Genomic signatures of local adaptation in recent invasive Aedes aegypti populations in California.

BACKGROUND: Rapid adaptation to new environments can facilitate species invasions and range expansions. Understanding the mechanisms of adaptation used by invasive disease vectors in new regions has key implications for mitigating the prevalence and spread of vector-borne disease, although they remain relatively unexplored. RESULTS: Here, we integrate whole-genome sequencing data from 96 Aedes aegypti mosquitoes collected from various sites in southern and central California with 25 annual topo-climate variables to investigate genome-wide signals of local adaptation among populations. Patterns of population structure, as inferred using principal components and admixture analysis, were consistent with three genetic clusters. Using various landscape genomics approaches, which all remove the confounding effects of shared ancestry on correlations between genetic and environmental variation, we identified 112 genes showing strong signals of local environmental adaptation associated with one or more topo-climate factors. Some of them have known effects in climate adaptation, such as heat-shock proteins, which shows selective sweep and recent positive selection acting on these genomic regions. CONCLUSIONS: Our results provide a genome wide perspective on the distribution of adaptive loci and lay the foundation for future work to understand how environmental adaptation in Ae. aegypti impacts the arboviral disease landscape and how such adaptation could help or hinder efforts at population control.

Population modification strategies for malaria vector control are uniquely resilient to observed levels of gene drive resistance alleles.

Cas9/guide RNA (gRNA)-based gene drive systems are expected to play a transformative role in malaria elimination efforts., whether through population modification, in which the drive system contains parasite-refractory genes, or population suppression, in which the drive system induces a severe fitness load resulting in population decline or extinction. DNA sequence polymorphisms representing alternate alleles at gRNA target sites may confer a drive-resistant phenotype in individuals carrying them. Modeling predicts that, for observed levels of SGV at potential target sites and observed rates of de novo DRA formation, population modification strategies are uniquely resilient to DRAs. We conclude that gene drives can succeed when fitness costs incurred by drive-carrying mosquitoes are low enough to prevent strong positive selection for DRAs produced de novo or as part of the SGV and that population modification strategies are less prone to failure due to drive resistance.

Genome-wide divergence among invasive populations of Aedes aegypti in California.

BACKGROUND: In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions. RESULTS: Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. Based on 4.65 million high quality biallelic SNPs, we identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California. CONCLUSIONS: A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.

The Genetic Basis of Host Preference and Resting Behavior in the Major African Malaria Vector, Anopheles arabiensis.

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.

The fate of genes that cross species boundaries after a major hybridization event in a natural mosquito population.

Animal species are able to acquire new genetic material via hybridization and subsequent introgression. However, little is known about how foreign genomic material is incorporated into a population over time and what genes are susceptible to introgression. Here, we follow the closely related mosquito sister species Anopheles coluzzii and Anopheles gambiae in a sympatric natural population in Mali at multiple time points spanning a period of 25 years. During this period, we observed the temporary breakdown of mating barriers, which allowed us to explore the fate of alleles that crossed the species boundary in a natural population. Whole genome sequencing of 74 individuals revealed introgression within only 34 genes (0.26% of total genes) from A. gambiae to A. coluzzii, the majority contained within a 4 Mb region on the 2L chromosome which includes the insecticide resistance gene (AGAP004707). We designed a genotyping assay to follow 25 of the 34 introgressed alleles over time and found that all A. gambiae alleles, except four, reached a frequency of 50% in the A. coluzzii population within 4 years (~50 generations) and increased to ~80% within 6 years (~75 generations). However, the frequency of all introgressed alleles, except three, decreased to ~60% in 2016. This suggests an ongoing process of purifying selection in the population against DNA of foreign ancestry, except for alleles that are under positive selection, resulting in a complex genomic landscape. This study shows that stable introgression is limited to only specific genes even within closely related species.

Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets.

Animal species adapt to changes in their environment, including man-made changes such as the introduction of insecticides, through selection for advantageous genes already present in populations or newly arisen through mutation. A possible alternative mechanism is the acquisition of adaptive genes from related species via a process known as adaptive introgression. Differing levels of insecticide resistance between two African malaria vectors, Anopheles coluzzii and Anopheles gambiae, have been attributed to assortative mating between the two species. In a previous study, we reported two bouts of hybridization observed in the town of Selinkenyi, Mali in 2002 and 2006. These hybridization events did not appear to be directly associated with insecticide-resistance genes. We demonstrate that during a brief breakdown in assortative mating in 2006, A. coluzzii inherited the entire A. gambiae-associated 2L divergence island, which includes a suite of insecticide-resistance alleles. In this case, introgression was coincident with the start of a major insecticide-treated bed net distribution campaign in Mali. This suggests that insecticide exposure altered the fitness landscape, favoring the survival of A. coluzzii/A. gambiae hybrids, and provided selection pressure that swept the 2L divergence island through A. coluzzii populations in Mali. We propose that the work described herein presents a unique description of the temporal dynamics of adaptive introgression in an animal species and represents a mechanism for the rapid evolution of insecticide resistance in this important vector of human malaria in Africa.

