Eastern Equine Encephalitis (EEE) (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne arbovirus that circulates in an enzootic cycle involving Culiseta melanura mosquitoes and wild Passeriformes birds in freshwater swamp habitats. Recently, the northeastern United States has experienced an intensification of virus activity with increased human involvement and northward expansion into new regions. In addition to its principal role in enzootic transmission of EEE virus among avian hosts, recent studies on the blood-feeding behavior of Cs. melanura throughout its geographic range suggest that this mosquito may also be involved in epizootic / epidemic transmission to equines and humans in certain locales. Variations in blood feeding behavior may be a function of host availability, environmental factors, and/or underlying genetic differences among regional populations. Despite the importance of Cs. melanura in transmission and maintenance of EEE virus, the genetics of this species remains largely unexplored. To investigate the occurrence of genetic variation in Cs. melanura, the genome of this mosquito vector was sequenced resulting in a draft genome assembly of 1. 28 gigabases with a contig N50 of 93. 36 kilobases. Populations of Cs. melanura from 10 EEE virus foci in the eastern North America were genotyped with double-digest RAD-seq. Following alignment of reads to the reference genome, variant calling, and filtering, 40,384 SNPs were retained for downstream analyses. Subsequent analyses revealed genetic differentiation between northern and southern populations of this mosquito species. Moreover, limited fine-scale population structure was detected throughout northeastern North America, suggesting local differentiation of populations but also a history of ancestral polymorphism or contemporary gene flow. Additionally, a genetically distinct cluster was identified predominantly at two northern sites. This study elucidates the first evidence of fine-scale population structure in Cs. melanura throughout its eastern range and detects evidence of gene flow between populations in northeastern North America. This investigation provides the groundwork for examining the consequences of genetic variations in the populations of this mosquito species that could influence vector-host interactions and the risk of human and equine infection with EEE virus.
Eastern equine encephalitis (EEE) virus (Alphavirus, Togaviridae) causes severe disease in humans and equines with high case mortality and persistent neurologic impairment in survivors [1]. Historically, outbreaks of this relatively rare but highly pathogenic arthropod-borne virus occurred intermittently in the eastern United States, predominantly in the mid-Atlantic and Gulf Coast states such as Florida, as well as in isolated foci in the northeast [2–6]. However, since the early 21st century, this region has experienced a recurring seasonal intensification of EEE virus activity [5,7], and a northward geographic expansion [8–11]. In the northeastern United States, EEE virus is maintained in an enzootic transmission cycle in freshwater swamp foci involving the ornithophilic mosquito Culiseta melanura (Coquillett) (Diptera: Culicidae) and passerine birds [12–15]. Human and equine disease cases occur predominantly in close proximity to the freshwater swamp habitats in which Cs. melanura breeds [2,12]. Culiseta melanura is distributed throughout eastern North America [16] and is widely considered the principal enzootic vector of EEE virus. This mosquito species exhibits considerable variability in avian host choice across geographic regions, favoring Passeriformes birds [6,15,17–20]. In addition to avian hosts, recent studies indicate that between 1 and 11% of Cs. melanura bloodmeals originate from mammalian hosts including humans [6,17–20], suggesting the involvement of this mosquito species in epidemic/epizootic transmission of EEE virus to humans and equines [17,21]. Moreover, in a survey of thirty-five mosquito species from the northeastern United States, Cs. melanura was the only species with the consistently high titers needed for transmission of the virus [21], and this species is among the predominant sources of virus isolations from field-collected mosquitoes in this region [21]. The apparent flexibility in host choice exhibited by Cs. melanura in various geographic regions [6,15,17–19] may be a function of environmental factors and/or underlying genetic variation that could influence vector-host interactions and potentially vectorial capacity. Despite the importance of Cs. melanura in transmission and maintenance of EEE virus, the genetics of this species remain largely unexplored. The present study was undertaken to gain insight into possible genetic variation in Cs. melanura populations that may contribute to its involvement in epidemic as well as enzootic transmission of EEE virus in certain locales. The specific objectives of the study were to: 1) characterize the genetic diversity of Cs. melanura in EEE virus foci across eastern North America, 2) investigate the occurrence of genetic structure among populations of Cs. melanura, and 3) examine patterns of gene flow among these populations.
