process of re-conquering certain regions of the Mediterranean basin and the Black Sea, from which it had been eliminated in the last century, mainly as a result of efforts to control the mosquitoes that transmit malaria. The incidence of Aedes-borne viral diseases, notably dengue, is on the increase worldwide and affecting new territories, which is favored by human mobility and climate change.

Controlling mosquito vectors with insecticides is failing to prevent and control these diseases, because the numerous and multiform breeding sites scattered on private property are difficult to reach; genetic resistance to insecticides is spreading in mosquito populations; and the use of insecticides is becoming more restrictive because of their impact on the environment and human health. To combat certain insect pests, the Sterile Insect Technique (SIT) is an old but exquisitely species-specific technology, which has scored many  successes: regional elimination of the New World Screwworm, of several species of fruit flies harmful to agriculture, and very recently of tsetse flies suppressed in an area of 1,375km2 around Dakar in Senegal. SIT consists in releasing large numbers of sterilized male insects that will seek out wild females, mate with them, and annihilate their progeny. Historically, SIT has not been developed for mosquitoes, considered too delicate creatures to withstand industrial production and handling, but this situation is changing. Numerous proof-of-principle studies now show that it is possible to mass produce, sterilize and release competitive male mosquitoes. More than forty SIT trials  against mosquitoes are currently ongoing worldwide. 

A major roadblock to the operationalisation of SIT against mosquitoes has been the separation of males from females, as it is highly undesirable to release any biting females, even if sterilized, along with their sterilized brothers. Indeed these females could still act as pathogen vectors  and would also reduce the acceptability of the technique. One way of overcoming this obstacle would be to use genetic sexing strains that simplify the sorting process. In this paper, we used transgenesis to produce fluorescent male mosquitoes. This approach relies on the insertion into the mosquito genome, in a heritable fashion, of fluorescence-encoding transgenes in close genetic proximity to the sex-determining genes. For the two main Aedes vector species, we now have transgenic lines whose males are brightly fluorescent, while females are non-transgenic. We have also produced genetic sexing strain versions in which fluorescence is visible in both sexes, but genetically linked to the Aedes equivalent of the X chromosome. Thus, with two copies of the genetic marker, females are more fluorescent than males. The two types of marked strains can be combined in genetic crosses of non-transgenic females with the less fluorescent males, in order to produce large populations of non-transgenic males that can be purified away from their fluorescent transgenic sisters. This would be a great asset for mosquito control interventions in regions where the use of genetically modified insects is considered undesirable. Some countries where legislation and public perception of GMOs is more favorable could also choose to release directly fluorescent males of the first strains described above, after sterilization by irradiation or by infection with specific strains of Wolbachia bacteria (incompatible insect technique). The status of these sterile fluorescent males will depend on local regulations, as they may not necessarily be considered “organisms” if they are completely sterile.

We have achieved efficient sex sorting by passing large numbers of small larvae of these fluorescent strains through flow cytometry machines that measure fluorescence levels and sort the desired objects as they are analyzed. We show that the sorting speed and the purity of the males obtained are compatible with the production rates and male quality required for the operationalisation of SIT against mosquitoes. Importantly, the removal of females at a very early stage of development allows important savings in production costs, by avoiding the rearing of unnecessary female larvae. We now hope that these tools will be adopted by mosquito control agencies keen to deploy innovative new approaches of vector control in parts of the world most affected by the threats posed by Aedes aegypti and Aedes albopictus.

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