Species distributed across heterogeneous environments often evolve locally adapted populations, but understanding how these persist in the presence of homogenizing gene flow remains puzzling. In Gabon, Anopheles coluzzii, a major African malaria mosquito, is found in various ecological settings, including urban areas, remote rural villages, and forested environments away from any human presence. This study investigates the genomic signatures of local adaptation in populations from distinct environments including the urban area of Libreville, and two proximate sites 10 km apart in the La Lopé National Park (LLP), a village and its sylvatic neighbourhood. Whole genome re-sequencing of 96 mosquitoes unveiled 5.9 million high-quality single nucleotide polymorphisms. Coalescent-based demographic analyses suggest an ∼12,000-year-old divergence between Libreville and La Lopé populations, followed by a secondary contact (∼4000 ybp) resulting in asymmetric effective gene flow. The urban population displayed reduced effective size, evidence of inbreeding, and strong selection pressures likely associated to insecticides or pollution present in urban settings, as suggested by the hard selective sweeps detected in genes involved in detoxification and insecticide resistance. In contrast, the two geographically proximate LLP populations showed larger effective sizes, and distinctive selective signals, notably soft-selective sweeps on the standing genetic variation. Although presumably neutral loci failed to discriminate between LLP populations, our findings support that microgeographic adaptation can swiftly emerge through selection on standing genetic variation despite gene flow. This study contributes to the growing understanding of evolution of populations in heterogeneous environments amid ongoing gene flow and how major malaria mosquitoes adapt to humans and its environment.

BACKGROUND:The mosquito Aedes aegypti is the primary vector of several medically important arboviruses, including dengue, Zika, chikungunya, and yellow fever. Characterizing the genetic diversity of Ae. aegypti is essential to understand its evolutionary history and population dynamics and to evaluate vector control strategies. However, whole-genome sequencing (WGS) is often cost-prohibitive at scale due to the species' large genome size. Reduced-representation approaches offer a cost-effective alternative, yet a standardized genome-wide marker set for population genetic studies of Ae. aegypti is lacking. METHODS:We developed a targeted amplicon sequencing panel for cost-effective, high-throughput genotyping across 291 loci distributed throughout the Ae. aegypti genome. We evaluated its performance by comparing patterns of population structure and genetic admixture inferred from the amplicon sequences with those obtained from WGS data. RESULTS:In silico analyses demonstrate that the amplicon panel reliably reproduces population structure patterns typically observed with WGS. It also effectively distinguishes among diverse laboratory colonies of Ae. aegypti and yields individual genetic admixture estimates consistent with WGS results. CONCLUSIONS:This targeted amplicon sequencing panel enables high-throughput genotyping at reduced cost and provides a practical alternative to WGS for population genetic and genomic surveillance studies. It should facilitate large-scale genotyping of Ae. aegypti, particularly in resource-limited settings.

Decoding mosquito genomes is essential for understanding their role as vectors of human pathogens. Transposable elements (TEs), once considered 'junk DNA', are now recognized as key drivers of genomic plasticity and evolution. These mobile DNA fragments are more abundant in culicine than anopheline mosquitoes, influencing their genome size and complexity. TEs may contribute to mosquito adaptation, influencing traits such as insecticide resistance and habitat expansion. Recent advances in long-read sequencing technologies and functional assays are uncovering TE dynamics, but challenges remain in measuring and manipulating their activity, which is critical to demonstrate their phenotypic effects. Further research into the environmental and biological conditions that activate TEs in mosquitoes, as well as the host mechanisms of TE regulation, such as small RNA pathways, will significantly enhance our understanding of mosquito biology.

The mosquito Aedes aegypti is the primary vector for dengue virus (DENV), which infects millions of people annually. Variability in DENV susceptibility among wild Ae. aegypti populations is governed by genetic factors, but specific causal variants are unknown. Here, we identify a cytochrome P450-encoding gene (CYP4G15) whose genetic variants drive differences in DENV susceptibility in a natural Ae. aegypti population. CYP4G15 is transiently upregulated in DENV-resistant midguts, while knockdown increases susceptibility, and transgenic overexpression enhances resistance. A naturally occurring 18-base-pair promoter deletion reduces CYP4G15 expression and confers higher DENV susceptibility. The unexpected role of a cytochrome P450 in DENV susceptibility challenges the long-standing focus on canonical immune pathways and opens new avenues for understanding antiviral defense and DENV transmission in mosquitoes.

