Molecular tools and genetic markers for the generation of transgenic sexing strains in Anopheline mosquitoes

Volume 11 Supplement 2 Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes Review Open Access Molecular tools and genetic markers for the generation of transgenic sexing strains in Anopheline mosquitoes Federica Bernardini1, Roya Elaine Haghighat-Khah1, Roberto Galizi1, Andrew Marc Hammond1, Tony Nolan1 and Andrea Crisanti1Email author Parasites & Vectors201811 (Suppl 2) :660 https://doi.org/10.1186/s13071-018-3207-8 ©  The Author(s). 2018 Published: 24 December 2018 Abstract Malaria is a serious global health burden, affecting more than 200 million people each year in over 90 countries, predominantly in Africa, Asia and the Americas. Since the year 2000, a concerted effort to combat malaria has reduced its incidence by more than 40%, primarily due to the use of insecticide-treated bednets, indoor residual spraying and artemisinin-based combination drug therapies. Nevertheless, the cost of control is expected to nearly triple over the next decade and the current downward trend in disease transmission is threatened by the rise of resistance to drugs and insecticides. Novel strategies that are sustainable and cost-effective are needed to help usher in an era of malaria elimination. The most effective strategies thus far have focussed on control of the mosquito vector. The sterile insect technique (SIT) is a potentially powerful strategy that aims to suppress mosquito populations through the unproductive mating of wild female mosquitoes with sterile males that are released en masse. The technique and its derivatives are currently not appropriate for malaria control because it is difficult to sterilise males without compromising their ability to mate, and because anopheline males cannot be easily separated from females, which if released, could contribute to disease transmission. Advances in genome sequencing technologies and the development of transgenic techniques provide the tools necessary to produce mosquito sexing strains, which promise to improve current malaria-control programs and pave the way for new ones. In this review, the progress made in the development of transgenic sexing strains for the control of Anopheles gambiae, a major vector of human malaria, is discussed. Keywords Malaria Anopheles mosquitoessex determinationgenetic engineeringvector control An evolutionary perspective of sex and sex determination in insects Reproduction is a fundamental feature for life to subsist. In the majority of eukaryotes, sexual reproduction has evolved to allow, through meiosis and fusion of gametes, the rapid appearance of new genetic traits [1]. Sex-specific life histories and adaptations can result in highly specialised sexes that act to maintain this reproduction strategy [2–4]. A common feature of sex determination is a signalling cascade that begins with a singular primary signal and results in the expression of several hundred sexually dimorphic traits, often via a set of sex-specifically spliced intermediates. In the model organism Drosophila melanogaster, sexual development is traditionally thought to be initiated by the balance of female determinants on the X chromosome and male determinants on the autosomes. A more recent work supports the alternative view that the primary sex determining signal in flies is represented by the number of X chromosomes in the embryo rather than the X:A ratio [5]. Around 2 hours after fertilisation, the primary signal sets the state of activity of the sex lethal (sxl) gene. Sxl is itself a splice factor that acts upon the transformer (tra) transcript to produce a female-specific form of the mRNA that encodes a functional Tra protein. In turn, Tra triggers splicing of doublesex (dsx) pre-mRNA resulting in expression of the female-specific isoform, DsxF protein. In its capacity as a transcription factor, DsxF leads to the development of females. In the absence of Sxl protein, the primary transformer transcript is spliced to produce an mRNA that does not encode functional Tra protein. This consequently determines splicing of the dsx primary transcript to produce a DsxM transcription factor that enable masculine features [6]. In other organisms, sex determination is due to a trans-acting male-determining gene located on the male sex chromosome, generally identified as the Y. When this gene is expressed, during early embryogenesis, it inhibits factors that destine the sex determination pathway to the default female form, thus determining male development [7]. In the organisms that fall into this category, unlike the situation in Drosophila, the presence of the male chromosome is a crucial factor for determining sex. Anopheles gambiae is a member of the Anopheles gambiae complex that includes the principal vectors of human malaria [8]. Due to the burden imposed by these mosquitoes, efforts have been made to characterise their biology and special attention has been given to sex determination. In 1979 Baker et al., found that triploid Anopheles culifacies individuals with 3 X chromosomes were phenotypically females whereas XXY individuals showed a male phenotype [9], thus indicating that in mosquitoes the primary signal is different from that which is observed in the Drosophila model. In addition, a number of experiments in several Anopheles species used male inheritance of translocations as evidence pointing to the involvement of the Y chromosome in sex determination [10–13]. Recently, Krzywinska et al., isolated and characterised a gene, Yob, which acts as a male determination factor in An. gambiae [14]. This gene is located on the Y chromosome and controls male-specific splicing of dsx, the only downstream member of the sex determination cascade known in anopheline mosquitoes [15]. In the Asian malaria vector An. stephensi, a small protein named GUY1 acts as a primary signal that affects embryonic development in a sex-specific manner. This protein is encoded by a Y-linked gene, Guy1, that represents the best candidate for the male-determining factor [16]. In Aedes aegypti, Nix, a dominant male-determining factor located within a Y chromosome-like region called M locus, has been recently identified [17]. Transgenic sexing strains for vector control Vector control is a crucial component of many disease control programmes. Sterile Insect Technique (SIT) is a species-specific and environmentally non-polluting method of insect control which aims to reduce the ability of the targeted species to produce viable offspring [18]. This technology requires repeated releases of mass reared male insects that are generally sterilised using irradiation. By competing with wild males and mating with females over time, sterilised males lead to reduction of the targeted insect population. SIT programs have been successfully used for the control or local elimination of some insect pests such as the tsetse fly [19], medfly [20], melon fly [21] and the screwworm [22]. A major (...truncated)