Sequencing of forty SMRT Cells yielded a total of 4. 2 million raw reads with an average read length of 9 kilobases (kb). These raw reads were assembled to produce an unpolished draft assembly of 1. 278 gigabases (Gb) across 25,500 contigs, with a contig N50 of 93. 23 kb. Subsequent polishing provided modest increase to assembly size and contig N50. The Quiver-polished assembly was 1. 279 Gb, with a contig N50 of 93. 29 kb, while the Arrow-polished assembly was a total of 1. 281 Gb, with a contig N50 of 93. 36 kb. Although polishing provided only minor improvements to assembly size and N50, improvement to the results of BUSCO analyses were more substantial. The unpolished genome assembly had evidence of 61. 3% of expected complete single copy orthologs, with 22. 4% missing entirely. More than 70% of complete single copy orthologs were detected in both polished genomes, with the Arrow-polished assembly performing the best in BUSCO analyses; BUSCO results for this assembly found more than 75% of expected single copy orthologs, and in total, at least partial evidence for 88. 7% of expected single copy orthologs (Full BUSCO Results: Complete, Single Copy: 75. 4%; Complete, Duplicate: 4. 5%; Fragmented: 8. 8%; Missing: 11. 3%; Fig B in S1 File). This Arrow-polished assembly performed similarity to other publicly available genomes on VectorBase (Fig 3). Polymerase chain reaction duplicates comprised 5. 29% (SD = 4. 0%) of reads across libraries. Of these, 71. 9% (SD = 10. 7%) were single duplications, although some reads were duplicated hundreds of times (Fig C in S1 File). Following removal of PCR duplicates and quality filtering, libraries resulted in 16. 4 (SD = 4. 3) million reads per mosquito, which aligned to the draft reference genome at a mean rate of 93. 2% (SD = 3. 8%). One mosquito from Canada had only 337,113 total reads and was removed from all downstream analyses, resulting in a total of 119 individuals from 10 geographic locations. Mean read depth across loci was 20. 4X (SD = 5. 8X) for the reference-aligned dataset, and 14. 9X (SD = 4. 3X) when Stacks was used for de novo assembly of loci (Table B in S1 File). In total, Stacks identified 3. 9 million variant loci across all samples for the reference-aligned dataset, and 8. 7 million variant loci for the de novo dataset. Datasets varied in number of SNPs and genotyping rates (Table 2). Datasets in which p was set contained the largest number of SNPs, with more overall SNPs recovered as p declined, but with a lower genotyping rate. Fewer SNPs were recovered with Stacks’ de novo assembly of loci. The results presented below are for the variants called from the reference-aligned dataset, and without a specification of the population parameter p, which contained a total of 40,384 SNPs following the filtering of minor alleles and linked loci; specification of the population parameter to different values resulted in qualitatively similar results in downstream analyses, as did the de novo-assembled dataset (see Supporting Information for examples).
