The number of dengue cases worldwide has increased ten-fold over the past decade as Aedes aegypti , the primary vector of this disease, thrives and expands its distribution, revealing limitations to current control methods. To better understand how Ae. aegypti evolved from a forest dwelling, generalist species to a highly anthropophilic urban species and the impact of contemporary gene flow on the future of dengue control, we sequenced 1,206 genomes from mosquitoes collected at 74 locations around the globe. Here we show that after evolving a preference for humans in the Sahel region of West Africa, the origin of the fully domesticated, anthropophilic subspecies Ae. aegypti aegypti ( Aaa ) occurred in the Americas during the Atlantic Slave Trade era and was followed by its explosive expansion around the globe. In recent decades, Aaa has invaded coastal Africa, the ancestral home range, introducing insecticide resistance mutations and an affinity for human hosts. Evidence of back-to-Africa migration is found in regions with recent dengue outbreaks, raising concern that global movement of Aaa could increase transmission risk of arboviruses including dengue in urban Africa. These data provide a platform to further study this important mosquito vector species and underscore developing complexity in the fight to limit the spread of dengue, Zika, and chikungunya diseases. ### Competing Interest Statement JEC and BJW report employment and equity ownership at Verily Life Sciences, a for-profit company developing new technologies for mosquito control.

Horizontal gene transfer from viruses to eukaryotic cells is a pervasive phenomenon. Somatic viral integrations are linked to persistent viral infection whereas integrations into germline cells are maintained in host genomes by vertical transmission and may be co-opted for host functions. In the arboviral vector Aedes aegypti, an endogenous viral element from a nonretroviral RNA virus (nrEVE) was shown to produce PIWI-interacting RNAs (piRNAs) to limit infection with a cognate virus. Thus, nrEVEs may constitute a heritable, sequence-specific mechanism for antiviral immunity, analogous to piRNA-mediated silencing of transposable elements. Here, we combine population genomics and evolutionary approaches to analyse the genomic architecture of nrEVEs in A. aegypti. We conducted a genome-wide screen for adaptive nrEVEs and searched for novel population-specific nrEVEs in the genomes of 80 individual wild-caught mosquitoes from five geographical populations. We show a dynamic landscape of nrEVEs in mosquito genomes and identified five novel nrEVEs derived from two currently circulating viruses, providing evidence of the environmental-dependent modification of a piRNA cluster. Overall, our results show that virus endogenization events are complex with only a few nrEVEs contributing to adaptive evolution in A. aegypti.

In the model organism , the PIWI-interacing RNA pathway contributes in silencing transposable elements (TEs) through smallRNAs (piRNAs), which arise from genomic loci (piRNA clusters) that contain sequences of previously-acquired TEs. As such, they are a functionally-immune archive of previous TE invasions that is passed to the offspring. In the arboviral vector , piRNA clusters contain TEs and endogenous viral elements from nonretroviral RNA viruses (nrEVEs) which produce piRNAs, supporting the hypothesis that nrEVEs are heritable immunity effectors. However, direct evidence that nrEVEs mediate adaptive immunity is lacking. Here, by using an analytic approach intersecting population genomics with molecular biology we demonstrate that the composition of piRNA clusters is modular through acquisition and absence of nrEVEs. We show that the genomes of wild-caught mosquitoes have a different set of nrEVEs than those annotated in the reference genome, including population-specific integrations. nrEVEs are not distributed in mosquito genomes only by genetic drift, but some show signs of positive selection. Moreover, by comparing natural mosquito populations expressing or lacking two newly characterised nrEVEs with high sequence complementarity to cell fusing agent virus, we show that nrEVEs confer antiviral immunity in ovaries against the cognate virus. Our results confirm that some nrEVEs have been co-opted for adaptive immunity to viral infections.

Background Aedes aegypti is the principal mosquito vector of Zika, dengue, and yellow fever viruses. Two subspecies of Ae. aegypti exhibit phenotypic divergence with regard to habitat, host preference, and vectorial capacity. Chromosomal inversions have been shown to play a major role in adaptation and speciation in dipteran insects and would be of great utility for studies of Ae. aegypti. However, the large and highly repetitive genome of Ae. aegypti makes it difficult to detect inversions with paired-end short-read sequencing data, and polytene chromosome analysis does not provide sufficient resolution to detect chromosome banding patterns indicative of inversions. Results To characterize chromosomal diversity in this species, we have carried out deep Illumina sequencing of linked-read (10X Genomics) libraries in order to discover inversion loci as well as SNPs. We analyzed individuals from colonies representing the geographic limits of each subspecies, one contact zone between subspecies, and a closely related sister species. Despite genome-wide SNP divergence and abundant microinversions, we do not find any inversions occurring as fixed differences between subspecies. Many microinversions are found in regions that have introgressed and have captured genes that could impact behavior, such as a cluster of odorant-binding proteins that may play a role in host feeding preference. Conclusions Our study shows that inversions are abundant and widely shared among subspecies of Aedes aegypti and that introgression has occurred in regions of secondary contact. This library of 32 novel chromosomal inversions demonstrates the capacity for linked-read sequencing to identify previously intractable genomic rearrangements and provides a foundation for future population genetics studies in this species.

