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Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting

['Luis E. Hernandez-Castro', 'Institute Of Biodiversity', 'Animal Health', 'Comparative Medicine', 'University Of Glasgow', 'Glasgow', 'United Kingdom', 'The Epidemiology', 'Economics', 'Risk Assessment Group']

Date: 2022-04

Abstract Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches.

Author summary Re-infestation of recently insecticide-treated houses by wild/secondary triatomine, their potential adaptation to this new environment and capabilities to geographically disperse across multiple human communities jeopardise sustainable Chagas disease control. This is the first study in Chagas disease vectors that identifies genomic regions possibly linked to adaptations to the built environment and describes landscape drivers for accurate prediction of geographic dispersal. We sampled multiple domestic and wild Rhodnius ecuadoriensis population pairs across a mountainous terrain in southern Ecuador. We evidenced that triatomine movement from forest to built enviroments does occur at a high rate. In these highly connected population pairs we detected loci possibly linked to local adaptation among the genomic makers we evaluated and in doing so we pave the way for future triatomine genomic research. We highlighted that current haphazardous vector control in the zone will be hindered by reinfestation of triatomines from the forest. Instead, we recommend frequent and spatially-targeted vector control and provided a landacape genomic model that identifies highly connected and isolated triatomine populations to facilitate efficient vector control.

Citation: Hernandez-Castro LE, Villacís AG, Jacobs A, Cheaib B, Day CC, Ocaña-Mayorga S, et al. (2022) Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting. PLoS Genet 18(2): e1010019. https://doi.org/10.1371/journal.pgen.1010019 Editor: Giorgio Sirugo, University of Pennsylvania, UNITED STATES Received: July 12, 2021; Accepted: January 6, 2022; Published: February 4, 2022 Copyright: © 2022 Hernandez-Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Rhodnius ecuadoriensis 2b-RAD raw sequence reads are stored in the Sequence Read Archive (SRA) repository accession number PRJNA797230 R code is available at the Github repository: github.com/lehernandezc/recuadoriensis. Funding: This work was possible thanks to the Mexican Council of Science and Technology (conacyt.mx/) doctorate scholarship (CVU Number 613766) awarded to LEHC., the National Institutes of Health (NIH - www.nih.gov/) grant number R15 AI105749-01A1 allocated to MJG who is PI, as well as together with MSL the UKRI (www.ukri.org/councils/) Engagement Network (EP/T003782/1) which supported co-author interactions. Funding was also received from Pontifical Catholic University of Ecuador (www.puce.edu.ec) to MJG (grant # C13025, E13027, E13037, H13174, I13048). ELL was supported by the National Institute of General Medical Sciences of the NIH (www.nih.gov), United States (Award Numbers P20GM130418). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The process by which insect vectors of human diseases adapt to survive and breed in human habitats is fundamental to the emergence and spread of vector-borne diseases (e.g., Aedes aegypti [1]). Relatively modest changes in vector host preference between ancestral (wild) and derived (domesticated) forms can drive devastating epidemics that result in millions of deaths [2]. Host preference variability in Culex pipiens of hybrid ancestry is thought to be genetically based and has contributed to local West Nile virus outbreaks in North America [3,4]. Similarly, host choice behaviour in Malaria mosquito Anopheles arabiensis has been linked to the allelic variation of a 3Ra chromosomal inversion [5]. Understanding the evolution and genetic bases of traits associated to the domestic habitat in disease vectors is, therefore, paramount and could inform control efforts and reveal the epidemic potential for new vector species [6,7]. Triatominae (Hemiptera: Reduviidae) are a group of hematophagous arthropods that transmit Trypanosoma cruzi, the parasite that causes Chagas disease, a fatal parasitic infection afflicting more than seven million people in Latin America [8]. Approximately 20 species are of public health concern due to their involvement in T. cruzi domestic transmission [9]. Eradication of ‘domesticated’ triatomines through insecticide spraying has been the mainstay of disease control in the past (e.g., Triatoma infestans [10], Rhodnius prolixus and Triatoma dimidiata [11]). However, wild (e.g., T. infestans [12] and R. prolixus [13]) and/or secondary competent species of triatomines (e.g., Triatoma sordida [14], Triatoma