Transposable elements (TEs) are DNA sequences that can change their position within a genome. In insects, small RNA pathways are central to the transcriptional and post-transcriptional regulation of TE expression. The Piwi-interacting RNA (piRNA) pathway is particularly important in germline tissues, where it silences TE transcripts via small RNAs of 24–30 nucleotides (nt) in length produced from genomic precursor transcripts as well as through a “ping-pong” amplification cycle. The small interfering RNA (siRNA) pathway helps restrict TE expression in somatic tissues via 21nt small RNAs produced from double-stranded RNA by the endonuclease Dicer2, which guide an RNA-induced silencing complex to degrade complementary RNAs. However, much of this knowledge comes from studies of the model insect Drosophila melanogaster. In the mosquito Aedes aegypti, a medically significant vector species, the siRNA pathway has mainly been investigated in connection with its antiviral role, leaving open whether it also regulates TE expression. We investigated the expression of TEs and small RNAs in both somatic and gonadal tissues of a Dicer2 mutant line of Ae. aegypti and its wild-type counterpart. Our results show a modified pattern of TE expression and a decrease in TE-derived 21nt RNAs in the Dicer2 mutant, but no major shift of TE transcript abundance. The lack of a functional siRNA pathway also causes perturbations in piRNA ping-pong signatures and the expression of certain piRNA-associated genes, but without clear evidence for compensation by increased piRNA pathway activity. The mosquito Ae. aegypti produces siRNAs derived from TEs but these lack a critical role in the regulation of TE expression both in somatic and in gonadal tissues.

BACKGROUND:Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control the spread of arboviruses focus on harnessing the antiviral immune system of their primary vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. RESULTS:We conducted a phylogenetic analysis of the Vago gene family in Diptera, which led us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti, which revealed a broad impact of VLG-1 on the mosquito transcriptome, affecting several biological processes potentially related to viral replication, including the oxidative stress response. Surprisingly, experimental viral challenge of the VLG-1 mutant line indicated a modest proviral role for this gene during DENV and ZIKV infections in vivo. In the absence of VLG-1, virus dissemination throughout the mosquito's body was slightly impaired, albeit not altering virus transmission rates. CONCLUSIONS:Our results challenge the conventional understanding of Vago-like genes as antiviral factors and underscore the need for further in vivo research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.

AbstractThe mosquitoAedes aegyptiis the primary vector for dengue virus (DENV), which infects millions of people annually. Variability in DENV susceptibility among wildAe. aegyptipopulations is governed by genetic factors, but specific causal variants are unknown. Here, we identify a cytochrome P450-encoding gene (CYP4G15) whose variants drive differences in DENV susceptibility in a naturalAe. aegyptipopulation.CYP4G15is transiently upregulated in DENV-resistant midguts, while knockdown increases susceptibility, and transgenic overexpression enhances resistance. A naturally occurring 18-base-pair promoter deletion reducesCYP4G15expression and confers higher DENV susceptibility. The unexpected role of a cytochrome P450 in DENV susceptibility challenges the long-standing focus on canonical immune pathways and opens new avenues for understanding antiviral defense and DENV transmission in mosquitoes.

Abstract The adaptation of Anopheles malaria vectors to domestic settings is directly linked to their ability to feed on humans. The strength of this species–habitat association is unequal across the species within the genus, with the major vectors being particularly dependent on humans. However, our understanding of how blood‐feeding behavior interacts with and adapts to environmental settings, including the presence of humans, remains limited. Using a field‐based approach, we first investigated Anopheles community structure and feeding behavior patterns in domestic and sylvatic settings in La Lopé National Park in Gabon, Central Africa. We characterized the preference indices using a dual‐host choice sampling approach across mosquito species, habitats, and seasons. We then quantified the plastic biting behavior of mosquito species in each habitat. We collected individuals from 16 Anopheles species that exhibited significant differences in species composition and abundance between sylvatic and domestic settings. The host‐seeking behavior also varied among the seven most abundant species. The general attractiveness to each host, human or animal, remained relatively constant for each species, but with significant variations between habitats across species. These variations, to more generalist and to more anthropophilic behavior, were related to seasonal changes and distance from the village, respectively. Finally, we pointed out that the host choice of major malaria vectors changed in the absence of humans, revealing a plastic feeding behavior of these species. This study highlights the effect of humans on Anopheles distribution and feeding evolution. The characterization of feeding behavior in wild and domestic settings provides opportunities to better understand the interplay between genetic determinants of host preference and ecological factors. Our findings suggest that protected areas may offer alternative thriving conditions to major malaria vectors.

Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control the spread of arboviruses focus on harnessing the antiviral immune system of their primary vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. We conducted a phylogenetic analysis of the Vago gene family in Diptera, which led us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti that revealed a proviral effect of this gene upon DENV and ZIKV infection. In the absence of VLG-1, virus dissemination throughout the mosquito's body was impaired, albeit not altering virus transmission rates. A tissue-specific transcriptome analysis revealed that the loss of VLG-1 impacted numerous biological processes potentially linked to viral replication, such as the oxidative stress response. Our results challenge the conventional understanding of Vago-like genes as antiviral factors and underscores the need for further research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions. ### Competing Interest Statement The authors have declared no competing interest.

In the last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulfite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, taking into account the structural complexity of genomes, their size, and their content in unique coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by sequencing capture bisulfite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea. We developed a strategy and a workflow for quantifying epigenetic diversity in natural populations combining whole-genome and targeted capture sequencing for DNA methylation.

We developed a strategy and a workflow for quantifying epigenetic diversity in natural populations combining whole genome and targeted capture sequencing for DNA methylation. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis).

Species distributed across heterogeneous environments often evolve locally adapted populations, but understanding how these persist in the presence of homogenizing gene flow remains puzzling. In Gabon, Anopheles coluzzii, a major African malaria mosquito is found along an ecological gradient, including a sylvatic population, away of any human presence. This study identifies into the genomic signatures of local adaptation in populations from distinct environments including the urban area of Libreville, and two proximate sites 10km apart in the La Lopé National Park (LLP), a village and its sylvatic neighborhood. Whole genome re-sequencing of 96 mosquitoes unveiled ∼ 5.7millions high-quality single nucleotide polymorphisms. Coalescent-based demographic analyses suggest an ∼ 8,000-year-old divergence between Libreville and La Lopé populations, followed by a secondary contact ( ∼ 4,000 ybp) resulting in asymmetric effective gene flow. The urban population displayed reduced effective size, evidence of inbreeding, and strong selection pressures for adaptation to urban settings, as suggested by the hard selective sweeps associated with genes involved in detoxification and insecticide resistance. In contrast, the two geographically proximate LLP populations showed larger effective sizes, and distinctive genomic differences in selective signals, notably soft-selective sweeps on the standing genetic variation. Although neutral loci and chromosomal inversions failed to discriminate between LLP populations, our findings support that microgeographic adaptation can swiftly emerge through selection on standing genetic variation despite high gene flow. This study contributes to the growing understanding of evolution of populations in heterogeneous environments amid ongoing gene flow and how major malaria mosquitoes adapt to human. Significance:Anopheles coluzzii , a major African malaria vector, thrives from humid rainforests to dry savannahs and coastal areas. This ecological success is linked to its close association with domestic settings, with human playing significant roles in driving the recent urban evolution of this mosquito. Our research explores the assumption that these mosquitoes are strictly dependent on human habitats, by conducting whole-genome sequencing on An. coluzzii specimens from urban, rural, and sylvatic sites in Gabon. We found that urban mosquitoes show de novo genetic signatures of human-driven vector control, while rural and sylvatic mosquitoes exhibit distinctive genetic evidence of local adaptations derived from standing genetic variation. Understanding adaptation mechanisms of this mosquito is therefore crucial to predict evolution of vector control strategies.