To facilitate the study of genetic variation in Cs. melanura, a draft genome was generated. This genome is comparable in presence of single copy orthologs to many other currently available assemblies of Dipteran vector genomes. The relatively large size of the Cs. melanura draft assembly, at over 1. 24 Gb, is consistent with a previous estimate of the genome size of this species based on cytophotometry at 1. 2 Gb [24]. Moreover, this is also consistent with the genome sizes of other mosquitoes in the subfamily Culicinae [24,60,61], such as Aedes aegypti where current genome assembly, AaegL5, is just under 1. 28 Gb [28,61]. By comparison, the vectors of human malaria in the genus Anopheles have significantly smaller genomes: The current Anopheles gambiae assembly, AgamP4, is under 0. 27 Gb. The draft assembly presented here was constructed principally as a reference for use in population studies of Cs. melanura, and further investigations are underway to improve the genome through additional sequencing and transcriptomics, a necessary first step to annotating the genome beyond the well-curated catalog of Dipteran single copy orthologs used in BUSCO analyses. As Cs. melanura is only the fourth non-Anopheline mosquito genome to be sequenced [60–62], further development of this genome could provide important insight into the ecology, evolution, and control of this and other vector species, as the availability of genomic data for vector species has enabled the characterization of pathways important to host seeking behavior (e. g. odorant receptors [63,64]), description of components of mosquito immunity [65–67], and the design of novel vector control methods [68–70]. The draft genome assembled in this study is a useful tool for population studies, as demonstrated here. Reads aligned to the reference at a mean rate greater than 93%, indicating the relative completeness as a reference. Utilization of the draft genome allowed the capture of a larger number of variants than under de novo variant calling with Stacks (Table 2), and with significantly less computation time. Thus, the draft assembly holds promise as a tool in future population studies, either as a reference for additional genotyping-by-sequencing, or for discovery of microsatellite markers, a low-cost alternative for the processing of larger sample sizes. The SNP dataset in this study recovered more than forty thousand markers passing filtering that were present in at least 75% of individuals. This dataset enabled the fine-scale differentiation of Cs. melanura populations (see Results, and below), despite the relatively small sample size. This is consistent with previous studies with empirical [71] and simulated [72] SNP data sets indicating that even sample sizes between two and ten [71–73] individuals per population can recover small degrees of significant genetic differentiation among populations, as long as at least 1500 SNPs are used [71]. Thus, the sample sizes used in this study should be adequate to evaluate even fine-scale genetic differentiation given the recovery of more than forty thousand SNPs. Moreover, this dataset provides an important framework for future studies that could expand on the sampling here, as RAD-seq datasets can be combined across laboratories with a high overlap of markers recovered, assuming identical library construction methods [74]. This study identified both regional differences between northern and southern populations of Cs. melanura, as well as fine scale population structure in this species. Pairwise Fst, AMOVA analyses, SNMF clusters, and PCA results all supported that populations within geographic regions of north (1-CAN, 2-ME, 3-NH, 4-VT, 5-NY, 6-MA, 7-CT) and south (9-VA and10-FL) were more akin to one another than to populations in other regions (Fig 4, Tables D and E in S1 File). These results suggest a degree of genetic differentiation at a geographic scale, although future studies are needed to confirm this differentiation and investigate its consequences with additional sampling, particularly in the southern United States, where our sampling was confined to two locations. FineRADstructure identified finer-scale population structure than did the estimation of individual ancestry coefficients via the least squares-based method SNMF, consistent with results in other systems (e. g. with human SNP data [48]), as well as with simulations [47,48], indicating that the MCMC algorithm used by fineSTRUCTURE and fineRADstructure can be more sensitive than other clustering algorithms. The fine-scale population structure detected by fineRADstructure is consistent with the significant molecular variation and pairwise Fst values among populations, as well as the ability of DAPC to assign most individuals back to their correct population. Although fineRADstructure was unable to cluster individuals from three populations into single population clusters (3-NH, 4-VT, 7-CT), DAPC successfully assigned many of these individuals to their respective populations at a rate similar to several other northern sites, suggesting underlying genetic variation differentiating these populations. The genetic