maculata and Rhodnius pallescens [15], Panstrongylus howardi [16] and P. chinai [17]) can continuously occupy empty domestic niches. Except from a few species that intrude houses seasonally (e.g., Triatoma dimidiata and other species in the Amazon basin [18,19]), constant triatomine house colonisation has historically jeopardised Chagas disease control strategies. Colonisation of the domestic niche may involve multiple, independent evolutionary processes across the geographic distribution of a given vector species [20,21], analogous to parallel trophic speciation observed in other arthropods [22]. Alternatively, domestication (hereafter, refers to the long-term evolutionary sense) of vectors with their associated zoonotic parasites may result from a single or limited number of independent colonisation events, followed by rapid and widespread dispersal within the domestic setting [23,24]. Domestication, and selection for domestic traits (e.g., pathogen resistance or efficient pollitators), in a given species may also represent a combination of these two scenarios, where multiple domesticated lineages serially introgress with wild lineages over evolutionary time, as has been elegantly demonstrated through analysis of the genomes of the Scutellata-European hybrid honey bees in America [25,26]. Disentangling these different scenarios in triatomine species, and their important implications for disease control, has been challenging due to a lack of genomic resources for these organisms which are only recently becoming available [27–29]. With adequate genomic tools; however, patterns of colonisation of the domestic niche can be established, and their underlying mechanisms unveiled. Models of ‘adaptation with gene flow’ (e.g., [30]) exploit standard population genetic metrics and theory to make generalisations about the genomic basis of adaptations (e.g., [22]). Such models can be deployed to study disease vector colonisation and reveal fundamental traits associated with the domestic niche. The genetic changes that allowed triatomines to thrive in the domestic niche may be related to feeding, reproduction and developmental performance. For instance, the development of potent saliva compounds that alter vertebrate host homeostatic, anti-inflamatory and immune responses was a crucial adaptation in triatomines for successful blood intake, and therefore, survival [31,32]. Saliva composition variation between domestic and wild populations has not been shown, yet saliva composition does play a role in highly ‘domesticated’ triatomines (e.g., R. prolixus and T. infestans) with exceptional feeding performace in humans [33,34]. Morphological changes such as reduced sexual dimorphism and body size have also been associated with the domestic habitat [35]. Egg development and viability are driven by neurohormonal signaling pathways starting soon after a female feeds on blood which results in yolk formation and supports embryonic development [36]. Under laboratory conditions, embryonic development of eggs collected inside houses was faster than those from the peridomicile [37]. Morphometric studies have attempted to develop phenotypic markers in triatomines associated with domestic or wild ecotopes with little (e.g., [38]) to moderate (e.g., [39]) success. Therefore, association of triatomine with the domestic niche is currently a qualitative concept with urgent need for quantitative foundations [40]. Identification of the ecological factors driving triatomine dispersal, with subsequent colonisation of a given niche, is necessary to predict complex triatomine population dynamics. High localised genetic structuring is expected in triatomine populations given their poor flying capabilities (< 2 Km), nymphs can only crawl short distances, and long-distance dispersal may sporadically occur via attachment to human cloths/bird feathers [41–43]. Models based on presence-only data have shown altitude, temperature, humidity, precipitation and vegetation as importat variables for triatomine distribution [44–46]. These models, however, represent broad spatial distribution rather than detailed local vector population dynamics and their accuracy requires extensive entomological records [47–49]. Instead, a landscape genomics framework (Fig 1) can accurately define landscape functional connectivity (the level at which the landscape heterogeneity facilitates or impedes a given organism’s movement from, and to, different habitat patches [50]) and shed light on the drivers of dispersal in a given vector species, and even assist in identifying poorly connected or isolated areas that can be easily targeted by eradication interventions [51–53]. Elevation may be a factor limiting R. ecuadoriensis dispersal given it limits the presence of other triatomine species [46]. Habitat fragmentation and human agricultural activities have shown to have an effect on triatomine population dynamics [54]. Human-mediated passive triatomine dispersal has been suggested elsewhere [11,41–43], and therefore, we assume roads might connect triatomine popuations (Fig 1D). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Step-by-step walk-through of the landscape genomics mixed modelling framework used to study the Chagas disease arthropod vector, Rhodnius ecuadoriensis. A, First, a research question is defined based on whether gene flow or adaptation processes are to be investigated and sampling design is established. B, In the field, triatomines are collected in different ecotopes in the spatial and temporal gradients defined in A. Different variables are recorded at this stage such as altitude and geographic coordinates. C, In the laboratory, triatomine next generation sequencing (NGS) libraries are prepared and sequenced in high-throughput platforms. NGS data is processed with bioinformatic tools, and each sample genotype information is used to obtain a matrix of pairwise populations (Pop) genetic distances. D, A hypothetical landscape model (1) is parametrised into a resistance surface (2) which is a spatial representation of a given species movement constraints at each grid cell on a digital layer. From this resistance surface, a matrix of pairwise population (Pop) effective distances is calculated (3). E, Finally, statistical methods are used to correlate pairwise population genetic and effective distance matrices to investigate whether isolation-by-resistance (landscape functional connectivity) is a fitted model of the genetic differentiation of triatomine populations. Source maps: www.usgs.gov/centers/eros/science/usgs-eros-archive-digital-elevation-global-multi-resolution-terrain-elevation, www.usgs.gov/media/images/south-america-land-cover-characteristics-data-base-version-20 and dataportaal.pbl.nl/downloads/GRIP4/GRIP4_Region2_vector_shp.zip. https://doi.org/10.1371/journal.pgen.1010019.g001 Rhodnius ecuadoriensis is the major vector for Chagas disease in Ecuador and Northern Peru [55]. Both domestic and wild populations of this species exist throughout its range [56]. Preliminary morphological and genetic evidence suggests some gene flow of R. ecuadoriensis between domestic and wild ecotopes [57,58]. By comparison, genetic studies of T. cruzi infecting the same vectors in Ecuador have shown strong to moderate differentiation between wild and domestic isolates [59,60]. As such there is a lack of a clear understanding of the micro and macro-evolutionary and ecological forces shaping R. ecuadoriensis domestic adaptation and dispersal capabilities, and those of the parasites they transmit. Our study represents an attempt to evidence gene flow from wild to domestic ecotopes in R. ecuadoriensis in Ecuador, a preliminary survey of any potential genomic signatures of adapation to the domestic niche in triatomines, as well as an assessment of the landscape drivers of vector dispersal. We used a reduced-representation sequencing approach (2b- RADseq) to recover genome-wide SNP variation in 272 Rhodnius ecuadoriensis individuals collected across ecological gradients in Loja, Ecuador. We confirmed R. ecuadoriensis do frequently invade houses from the forest in southern Ecuador. Significantly elevated allelic richness in wild sites by comparison to nearby domestic foci clearly confirmed that dispersal occurred most frequently from wild ecotopes into domestic structures. Genome scans across multiple parallel colonisation events revealed possible evidence of ‘adaptation with geneflow’, with key outlier loci associated with colonisation of built domestic structures and, presumably, human blood feeding. Several outlier loci were mapped to the annotated regions of the R. prolixus genome. A strong signature of isolation-by-distance (IBD) was observable throughout the dataset, an effect less pronounced between domestic sites than between wild foci. Formal landscape genomic analyses revealed elevation surface as the major barrier to genetic connectivity between populations. Landscape genomic analysis enabled a spatial model of vector connectivity to be elaborated, informing ongoing control efforts in the region and providing a model for mapping the dispersal potential of triatomines and other disease vectors. Our findings suggest frequent and spatially targeted interventions, to cope with high gene flow and fragmented populations, are necessary to suppress Chagas disease transmission in Loja. Moreover, the discovery of signatures of possible local adaptation shed the first light on the genomic basis of domestication in triatomines.