Background: Anopheles funestus is one of the three most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito. Findings: Here we present a new high-quality An. funestus reference genome (AfunF3) assembled using 240x coverage of long-read single-molecule sequencing for contigging, combined with 100x coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using alignment and depth-of-coverage information, these contigs were deduplicated to a 211 Mbp primary assembly, which is closer to the expected haploid genome size of 250 Mbp. This primary assembly consists of 1,053 contigs organized into 3 chromosome-scale scaffolds with an N50 contig size of 632 kbp and an N50 scaffold size of 93.811 Mbp, representing a 100-fold improvement in continuity versus the current reference assembly, AfunF1. Conclusion: This highly contiguous and complete An. funestus reference genome assembly will serve as an improved basis for future studies of genomic variation and organization in this important disease vector.

Chromosomal inversion polymorphisms play an important role in adaptation to environmental heterogeneities. For mosquito species in the Anopheles gambiae complex that are significant vectors of human malaria, paracentric inversion polymorphisms are abundant and are associated with ecologically and epidemiologically important phenotypes. Improved understanding of these traits relies on determining mosquito karyotype, which currently depends upon laborious cytogenetic methods whose application is limited both by the requirement for specialized expertise and for properly preserved adult females at specific gonotrophic stages. To overcome this limitation, we developed sets of tag single nucleotide polymorphisms (SNPs) inside inversions whose biallelic genotype is strongly correlated with inversion genotype. We leveraged 1,347 fully sequenced An. gambiae and Anopheles coluzzii genomes in the Ag1000G database of natural variation. Beginning with principal components analysis (PCA) of population samples, applied to windows of the genome containing individual chromosomal rearrangements, we classified samples into three inversion genotypes, distinguishing homozygous inverted and homozygous uninverted groups by inclusion of the small subset of specimens in Ag1000G that are associated with cytogenetic metadata. We then assessed the correlation between candidate tag SNP genotypes and PCA-based inversion genotypes in our training sets, selecting those candidates with >80% agreement. Our initial tests both in held-back validation samples from Ag1000G and in data independent of Ag1000G suggest that when used for in silico inversion genotyping of sequenced mosquitoes, these tags perform better than traditional cytogenetics, even for specimens where only a small subset of the tag SNPs can be successfully ascertained.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S -transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a set of clinical samples and novel methods of sequencing, library preparation, and genotyping, we have genotyped low-complexity regions of the genome with a high degree of accuracy. Despite its slower evolutionary rate compared to bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2 years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

Drug resistance is an obstacle to global malaria control, as evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of the PfKelch13 protein in Southeast Asia. Identifying genetic determinants of ART resistance in African-derived parasites is important for surveillance and for understanding the mechanism of resistance. In this study, we carried out long-term in vitro selection of two recently isolated West African parasites (from Pikine and Thiès, Senegal) with increasing concentrations of dihydroartemisinin (DHA), the biologically active form of ART, over a 4-y period. We isolated two parasite clones, one from each original isolate, that exhibited enhanced survival to DHA in the ring-stage survival assay. Whole-genome sequence analysis identified 10 mutations in seven different genes. We chose to focus on the gene encoding PfCoronin, a member of the WD40-propeller domain protein family, because mutations in this gene occurred in both independent selections, and the protein shares the β-propeller motif with PfKelch13 protein. For functional validation, when pfcoronin mutations were introduced into the parental parasites by CRISPR/Cas9-mediated gene editing, these mutations were sufficient to reduce ART susceptibility in the parental lines. The discovery of a second gene for ART resistance may yield insights into the molecular mechanisms of resistance. It also suggests that pfcoronin mutants could emerge as a nonkelch13 type of resistance to ART in natural settings.

Female Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, has been slowed by the lack of a high-quality genome assembly. Here we combine diverse genome technologies to produce AaegL5, a dramatically improved and annotated assembly, and demonstrate how it accelerates mosquito science and control. We anchored the physical and cytogenetic maps, resolved the size and composition of the elusive sex-determining “M locus”, significantly increased the known members of the glutathione-S-transferase genes important for insecticide resistance, and doubled the number of chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites. Using high-resolution QTL and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. We predict that AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly arboviral vector.