Whole-genome assembly of Culex tarsalis.

The mosquito, Culex tarsalis, is a key vector in the western United States due to its role in transmission of zoonotic arboviruses that affect human health. Extensive research has been conducted on Cx. tarsalis ecology, feeding behavior, vector competence, autogeny, diapause, genetics, and insecticide resistance. Population genetic analyses in the western U.S. have identified at least three genetic clusters that are geographically distinct. However, in-depth genetic studies have been hindered by the lack of a reference genome. In this study, we present the first whole-genome assembly of this mosquito species (CtarK1) based on PacBio HiFi reads from high-molecular-weight DNA extracted from a single male. The CtarK1 assembly is 790 Mb with an N50 of 58 kb, which is 27% larger than Culex quinquefasciatus (578 Mb). This difference appears to be mostly composed of transposable elements. To annotate CtarK1, we used a previously assembled Cx. tarsalis transcriptome and approximately 17,456 protein genes from Cx. quinquefasciatus (N = 17,456). Genome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool, which identified 84.8% of the 2799 Dipteran BUSCO genes. Using a Bayesian phylogeny based on mitochondrial genomes, we place Cx. tarsalis in the context of other mosquito species and estimate the divergence between Cx. tarsalis and Cx. quinquefasciatus to be between 15.8 and 22.2 million years ago (MYA). Important next steps from this work include characterizing the genetic basis of diapause and sex determination in Culex mosquitoes.

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.

Impact of temperature on the extrinsic incubation period of Zika virus in Aedes aegypti.

Since Zika virus (ZIKV) emerged as a global human health threat, numerous studies have pointed to Aedes aegypti as the primary vector due to its high competence and propensity to feed on humans. The majority of vector competence studies have been conducted between 26-28°C, but arboviral extrinsic incubation periods (EIPs), and therefore transmission efficiency, are known to be affected strongly by temperature. To better understand the relationship between ZIKV EIPs and temperature, we evaluated the effect of adult mosquito exposure temperature on ZIKV infection, dissemination, and transmission in Ae. aegypti at four temperatures: 18°C, 21°C, 26°C, and 30°C. Mosquitoes were exposed to viremic mice infected with a 2015 Puerto Rican ZIKV strain, and engorged mosquitoes were sorted into the four temperatures with 80% RH and constant access to 10% sucrose. ZIKV infection, dissemination, and transmission rates were assessed via RT-qPCR from individual mosquito bodies, legs and wings, and saliva, respectively, at three to five time points per temperature from three to 31 days, based on expectations from other flavivirus EIPs. The median time from ZIKV ingestion to transmission (median EIP, EIP50) at each temperature was estimated by fitting a generalized linear mixed model for each temperature. EIP50 ranged from 5.1 days at 30°C to 24.2 days at 21°C. At 26°C, EIP50 was 9.6 days. At 18°C, only 15% transmitted by day 31 so EIP50 could not be estimated. This is among the first studies to characterize the effects of temperature on ZIKV EIP in Ae. aegypti, and the first to do so based on feeding of mosquitoes on a live, viremic host. This information is critical for modeling ZIKV transmission dynamics to understand geographic and seasonal limits of ZIKV risk; it is especially relevant for determining risk in subtropical regions with established Ae. aegypti populations and relatively high rates of return travel from the tropics (e.g. California or Florida), as these regions typically experience cooler temperature ranges than tropical regions.

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.

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.

How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluate our multiplexed amplicon approach, PrimalSeq, to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We develop an experimental protocol and computational tool, iVar, for using PrimalSeq to measure virus diversity using Illumina and compare the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.

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.

Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus.

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher's exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Genetic variation associated with increased insecticide resistance in the malaria mosquito, Anopheles coluzzii.

BACKGROUND: Malaria mortality rates in sub-Saharan Africa have declined significantly in recent years as a result of increased insecticide-treated bed net (ITN) usage. A major challenge to further progress is the emergence and spread of insecticide resistance alleles in the Anopheles mosquito vectors, like An. coluzzii. A non-synonymous mutation in the para voltage-gated sodium channel gene reduces pyrethroid-binding affinity, resulting in knockdown resistance (kdr). Metabolic mechanisms of insecticide resistance involving detoxification genes like cytochrome P450 genes, carboxylesterases, and glutathione S-transferases are also important. As some gene activity is tissue-specific and/or environmentally induced, gene regulatory variation may be overlooked when comparing expression from whole mosquito bodies under standard rearing conditions. RESULTS: We detected complex insecticide resistance in a 2014 An. coluzzii colony from southern Mali using bottle bioassays. Additional bioassays involving recombinant genotypes from a cross with a relatively susceptible 1995 An. coluzzii colony from Mali confirmed the importance of kdr and associated increased permethrin resistance to the CYP9K1 locus on the X chromosome. Significant differential expression of CYP9K1 was not observed among these colonies in Malpighian tubules. However, the P450 gene CYP6Z1 was overexpressed in resistant individuals following sublethal permethrin exposure and the carboxylesterase gene COEAE5G was constitutively overexpressed. CONCLUSIONS: The significant P450-related insecticide resistance observed in the 2014 An. coluzzii colony indicates that ITNs treated with the P450 inhibitor piperonyl butoxide (PBO) would be more effective in this region. The known insecticide resistance gene CYP6Z1 was differentially expressed exclusively in the context of sublethal permethrin exposure, highlighting the importance of tissue-specificity and environmental conditions in gene expression studies. The increased activity of the carboxylesterase COEAE5G in the resistant An. coluzzii colony suggests resistance to other insecticides like organophosphates. Additional gene expression studies involving other tissues (e.g. fat body) would provide a more comprehensive view of genes underlying metabolic insecticide resistance in An. coluzzii from Mali. Identifying genetic markers linked to these regulatory alleles is an important next step that would substantially improve insecticide resistance surveillance and population genetic studies in this important vector species.