Transposable elements (TEs) contribute significantly to variation in genome size among eukaryotic species, but the factors influencing TE accumulation and diversification are only partially understood. Most of our current knowledge about TE organization, dynamics and evolution derives from investigations in model organisms with a relatively small genome size such as Drosophila melanogaster or Arabidopsis thaliana . Whether the observed patterns hold true in larger genomes remains to be determined. The Diptera order is an ideal taxon to address this question, because it includes a forty-year model of TE biology ( D. melanogaster ) as well as mosquito species with significantly larger genomes. Here, we use a comparative genomics approach to characterize the genomic forces that have shaped the TE content of the Aedes aegypti genome (1.3 Gb) relative to the Anopheles coluzzii genome (300 Mb) and the D. melanogaster genome (180 Mb). Leveraging a newly developed high-quality TE library for Ae. aegypti , our results reveal a contrasted pattern of TE organization in Ae. aegypti compared to An. coluzzii and D. melanogaster . Our analyses suggest that the substantial TE fraction observed in the Ae. aegypti genome reflect both a high rate of TE transposition and a low rate of TE elimination. Together, our results indicate that TE organization and evolutionary dynamics in the large genome of Ae. aegypti are distinct from those of other dipterans with smaller genomes.### Competing Interest StatementThe authors have declared no competing interest.

AbstractSince the discovery of natural malaria vector populations infected by the endosymbiont bacteriumWolbachia, a renewed interest has arisen for using this bacterium as an alternative for malaria control. Among naturally infected mosquitoes,Anopheles moucheti, a major malaria mosquito in Central Africa, exhibits one of the highest prevalences ofWolbachiainfection. To better understand whether this maternally inherited bacterium could be used for malaria control, we investigatedWolbachiainfluence inAn. mouchetipopulations naturally infected by the malaria parasitePlasmodium falciparum. To this end, we collected mosquitoes in a village from Cameroon, Central Africa, where this mosquito is the main malaria vector. We found that the prevalence ofWolbachiabacterium was almost fixed in the studied mosquito population, and was higher than previously recorded. We also quantifiedWolbachiain whole mosquitoes and dissected abdomens, confirming that the bacterium is also elsewhere than in the abdomen, but at lower density. Finally, we analyzed the impact ofWolbachiapresence and density onP. falciparuminfection.Wolbachiadensity was slightly higher in mosquitoes infected with the malaria parasite than in uninfected mosquitoes. However, we observed no correlation between theP. falciparumandWolbachiadensities. In conclusion, our study indicates that naturally occurringWolbachiainfection does not affectP. falciparumdevelopment withinAn. mouchetimosquitoes.

Differences in codon frequency between genomes, genes, or positions along a gene, modulate transcription and translation efficiency, leading to phenotypic and functional differences. Here, we present a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells, quantifying the phenotypic consequences of codon usage bias at different molecular and cellular levels, with an emphasis on translation elongation. Six synonymous versions of an antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was analyzed by means of quantitative transcriptome and proteome assessment, as proxies for gene expression; cellular fluorescence, as a proxy for single-cell level expression; and real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness. We show that differences in codon usage bias strongly impact the molecular and cellular phenotype: (i) they result in large differences in mRNA levels and protein levels, leading to differences of over 15 times in translation efficiency; (ii) they introduce unpredicted splicing events; (iii) they lead to reproducible phenotypic heterogeneity; and (iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression. In human cells in culture, codon usage bias modulates gene expression by modifying mRNA availability and suitability for translation, leading to differences in protein levels and eventually eliciting functional phenotypic changes.

Plasmodium falciparum, the most virulent agent of human malaria, spread from Africa to all continents following the out-of-Africa human migrations. During the transatlantic slave trade between the 16th and 19th centuries, it was introduced twice independently to the Americas where it adapted to new environmental conditions (new human populations and mosquito species). Here, we analyzed the genome-wide polymorphisms of 2,635 isolates across the current P. falciparum distribution range in Africa, Asia, Oceania, and the Americas to investigate its genetic structure, invasion history, and selective pressures associated with its adaptation to the American environment. We confirmed that American populations originated from Africa with at least two independent introductions that led to two genetically distinct clusters, one in the North (Haiti and Colombia) and one in the South (French Guiana and Brazil), and an admixed Peruvian group. Genome scans revealed recent and more ancient signals of positive selection in the American populations. Particularly, we detected positive selection signals in genes involved in interactions with hosts (human and mosquito) cells and in genes involved in resistance to malaria drugs in both clusters. Analyses suggested that for five genes, adaptive introgression between clusters or selection on standing variation was at the origin of this repeated evolution. This study provides new genetic evidence on P. falciparum colonization history and on its local adaptation in the Americas.