variation among populations identified here could, among other factors, influence behavioral characteristics exhibited by Cs. melanura such as heterogeneities in host choice across regions [6,15,18,19], but correlating the fine-scale population structure reported here with previously observed host feeding patterns requires further investigations. Of note, human-derived blood meals from Cs. melanura have been identified in populations across the eastern United States, including from mosquitoes trapped near several locations utilized in this study, albeit at low frequencies (e. g. near 4-VT [17], 6-MA [19], 8-NJ [75], and 10-FL [18]). However, these populations belong to multiple genetic clusters identified in the present study, indicating that a propensity to feed on humans might be widespread across genetic clusters. Given the importance of this mosquito vector, the influence of population structure warrants further investigation, such as through the simultaneous sampling of engorged mosquitoes and SNP data across populations. Moreover, our results are consistent with a post glacial recolonization of the northeast from southern populations following the retreat of the Laurentide ice sheet twelve to eighteen thousand years ago [76,77], and the melting of permafrost from the mid-Atlantic at this time period (e. g. near southern New Jersey [78]). The genetic cluster associated with southern populations (9-VA and 10-FL in Cluster B, Figs 4 and 5) is basal to Clusters C and D containing northern populations (1-CAN, 2-ME, 3-NH, 4-VT, 5-NY, 6-MA, 7-CT) and the mid-Atlantic population (8-NJ) in the MAP tree (Fig 5). Although the MAP tree is only an approximate guide to relatedness of populations [47,48], MAP trees perform well in simulations at inferring ancestral history between populations [48]. Moreover, southern populations (9-VA, 10-FL) had more private alleles than northern populations (1-CAN, 2-ME, 3-NH, 4-VT, 5-NY, 6-MA, 7-CT), indicating a higher genetic diversity (Table 3). The decline in private alleles south to north, as well as limited genetic variation among northern populations (e. g. the limited population structure, low pairwise Fst values, and lower number of private alleles), would be consistent with colonization northward and subsequent loss of genetic diversity through founder effects and/or bottlenecking events, the occurrence of which could be investigated in future studies. Thus, genomic evidence is consistent with the northward expansion of Cs. melanura from southern source populations. To confirm the pattern observed here, future studies with additional sampling throughout the range of this mosquito species could infer evolutionary history of these populations through Bayesian analyses estimating divergence time and biogeographic/demographic history [79,80]. Substantial evidence of shared ancestry among populations was detected, likely indicating the presence of gene flow and/or shared ancestral polymorphism, particularly at the regional scales of northern (1-CAN, 2-ME, 3-NH, 4-VT, 5-NY, 6-MA, 7-CT) and southern (9-VA, 10-FL) populations. A lack of isolation by distance (Table C in S1 File), as well as lower pairwise Fst values among populations within a region compared to pairwise Fst values between northern and southern regions suggest not only a degree of differentiation between regions but could also indicate contemporary gene flow or a history of connectivity among populations [81,82]. At least one individual from northern sampling sites (1-CAN, 2-ME, 3-NH, 4-VT, 5-NY, 6-MA, 7-CT) had high ancestry with a different genetic cluster from the majority of those mosquitoes sampled at the same site, suggesting a degree of gene flow among genetic clusters (Fig 4). Moreover, the coancestry matrix and associated MAP tree provides additional evidence of gene flow among populations, as several individuals had higher coancestry with alternative genetic clusters and geographically distant populations in the north compared with other individuals from the same sampling location (e. g. two individuals from 1-CAN with higher coancestry to mosquitoes from 5-NY in Cluster C, Fig 5). It is unlikely that this signal of gene flow is the result of direct dispersal among populations sampled in this study, as the mean flight range of Cs. melanura, estimated via mark and recapture, ranged from 4 to 9 kilometers [83]. Instead, these results suggest Cs. melanura in swamp habitats are not isolated at a regional scale, and there exists a large degree of connectivity among populations in northeastern North America. In addition to the aforementioned fine-scale population structure, an unexpected genetic cluster was identified (Fig 4, Figs D, F, G in S1 File, Table E in S1 File). This cluster was comprised primarily of a small number of individuals from 2-ME and 3-NH; however, it also contributed substantial ancestry to individuals in other northern populations (particularly 1-CAN, 5-NY; Fig E in S1 File). As 2-ME and 3-NH were sequenced in separate sequencing lanes (Table 1) and had similar coverage and rates of alignment to the reference genome (Table B in S1 File) it seems unlikely this