Spatiotemporal dynamics of gene flow and hybrid fitness between the M and S forms of the malaria mosquito, Anopheles gambiae.

The M and S forms of Anopheles gambiae have been the focus of intense study by malaria researchers and evolutionary biologists interested in ecological speciation. Divergence occurs at three discrete islands in genomes that are otherwise nearly identical. An "islands of speciation" model proposes that diverged regions contain genes that are maintained by selection in the face of gene flow. An alternative "incidental island" model maintains that gene flow between M and S is effectively zero and that divergence islands are unrelated to speciation. A "divergence island SNP" assay was used to explore the spatial and temporal distributions of hybrid genotypes. Results revealed that hybrid individuals occur at frequencies ranging between 5% and 97% in every population examined. A temporal analysis revealed that assortative mating is unstable and periodically breaks down, resulting in extensive hybridization. Results suggest that hybrids suffer a fitness disadvantage, but at least some hybrid genotypes are viable. Stable introgression of the 2L speciation island occurred at one site following a hybridization event.

Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali.

In certain cases, a species may have access to important genetic variation present in a related species via adaptive introgression. These novel alleles may interact with their new genetic background, resulting in unexpected phenotypes. In this study, we describe a selective sweep on standing variation on the X chromosome in the mosquito Anopheles coluzzii, a principal malaria vector in West Africa. This event may have been influenced by the recent adaptive introgression of the insecticide resistance gene known as kdr from the sister species Anopheles gambiae. Individuals carrying both kdr and a nearly fixed X-linked haplotype, encompassing at least four genes including the P450 gene CYP9K1 and the cuticular protein CPR125, have rapidly increased in relative frequency. In parallel, a reproductively isolated insecticide-susceptible A. gambiae population (Bamako form) has been driven to local extinction, likely due to strong selection from increased insecticide-treated bed net usage.

Defining Genetic, Taxonomic, and Geographic Boundaries Among Species of the Psorophora confinnis (Diptera: Culicidae) Complex in North and South America.

The Psorophora confinnis complex is currently composed of three species--Psorophora confinnis sensu stricto (Lynch Arribalzaga) in South America, Psorophora columbiae (Dyar and Knab) in North America, and Psorophora jamaicensis (Theobald) in the Caribbean. Members of the complex are of considerable importance as vectors of arboviruses, for example, Venezuelan equine encephalitis virus, and are significant biting pests throughout their range. The biological and geographic boundaries of Ps. confinnis and Ps. columbiae are unclear. In fact, the name Ps. columbiae is presently designated as "provisional." In this article, we aim to clarify the taxonomy and geographic distributions of species within the Ps. confinnis complex. A population genetics approach was employed using gene and genotypic frequency data at 26 isozyme loci. The results suggest that the Ps. confinnis complex in North and South America is composed of four species. Ps. confinnis s.s. and Ps. columbiae are distinct species in South and North America, respectively. Populations in Colombia, South America, formally designated as Ps. funiculus (Dyar) and populations in the southwestern United States and western Mexico, formally designated Ps. toltecum (Dyar and Knab), are distinct species. Psorophora toltecum and Psorophora funiculus species names should be resurrected from synonymy. In addition we identified a Ps. columbiae and Ps. toltecum hybrid zone in central Texas in a region described as being one of 13 North American suture zones, being geographical areas in which closely related species occur in sympatry and frequently hybridize.

The first genome sequence of Anopheles squamous from Macha, Zambia.