Since the discovery of natural malaria vector populations infected by the endosymbiont bacterium Wolbachia, a renewed interest has arisen for using this bacterium as an alternative for malaria control. Among naturally infected mosquitoes, Anopheles moucheti, a major malaria mosquito in Central Africa, exhibits one of the highest prevalences of Wolbachia infection. To better understand whether this maternally inherited bacterium could be used for malaria control, we investigated Wolbachia influence in An. moucheti populations naturally infected by the malaria parasite Plasmodium falciparum. To this end, we collected mosquitoes in a village from Cameroon, Central Africa, where this mosquito is the main malaria vector. We found that the prevalence of Wolbachia bacterium was almost fixed in the studied mosquito population, and was higher than previously recorded. We also quantified Wolbachia in whole mosquitoes and dissected abdomens, confirming that the bacterium is also elsewhere than in the abdomen, but at lower density. Finally, we analyzed the association of Wolbachia presence and density on P. falciparum infection. Wolbachia density was slightly higher in mosquitoes infected with the malaria parasite than in uninfected mosquitoes. However, we observed no correlation between the P. falciparum and Wolbachia densities. In conclusion, our study indicates that naturally occurring Wolbachia infection is not associated to P. falciparum development within An. moucheti mosquitoes.

Malaria is a vector-borne disease caused by protozoan parasites of the genus Plasmodium. Plasmodium vivax is the most prevalent human-infecting species in the Americas. However, the origins of this parasite in this continent are still debated. Similarly, it is now accepted that the existence of Plasmodium simium is explained by a P. vivax transfer from humans to monkey in America. However, many uncertainties still exist concerning the origin of the transfer and whether several transfers occurred. In this review, the most recent studies that addressed these questions using genetic and genomic approaches are presented.

Plasmodium vivax is the most common and widespread human malaria parasite. It was recently proposed that P. vivax originates from sub-Saharan Africa based on the circulation of its closest genetic relatives (P. vivax-like) among African great apes. However, the limited number of genetic markers and samples investigated questions the robustness of this hypothesis. Here, we extensively characterized the genomic variations of 447 human P. vivax strains and 19 ape P. vivax-like strains collected worldwide. Phylogenetic relationships between human and ape Plasmodium strains revealed that P. vivax is a sister clade of P. vivax-like, not included within the radiation of P. vivax-like By investigating various aspects of P. vivax genetic variation, we identified several notable geographical patterns in summary statistics in function of the increasing geographic distance from Southeast Asia, suggesting that P. vivax may have derived from a single area in Asia through serial founder effects.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human-emerged virus of the 21st century from the Coronaviridae family, causing the ongoing coronavirus disease 2019 (COVID-19) pandemic. Due to the high zoonotic potential of coronaviruses, it is critical to unravel their evolutionary history of host species breadth, host-switch potential, adaptation and emergence, to identify viruses posing a pandemic risk in humans. We present here a comprehensive analysis of the composition and codon usage bias of the 82 Orthocoronavirinae members, infecting 47 different avian and mammalian hosts. Our results clearly establish that synonymous codon usage varies widely among viruses, is only weakly dependent on their primary host, and is dominated by mutational bias towards AU-enrichment and by CpG avoidance. Indeed, variation in GC3 explains around 34%, while variation in CpG frequency explains around 14% of total variation in codon usage bias. Further insight on the mutational equilibrium within Orthocoronavirinae revealed that most coronavirus genomes are close to their neutral equilibrium, the exception being the three recently infecting human coronaviruses, which lie further away from the mutational equilibrium than their endemic human coronavirus counterparts. Finally, our results suggest that, while replicating in humans, SARS-CoV-2 is slowly becoming AU-richer, likely until attaining a new mutational equilibrium.