cluster is a sequencing artifact. Subsequent sequencing of ITS2 confirmed previous morphological identification that individuals belonging to Cluster A were indeed Cs. melanura, as pairwise identity of ITS2 sequences was >99% between individuals belonging to this cluster and all other Cs. melanura sequenced. Although the similarity in pairwise ITS2 sequence identity between members of Cluster A and other Cs. melanura may seem contrary to genomic results, this discrepancy is likely due to the greater number of variable sites in the SNP dataset, compared to only 297 nucleotide positions in the ITS2 alignment. Mosquitoes belonging to Cluster A were relatively differentiated compared to mosquitoes belonging to other genetic clusters, with pairwise Fst values greatly exceeding those among other populations (Table E in S1 File). In addition, this cluster had far more private alleles than any population (Table 3). These results are consistent with evidence of an influx of genetic variation from a source not sampled in this study, such as admixture between northern populations of Cs. melanura and an unsampled and highly diverged population of this species, or interspecific hybridization with another closely related mosquito. However, due to the small number of individuals in this cluster, the lack of any previous evidence of hybridization between this species and others, and the paucity of sequencing information for other Culiseta species, it is not feasible with the available data to assess hypotheses regarding this cluster. Further studies investigating northeastern North American Cs. melanura populations with a larger sampling of mosquitoes, particularly targeting populations near 2-ME and 3-NH, may elucidate the origins of this cluster and any potential impacts it may have on behavioral characteristics of this mosquito species e. g. host feeding pattern and the risk of human infection with EEE virus. The results presented here provide the first insight into genetic variation in Cs. melanura. Although the existence of fine-scale structure and gene flow among populations was detected in these analyses, correlation between our findings and observed human and equine disease risk is difficult at this time. Future studies integrating sampling of populations along with mosquito bloodmeal analysis and observations of host diversity and abundance would be more informative to answering whether the genetic structure identified here is correlated with heterogeneities in blood host choice and/or disease risk across the region. By utilizing the new genomic resources available from this study, future investigations may build on the framework this study represents and provide greater insight into the vector-host interactions in Cs. melanura and its associated disease risk to humans and equines.
Eastern equine encephalitis (EEE) is a highly pathogenic mosquito-borne virus responsible for outbreaks of severe disease in humans and equines, causing high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern United States occurred intermittently; however, recently, this region has experienced a recurring seasonal intensification of EEE virus activity with expansion into more northerly locales. Eastern equine encephalitis virus is maintained in a transmission cycle involving the ornithophilic mosquito, Culiseta melanura, and wild passerine birds in freshwater swamp foci. Recent studies on the blood feeding behavior of Cs. melanura suggest this mosquito species could also be involved in transmission of EEE virus to humans and equines. Since variation in host feeding of this mosquito vector may be a function of environmental factors and/or genetic differences among regional populations, we examined the population genetics of Cs. melanura in order to: 1) characterize the genetic diversity of Cs. melanura at EEE virus foci across eastern North America, 2) investigate the occurrence of genetic structure among populations of Cs. melanura, and 3) examine patterns of gene flow among these populations. We generated a draft genome of this species as a reference for population studies, identified evidence of significant genetic differentiation and fine-scale genetic structure among populations, and found evidence of gene flow among northeastern populations. This study provides the molecular basis for future investigations on the causes and consequences of the genomic variation in Cs. melanura on the risk of human and equine infection with EEE virus.
invertebrates
medicine and health sciences
population genetics
genomic library construction
animals
invertebrate genomics
genome analysis
dna construction
molecular biology techniques
population biology
insect vectors
research and analysis methods
infectious diseases
genetic polymorphism
comparative genomics
molecular biology
disease vectors
insects
animal genomics
arthropoda
mosquitoes
eukaryota
dna library construction
heredity
genetics
biology and life sciences
species interactions
genomics
evolutionary biology
gene flow
computational biology
organisms
Population genomics of Culiseta melanura, the principal vector of Eastern equine encephalitis virus in the United States