Despite efforts to minimize the impacts of malaria and reduce the number of primary vectors, malaria has yet to be eliminated in Zambia. Understudied vector species may perpetuate malaria transmission in pre-elimination settings. Anopheles squamosus is one of the most abundantly caught mosquito species in southern Zambia and has previously been found with Plasmodium falciparum sporozoites, a causal agent of human malaria. This species may be a critical vector of malaria transmission, however, there is a lack of genetic information available for An. squamosus. We report the first genome data and the first complete mitogenome (Mt) sequence of An. squamosus. The sequence was extracted from one individual mosquito from the Chidakwa area in Macha, Zambia. The raw reads were obtained using Illumina Novaseq 6000 and assembled through NOVOplasty alignment with related species. The length of the An. squamosus Mt was 15,351 bp, with 77.9 % AT content. The closest match to the whole mitochondrial genome in the phylogenetic tree is the African malaria mosquito, Anopheles gambiae. Its genome data is available through National Center for Biotechnology Information (NCBI) Sequencing Reads Archive (SRA) with accession number SRR22114392. The mitochondrial genome was deposited in NCBI GenBank with the accession number OP776919. The ITS2 containing contig sequence was deposited in GenBank with the accession number OQ241725. Mitogenome annotation and a phylogenetic tree with related Anopheles mosquito species are provided.

Complete mitogenome sequence of Aedes (Hulecoeteomyia) japonicus japonicus from Hawai'i Island.

We report the first complete mitogenome (Mt) sequence of Aedes japonicus japonicus (Diptera: Culicidae). The sequence was extracted from one adult from the Big Island of Hawai'i Island. The length of the Ae. japonicus japonicus Mt was 16,528bp with 78.1% AT content. Its sequence is most similar to the Mt sequence of Aedes koreicus with 90.81% sequence identity. This is the first full Mt sequence available for this species and provides important genetic resource for studying population genetics and dynamics of this important invasive mosquito species.

The origin of island populations of the African malaria mosquito, Anopheles coluzzii.

Anopheles coluzzii is a major malaria vector throughout its distribution in west-central Africa. Here we present a whole-genome study of 142 specimens from nine countries in continental Africa and three islands in the Gulf of Guinea. This sample set covers a large part of this species' geographic range. Our population genomic analyses included a description of the structure of mainland populations, island populations, and connectivity between them. Three genetic clusters are identified among mainland populations and genetic distances (FST) fits an isolation-by-distance model. Genomic analyses are applied to estimate the demographic history and ancestry for each island. Taken together with the unique biogeography and history of human occupation for each island, they present a coherent explanation underlying levels of genetic isolation between mainland and island populations. We discuss the relationship of our findings to the suitability of São Tomé and Príncipe islands as candidate sites for potential field trials of genetic-based malaria control strategies.

Acoustic localization of antbirds in a Mexican rainforest using a wireless sensor network.

Acoustic localization is a promising method to passively observe vocal animal species, but remains difficult and time consuming to employ. To reduce the labor intensity and impact of deployment, an acoustic localization system has been developed consisting of battery powered wireless sensor nodes. The system also has the ability to perform an acoustic self-survey, which compares favorably in accuracy to global positioning system survey methods, especially in environments such as forest. The self-survey and localization accuracy of the system was tested in the neotropical rainforest of Chiapas, Mexico. A straight-forward and robust correlation sum localization computation method was utilized and is described in detail. Both free-ranging wild antbird songs and songs played from a speaker were localized with mean errors of 0.199 m and 0.445 m, respectively. Finally, additional tests utilizing only a short segment of each song or a subset of sensor nodes were performed and found to minimally affect localization accuracy. The use of a wireless sensor network for acoustic localization of animal vocalizations offers greater ease and flexibility of deployment than wired microphone arrays without sacrificing accuracy.

Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.

A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.

Evidence for Divergent Selection on Immune Genes between the African Malaria Vectors, Anopheles coluzzii and A. gambiae.

During their life cycles, microbes infecting mosquitoes encounter components of the mosquito anti-microbial innate immune defenses. Many of these immune responses also mediate susceptibility to malaria parasite infection. In West Africa, the primary malaria vectors are Anopheles coluzzii and A. gambiae sensu stricto, which is subdivided into the Bamako and Savanna sub-taxa. Here, we performed whole genome comparisons of the three taxa as well as genotyping of 333 putatively functional SNPs located in 58 immune signaling genes. Genome data support significantly higher differentiation in immune genes compared with a randomly selected set of non-immune genes among the three taxa (permutation test p < 0.001). Among the 58 genes studied, the majority had one or more segregating mutations (72.9%) that were significantly diverged among the three taxa. Genes detected to be under selection include MAP2K4 and Raf. Despite the genome-wide distribution of immune genes, a high level of linkage disequilibrium (r2 > 0.8) was detected in over 27% of SNP pairs. We discuss the potential role of immune gene divergence as adaptations to the different larval habitats associated with A. gambiae taxa and as a potential force driving ecological speciation in this group of mosquitoes.

Transcontinental dispersal of Anopheles gambiae occurred from West African origin via serial founder events.