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.

Salinity Adaptation and the Contribution of Parental Environmental Effects in Medicago truncatula.

High soil salinity negatively influences plant growth and yield. Some taxa have evolved mechanisms for avoiding or tolerating elevated soil salinity, which can be modulated by the environment experienced by parents or offspring. We tested the contribution of the parental and offspring environments on salinity adaptation and their potential underlying mechanisms. In a two-generation greenhouse experiment, we factorially manipulated salinity concentrations for genotypes of Medicago truncatula that were originally collected from natural populations that differed in soil salinity. To compare population level adaptation to soil salinity and to test the potential mechanisms involved we measured two aspects of plant performance, reproduction and vegetative biomass, and phenological and physiological traits associated with salinity avoidance and tolerance. Saline-origin populations had greater biomass and reproduction under saline conditions than non-saline populations, consistent with local adaptation to saline soils. Additionally, parental environmental exposure to salt increased this difference in performance. In terms of environmental effects on mechanisms of salinity adaptation, parental exposure to salt spurred phenological differences that facilitated salt avoidance, while offspring exposure to salt resulted in traits associated with greater salt tolerance. Non-saline origin populations expressed traits associated with greater growth in the absence of salt while, for saline adapted populations, the ability to maintain greater performance in saline environments was also associated with lower growth potential in the absence of salt. Plastic responses induced by parental and offspring environments in phenology, leaf traits, and gas exchange contribute to salinity adaptation in M. truncatula. The ability of plants to tolerate environmental stress, such as high soil salinity, is likely modulated by a combination of parental effects and within-generation phenotypic plasticity, which are likely to vary in populations from contrasting environments.

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.

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.

GBshape: a genome browser database for DNA shape annotations.

Many regulatory mechanisms require a high degree of specificity in protein-DNA binding. Nucleotide sequence does not provide an answer to the question of why a protein binds only to a small subset of the many putative binding sites in the genome that share the same core motif. Whereas higher-order effects, such as chromatin accessibility, cooperativity and cofactors, have been described, DNA shape recently gained attention as another feature that fine-tunes the DNA binding specificities of some transcription factor families. Our Genome Browser for DNA shape annotations (GBshape; freely available at http://rohslab.cmb.usc.edu/GBshape/) provides minor groove width, propeller twist, roll, helix twist and hydroxyl radical cleavage predictions for the entire genomes of 94 organisms. Additional genomes can easily be added using the GBshape framework. GBshape can be used to visualize DNA shape annotations qualitatively in a genome browser track format, and to download quantitative values of DNA shape features as a function of genomic position at nucleotide resolution. As biological applications, we illustrate the periodicity of DNA shape features that are present in nucleosome-occupied sequences from human, fly and worm, and we demonstrate structural similarities between transcription start sites in the genomes of four Drosophila species.

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.

Transcription start site evolution in Drosophila.

Transcription start site (TSS) evolution remains largely undescribed in Drosophila, likely due to limited annotations in non-melanogaster species. In this study, we introduce a concise new method that selectively sequences from the 5'-end of mRNA and used it to identify TSS in four Drosophila species, including Drosophila melanogaster, D. simulans, D. sechellia, and D. pseudoobscura. For verification, we compared our results in D. melanogaster with known annotations, published 5'-rapid amplification of cDNA ends data, and with RNAseq from the same mRNA pool. Then, we paired 2,849 D. melanogaster TSS with its closest equivalent TSS in each species (likely to be its true ortholog) using the available multiple sequence alignments. Most of the D. melanogaster TSSs were successfully paired with an ortholog in each species (83%, 86%, and 55% for D. simulans, D. sechellia, and D. pseudoobscura, respectively). On the basis of the number and distribution of reads mapped at each TSS, we also estimated promoter-specific expression (PSE) and TSS peak shape, respectively. Among paired TSS orthologs, the location and promoter activity were largely conserved. TSS location appears important as PSE, and TSS peak shape was more frequently divergent among TSS that had moved. Unpaired TSS were surprisingly common in D. pseudoobscura. An increased mutation rate upstream of TSS might explain this pattern. We found an enrichment of ribosomal protein genes among diverged TSS, suggesting that TSS evolution is not uniform across the genome.

Allele-specific expression assays using Solexa.

BACKGROUND: Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts. RESULTS: We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation. CONCLUSION: We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.

Regulatory divergence in Drosophila melanogaster and D. simulans, a genomewide analysis of allele-specific expression.

Species-specific regulation of gene expression contributes to the development and maintenance of reproductive isolation and to species differences in ecologically important traits. A better understanding of the evolutionary forces that shape regulatory variation and divergence can be developed by comparing expression differences among species and interspecific hybrids. Once expression differences are identified, the underlying genetics of regulatory variation or divergence can be explored. With the goal of associating cis and/or trans components of regulatory divergence with differences in gene expression, overall and allele-specific expression levels were assayed genomewide in female adult heads of Drosophila melanogaster, D. simulans, and their F1 hybrids. A greater proportion of cis differences than trans differences were identified for genes expressed in heads and, in accordance with previous studies, cis differences also explained a larger number of species differences in overall expression level. Regulatory divergence was found to be prevalent among genes associated with defense, olfaction, and among genes downstream of the Drosophila sex determination hierarchy. In addition, two genes, with critical roles in sex determination and micro RNA processing, Sxl and loqs, were identified as misexpressed in hybrid female heads, potentially contributing to hybrid incompatibility.