Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever1. Specific host odours likely drive this strong preference since other attractive cues, including body heat and exhaled carbon dioxide (CO2) are common to all warm-blooded hosts2, 3. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs). ORs are membrane proteins that form heteromeric odour-gated ion channels4, 5 comprised of a variable ligand-selective subunit and an obligate co-receptor called Orco6. Here we use zinc-finger nucleases to generate targeted mutations in the Ae. aegypti orco gene to examine the contribution of Orco and the OR pathway to mosquito host selection and sensitivity to the insect repellent DEET. orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the OR pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50–100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A “sterile-male-release” strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release (“genetic sexing”), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.

The mariner transposable element is capable of interplasmid transposition in the embryonic soma of the yellow fever mosquito, Aedes aegypti. To determine if this demonstrated mobility could be utilized to genetically transform the mosquito, a modified mariner element marked with a wild-type allele of the Drosophila melanogaster cinnabar gene was microinjected into embryos of a kynurenine hydroxylasedeficient, white-eyed recipient strain. Three of 69 fertile male founders resulting from the microinjected embryos produced families with colored-eyed progeny individuals, a transformation rate of 4%. The transgene-mediated complementation of eye color was observed to segregate in a Mendelian manner, although one insertion segregates with the recessive allele (female-determining) of the sex-determining locus, and a separate insertion is homozygous lethal. Molecular analysis of selected transformed families demonstrated that a single complete copy of the construct had integrated independently in each case and that it had done so in a transposase-mediated manner. The availability of a mariner transformation system greatly enhances our ability to study and manipulate this important vector species.The incidence of vector-borne diseases is on the rise. As part of a multifaceted effort to control the transmission of diseases, we are developing tools for the molecular genetic manipulation of mosquitoes (1). We intend to use the tools and techniques of modern molecular biology to generate strains of mosquitoes that are incapable of transmitting a specific pathogen. These strains will be used selectively in release programs to reduce disease transmission. One of the key requirements for this effort is a method for introducing genes into mosquitoes. Recently, the Hermes transposable element from the housefly, Musca domestica, was shown to integrate into the germ line of the yellow fever mosquito, Aedes aegypti (2). The identification of Hermes as a viable candidate for mosquito transgenesis resulted from a strategy that first evaluated the ability of the element to mobilize (excise and insert) in the embryonic soma of the mosquito (3), followed by demonstration that it would integrate into the germ line (2). By using this approach, we show that a modified mariner transposable element efficiently and stably integrates into the germ line of Ae. aegypti.Transposition assays based on the mobilization of a marked transposon from a donor to a target plasmid (4) were used to show that the mariner element, Mos1, from Drosophila mauritiana (5), was capable of mobility in embryos of Ae. aegypti. Subsequently, a genetic transformation experiment showed that Mos1 could integrate into the germ line of the mosquito. This experiment exploited the recently demonstrated ability of a wild-type copy of the Drosophila melanogaster cinnabar (cn ϩ ) gene to complement the white-eye phenotype of the kynurenine hydroxylase-white (kh w ) strain of Ae. aegypti (6-8). We report the successful generation of transgenic Ae. aegypti lines that contai...

The mosquito Aedes aegypti is the world's most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germline transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G 0 adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn ؉ -carrying transposon. The cn ؉ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.