The mosquito Anopheles gambiae s.s. is distributed across most of sub-Saharan Africa and is of major scientific and public health interest for being an African malaria vector. Here we present population genomic analyses of 111 specimens sampled from west to east Africa, including the first whole genome sequences from oceanic islands, the Comoros. Genetic distances between populations of A. gambiae are discordant with geographic distances but are consistent with a stepwise migration scenario in which the species increases its range from west to east Africa through consecutive founder events over the last ~200,000 years. Geological barriers like the Congo River basin and the East African rift seem to play an important role in shaping this process. Moreover, we find a high degree of genetic isolation of populations on the Comoros, confirming the potential of these islands as candidate sites for potential field trials of genetically engineered mosquitoes for malaria control.

Characterization of the complete mitogenome of Anopheles aquasalis, and phylogenetic divergences among Anopheles from diverse geographic zones.

Whole mitogenome sequences (mtDNA) have been exploited for insect ecology studies, using them as molecular markers to reconstruct phylogenies, or to infer phylogeographic relationships and gene flow. Recent Anopheles phylogenomic studies have provided information regarding the time of deep lineage divergences within the genus. Here we report the complete 15,393 bp mtDNA sequences of Anopheles aquasalis, a Neotropical human malaria vector. When comparing its structure and base composition with other relevant and available anopheline mitogenomes, high similarity and conserved genomic features were observed. Furthermore, 22 mtDNA sequences comprising anopheline and Dipteran sibling species were analyzed to reconstruct phylogenies and estimate dates of divergence between taxa. Phylogenetic analysis using complete mtDNA sequences suggests that A. aquasalis diverged from the Anopheles albitarsis complex ~28 million years ago (MYA), and ~38 MYA from Anopheles darlingi. Bayesian analysis suggests that the most recent ancestor of Nyssorhynchus and Anopheles + Cellia was extant ~83 MYA, corroborating current estimates of ~79-100 MYA. Additional sampling and publication of African, Asian, and North American anopheline mitogenomes would improve the resolution of the Anopheles phylogeny and clarify early continental dispersal routes.

Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division.

Here we report the complete mitochondrial sequences of 70 individual field collected mosquito specimens from throughout Sub-Saharan Africa. We generated this dataset to identify species specific markers for the following Anopheles species and chromosomal forms: An. arabiensis, An. coluzzii (The Forest and Mopti chromosomal forms) and An. gambiae (The Bamako and Savannah chromosomal forms).  The raw Illumina sequencing reads were mapped to the NC_002084 reference mitogenome sequence. A total of 783 single nucleotide polymorphisms (SNPs) were detected on the mitochondrial genome, of which 460 are singletons (58.7%). None of these SNPs are suitable as molecular markers to distinguish among An. arabiensis, An. coluzzii and An. gambiae or any of the chromosomal forms. The lack of species or chromosomal form specific markers is also reflected in the constructed phylogenetic tree, which shows no clear division among the operational taxonomic units considered here.

A DNA extraction protocol for improved DNA yield from individual mosquitoes.

Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people's own DNA extraction protocols and not as an advertisement of a commercially available product.

Chromosome inversions, genomic differentiation and speciation in the African malaria mosquito Anopheles gambiae.

The African malaria vector, Anopheles gambiae, is characterized by multiple polymorphic chromosomal inversions and has become widely studied as a system for exploring models of speciation. Near complete reproductive isolation between different inversion types, known as chromosomal forms, has led to the suggestion that A. gambiae is in early stages of speciation, with divergence evolving in the face of considerable gene flow. We compared the standard chromosomal arrangement (Savanna form) with genomes homozygous for j, b, c, and u inversions (Bamako form) in order to identify regions of genomic divergence with respect to inversion polymorphism. We found levels of divergence between the two sub-taxa within some of these inversions (2Rj and 2Rb), but at a level lower than expected and confined near the inversion breakpoints, consistent with a gene flux model. Unexpectedly, we found that the majority of diverged regions were located on the X chromosome, which contained half of all significantly diverged regions, with much of this divergence located within exons. This is surprising given that the Bamako and Savanna chromosomal forms are both within the S molecular form that is defined by a locus near centromere of X chromosome. Two X-linked genes (a heat shock protein and P450 encoding genes) involved in reproductive isolation between the M and S molecular forms of A. gambiae were also significantly diverged between the two chromosomal forms. These results suggest that genes mediating reproductive isolation are likely located on the X chromosome, as is thought to be the case for the M and S molecular forms. We conclude that genes located on the sex chromosome may be the major force driving speciation between these chromosomal forms of A. gambiae.