Significance: Genetic systems for controlling transmission of vector-borne diseases are moving from discovery-stage demonstrations of proofs-of-principle to the next phases of development. A successful transition requires meeting safety and efficacy criteria defined in target product profiles. We show here that the Cas9/guide RNA-based gene-drive components of a genetically-engineered malaria mosquito vector, Anopheles gambiae , achieve key target product profile requirements for efficacy and performance. This system is designed to achieve mosquito population modification when coupled with genes encoding antiparasite effector molecules and result in stable and sustainable blocking of malaria parasite transmission.

Significance: We report that during a recent period of hybridization between two major African malaria mosquitoes, Anopheles gambiae and Anopheles coluzzii , an island of divergence on chromosome 2 introgressed from the A. gambiae into the A. coluzzii genome and its frequency subsequently increased. This introgression was coincident with the start of a major insecticide-treated bed net campaigns in Mali. These observations suggest that increased insecticide exposure acted as a selective force sufficient to drive introgression of an entire genomic island of divergence across the reproductive barrier separating these two species. This study provides a rare example of adaptive introgression in an animal species and elucidates the dynamics of how insecticide resistance evolved in A. coluzzii .

Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.

Significance: Populations of the African malaria vector, Anopheles gambiae , are structured into M and S forms. All current work assumes the two rarely hybridize. Here we show this assumption is false. We demonstrate ( i ) significant exchange of genes between the two forms, even though ( ii ) hybrids have reduced fitness and ( iii ) the gene exchange process is spatially and temporally dynamic. For malaria, it is important to determine if genes for traits like insecticide resistance are shared between forms. For evolutionary biologists, this work confirms that this mosquito is a good model for studying if and how species may evolve in cases where there is ongoing gene flow.

Malaria transmission is dependent on the propensity of Anopheles mosquitoes to bite humans (anthropophily) instead of other dead end hosts. Recent increases in the usage of Long Lasting Insecticide Treated Nets (LLINs) in Africa have been associated with reductions in highly anthropophilic and endophilic vectors such as Anopheles gambiae s.s., leaving species with a broader host range, such as Anopheles arabiensis, as the most prominent remaining source of transmission in many settings. An. arabiensis appears to be more of a generalist in terms of its host choice and resting behavior, which may be due to phenotypic plasticity and/or segregating allelic variation. To investigate the genetic basis of host choice and resting behavior in An. arabiensis we sequenced the genomes of 23 human-fed and 25 cattle-fed mosquitoes collected both in-doors and out-doors in the Kilombero Valley, Tanzania. We identified a total of 4,820,851 SNPs, which were used to conduct the first genome-wide estimates of “SNP heritability” for host choice and resting behavior in this species. A genetic component was detected for host choice (human vs cow fed; permuted P = 0.002), but there was no evidence of a genetic component for resting behavior (indoors versus outside; permuted P = 0.465). A principal component analysis (PCA) segregated individuals based on genomic variation into three groups which were characterized by differences at the 2Rb and/or 3Ra paracentromeric chromosome inversions. There was a non-random distribution of cattle-fed mosquitoes between the PCA clusters, suggesting that alleles linked to the 2Rb and/or 3Ra inversions may influence host choice. Using a novel inversion genotyping assay, we detected a significant enrichment of the standard arrangement (non-inverted) of 3Ra among cattle-fed mosquitoes (N = 129) versus all non-cattle-fed individuals (N = 234; χ², p = 0.007). Thus, tracking the frequency of the 3Ra in An. arabiensis populations may be of use to infer selection on host choice behavior within these vector populations; possibly in response to vector control. Controlled host-choice assays are needed to discern whether the observed genetic component has a direct relationship with innate host preference. A better understanding of the genetic basis for host feeding behavior in An. arabiensis may also open avenues for novel vector control strategies based on driving genes for zoophily into wild mosquito populations. Malaria transmission is driven by the propensity for mosquito vectors to bite people, while its control depends on the tendency of mosquitoes to bite and rest in places where they will come into contact with insecticides. In many parts of Africa, where coverage with Long Lasting Insecticide Treated Nets is high, Anopheles arabiensis is the only remaing malaria vector. We sought to assess the potential for An. arabiensis to adapt its behavior to avoid control measures by investigating the genetic basis for its host choice and resting behavior. Blood fed An. arabiensis were collected resting indoors and outdoors in the Kilombero Valley, Tanzania. We sequenced a total of 48 genomes representing 4 phenotypes (human or cow fed, resting in or outdoors) and tested for genetic associations with each phenotype. Genomic analysis followed up by application of a novel molecular karyotyping assay revealed a relationship between An. arabiensis that fed on cattle and the standard arrangement of the 3Ra inversion. This is strong support that An. arabiensis blood-feeding behavior has a substantial genetic component. Controlled host choice assays are needed to confirm a direct link between allelic variation within the 3Ra inversion and host preference.

Despite significant reductions in malaria transmission across Africa since 2000, progress is stalling. This has been attributed to the development of insecticide resistance and behavioural adaptations in malaria vectors. Whilst insecticide resistance has been widely investigated, there is poorer understanding of the emergence, dynamics and impact of mosquito behavioural adaptations. We conducted a longitudinal investigation of malaria vector host choice over 3 years and resting behaviour over 4 years following a mass long-lasting insecticidal nets (LLINs) distribution in Tanzania. By pairing observations of mosquito ecology with environmental monitoring, we quantified longitudinal shifts in host-choice and resting behaviour that are consistent with adaptation to evade LLINs. The density of An. funestuss.l., declined significantly through time. In tandem, An. arabiensis and An. funestuss.l. exhibited an increased rate of outdoor relative to indoor resting; with An. arabiensis reducing the proportion of blood meals taken from humans in favour of cattle. By accounting for environmental variation, this study detected clear evidence of intra-specific shifts in mosquito behaviour that could be obscured in shorter-term or temporally-coarse surveys. This highlights the importance of mosquito behavioural adaptations to vector control, and the value of longer-term behavioural studies.