Aedes. albopictus or tiger mosquito is an invasive species of global medical concern known to transmit diseases such as dengue or chikungunya virus to human via the bite of an infected female. As a result of its recent spreading in Africa, America and now in South-Europa where it is now established, this mosquito represents now a health threat which is necessary to control by control methods.
Nevertheless, the recurrent selection of insecticide resistance in vector natural populations , together with unwanted effects on non-target species has conducted restriction uses of synthetic pesticides and stimulated the development of innovative vector control strategies .
Among these strategies, the sterilising technologies such as sterile insect technique (SIT) is based on the massive release of sterile males in order to control insect via the population suppression .
This technique has been experiencing with success to control different insect pests at large-scale in North and Central America, such as the screwworm Cochliomyia. hominivorax  and the Mediterranean fruit fly, Ceratitis. capitata ,  but also in Africa where SIT allow to control the Tse-Tse fly –, the vector of sleeping sickness.
It has been also executed against different mosquito species over the past 40 years, but not at large scale, showing varying degrees of success  due to the fact that the principle of this technique needs to target isolated insect populations or to treat an important geographic area to be efficient. Therefore, field-trials at small-scales didn’t allow to show all the potential of this method .
The SIT method consists to raise en masse males of the target insect and to sterilise them by irradiation before their release in nature where they will become competitive for the reproduction with their wild counterparts which are not sterile. Thus, this technique aims to decrease the population fecundity in order to control it , .
Considering that it is only mosquito females which can bite and transmit diseases, the challenge of the SIT is based on the necessity to release a large number of sterile males, imposing a manual and mechanical sorting of insect nymphs thanks to the sexual dimorphism* observable in some mosquito species such as Aedes aegypti.
Nevertheless, when the size difference between males and females is not sufficient as in Aedes. albopictus, this sorting work is tedious and time-consuming. Therefore, it does not allow a perfect selection nor a massive and sufficient production of sterile male to impose an adequate ratio sterile males/wild males in order to reduce or eradicate these mosquito populations once they are released in nature.
Moreover, the accidental release of the female mosquito is a problem regarding the use of the SIT with mosquitoes. Indeed, even if these females are sterile, they could transmit pathogen agents. So, it is essential to be able to do an efficient sex-sorting to remove females before the release of these mosquitoes in nature.
The use of sex-sorting systems based primarily on the sex linkage**, also known as genetic sexing mechanisms, arguably remains the best approach currently available to allow a sexual separation of males and females at industrial scales.
This technique uses existing genes in the wild insect population, as selectable markers conferring a resistance to a chemical (insecticide for example) or physical treatment (the temperature for example) and requires the construction of homozygous resistant and susceptible mosquito lines for this selectable marker. Then, this latter will be translocated on only one of male sexual chromosome via an irradiation of the resistant males in order to create mutations. Then, different crosses between these resistant males and susceptible females will allow the development of a progeny where only males will be resistant for this selectable marker allowing the elimination of females via the chemical or physical treatment before the release in nature.
This allow the production of genetic sexing strain (GSS) of mosquitoes under conditions that genetically favour production of males and provide the basis for separating males from females .
Currently, GSSs are available for different insect species such as Cochliomyia. hominivorax  and Ceratitis. capitata ,  but also for mosquito species such as Anopheles. arabiensis , Anopheles. gambiae  and Culex. tarsalis .
However, no GSS were available for Aedes. albopictus up to the recently creation of a GSS for this mosquito species, named TiCoq, by the team of Dr Pablo Tortosa from the university of La Réunion .
Researchers constructed in 3 steps, the GSS TiCoq using the rdl gene, a selectable marker conferring a resistance to the dieldrin insecticide.
The first step consisted to construct dieldrin resistant (R) and susceptible (S) mosquito lines using two wild Ae. albopictus lines from La Réunion Island, displaying low and high rdl resistance allelic frequencies respectively .
The dieldrin sensitive strain was constructed using the crosses between homozygous rdl(S) females and male allowing to lay eggs which were pooled and immersed in water for hatching, providing a homozygous sensitive rdl(S) line named S-Run [SS].
The dieldrin resistant strain required two rounds of dieldrin selection to increase the frequency of the rdl(R) allele in mosquitoes. Researchers used third and fourth instar mosquito larvae that they exposed to increasing dieldrin concentrations.
At the last round, surviving larvae were then allowed to moult and nymphs were sex separated. Then, the lines were constructed following the same previous procedure and eggs resulting from copulations between homozygous rdl(R) male and female mosquitoes were pooled for hatching eggs providing an homozygous resistant rdl(R) line named R-Run [RR].
The second step consisted to determine the dieldrin diagnostic selective dose, via a cross between R-Run males with S-Run females to obtain heterozygous larvae [RS].
Then, researchers exposed third instar larvae of [SS], [RR] and [RS] to different insecticide doses and recorded their survival rate.
Results showed that a dieldrin concentration of 0.1 ppm killed 100% of S-Run larvae but none of the heterozygous or R-Run larvae, and was thus selected as the diagnostic/selective dose (Figure 1).
Figure 1: Survival of R-Run (RR), S-Run (SS) and heterozygous (RS) third instar larvae following 24h treatment with increasing dieldrin concentrations
Source: Construction of a Genetic Sexing Strain for Aedes albopictus: a promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito (Lebon et al. 2018)
The third step consisted to obtain the sex-linkage of the rdl gene through X-ray irradiation and selection of the sex biased mosquito lines.
After the irradiation, R-Run male pupae were allowed to emerge and then crossed en masse with virgin S-Run females which were then fed and allowed to lay eggs.
After an exposition to the selective dose of dieldrin (0,1 ppm), dieldrin resistant males issued from these crosses were then individually crossed with S-Run females which were fed and allowed to lay eggs.
Results showed that only 4 males named TiCoq led to progenies exhibiting male biases exceeding 60% and were further backcrossed with S-Run females during twelve generations. Larvae issued from each cross were exposed to the dieldrin at each generation.
The hatching rate, larval survival and sex ratio were followed for each generation and researchers observed that the hatching rate averaged 30% over the entire experiment, larvae surviving rate following dieldrin treatment averaged 50% and sex ratio bias was stable at 97.8%.
Moreover, results showed that the sex bias was not perfect and 2% females descending from crosses between TiCoq males and S-Run females survived to the dieldrin treatment at larval stage.
In order to evaluate the stability of TiCoq, a part of layers was not treated with dieldrin after the 7th generation in order to measure the rdl(R) frequency. Results showed that the hatching rate tripled during 4 generations while larvae survival following dieldrin treatments decreased from about 50% to about 3% in the meantime, and no resistant larvae were observed after 5 generations in the absence of insecticide pressure.
This study showed that this GSS construction using the dieldrin treatment allowed selecting about 98% of males and seems adapted for control method based on the mass production of Aedes albopictus sterile males.
However, it’s necessary to improve it because it displayes a strong bias in sex ratio and uses a selectable resistance marker to the dieldrin, an insecticide which is forbidden in several countries since a long time and obligating its use only in the laboratory.
Despite of these limitations, the construction of this GSS paves the way for industrial sex separation of Aedes. albopictus.
Currently, another project named “Revolutionizing insect control” (REVOLINC) aims to develop a new method of male mass production for Aedes albopictus to improve the sterile insect technique.
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*Sexual dimorphism: Condition occurring in many animals and some plants, where the two sexes of the same species exhibit different characteristics beyond the differences in their sexual organs.
**Sex linkage: Phenotypic expression of an allele related to the sex chromosome of the individual.
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