Discussion In this study we make several core observations: R. ecuadoriensis do invade houses from wild populations, R. ecuadoriensis loci associated with the domestic niche can be identified within our limited marker set and mapped to annotated triatomine genomic regions, and the landscape drivers of vector dispersal can be identified. Consistent with frequent house invasion, high levels of gene flow between multiple domestic and wild R. ecuadoriensis populations were detected by hierarchical analysis. Low and largely non-significant pairwise F ST values, as well as interleaved sample clustering based on phylogenetic and discriminant analyses were also consistent with house invasion. Significantly elevated allelic richness in wild sites by comparison to nearby domestic foci clearly confirmed that dispersal occurred most frequently from wild ecotopes into domestic structures. Genome scans across these parallel events of colonisation to the domestic niche revealed possible evidence of ‘adaptation with geneflow’, with key outlier loci associated with colonisation of built domestic structures and, presumably, human blood feeding—several of which mapped to the R. prolixus genome. A strong signature of isolation-by-distance (IBD) was observable throughout the dataset, an effect less pronounced between domestic sites than between wild foci. Formal landscape genomic analyses revealed elevation surface as the major barrier to genetic connectivity between populations. Landscape genomic analysis enabled a spatial model of vector connectivity to be elaborated, informing ongoing control efforts in the region and providing a model for mapping the dispersal potential of triatomines and other disease vectors. Vector control is the mainstay of Chagas Disease control [11]. Widespread wild reservoir hosts, as well as a lack of safe treatment options [73,74] and associated healthcare infrastructure, mean that transmission cannot be blocked by reducing parasite prevalence in human and animal hosts [75]. Our data indicate that elimination of domesticated R. ecuadoriensis in Ecuador will be frustrated by repeated re-invasion from the wild environment. Similar risks to effective control are posed by wild T. infestans in the southern cone region [12], R. prolixus in Los Llanos of Colombia and Venezuela [13] and potentially elsewhere in Latin America where competent vectors are present in the wild environment and nearby domestic locales (e.g., T. sordida, T. maculata, R. pallescens and others [14,15]). Understanding evolutionary processes that underpin the colonisation of the domestic environment by arthropod vectors, and their specialisation to feeding on humans, is required to characterize their vectorial capacity. Hybrid ancestry in Culex pipiens, for example, is thought to contribute to the biting preference for humans [3]. Human feeding preference can be rapidly genetically selected for in Anopheles gambiae [76]. Specialisation of Aedes aegypti on humans, and resultant global outbreaks of dengue, yellow fever, and Chikungunya viruses, may be traceable to SNPs associated with the emergence of differential ligand-sensitivity of the odorant receptor AaegOr4 in East Africa [2]. In triatomines, the nature of genetic adaptations that have enabled the widespread dispersal of successful lineages are far from clear. T. infestans, thought to have originated in the Western Andean region of Bolivia, spread rapidly among human dwellings in the Southern Cone region of South America before its near eradication in the 1990s [10]. Cytogenetic analyses suggest this early expansion was accompanied by a substantial reduction in genome size [77], but the significance of such a change is not clear. The advantage of the R. ecuadoriensis system we describe is that it may be able to capture multiple parallel adaptive processes and; therefore, can assist in the identification of common evolutionary features associated with colonisation of the domestic environment. Despite limited genomic coverage, and with no R. ecuadoriensis reference genome available, we mapped outlier loci to genes in the R. prolixus draft genome, and found hits related to salivary enzyme production [65], as well as embryonic development [63]. However, these findings represent only a small first step towards undertstaning domestic adaptation in triatomines. Our methodological pathway was limited to comparing allele frequencies at a relatively small fraction of genomic loci between triatomine natural populations in order to identify oulier loci associated with a given niche, and map them to genomic regions in R. prolixus ([30,78]). Although, these genes may have a role in domestic adaptation in triatomines, genome-wide association studies, quantitative trait locus mapping or CRISPR/Cas9 gene knockout approaches are necessary to fully reveal the genomic architecture of adaptation to the domestic setting. Nevertheless, these findings motivate us to investigate further putative genes involved in local adaptation to the domestic environment such as blood-feeding [79], sensory cues and host-seeking behaviour [28,80], as well as human blood detoxification [79,81]. Recent data from our group in Loja province shows that, without doubt, domestic R. ecuadoriensis feed extensively on human blood [82]. To adequately explore the genomic bases of adaptive traits in triatomines, future work should focus not only on improving functional annotation of triatomine genomes, but also robust experimental designs (e.g., common-garden or recriprocal transplant experiments [83,84]), to enable genotype and phenotype to be linked. Our analyses identified a strong signal of genetic IBD among R. ecuadoriensis populations across our study area. Geographic partitioning at this scale is consistent with limited autonomous dispersal capabilities of triatomines which are, in the main, poor fliers [41]. Wind-blown dispersal observed in smaller vector species is unlikely in triatomines [85]. Passive dispersal of triatomine vectors alongside the movements of their human hosts, which certainly underpins the successful dispersal of other domesticated vector species, is more likely (e.g., Aedes spp. [86,87]). Lower IBD observed among domestic sites than wild sites may be consistent with passive dispersal alongside humans in the former. We observed a similar phenomenon among parasite isolates from the same region in a previous study [59] in which T. cruzi domestic/peridomicile isolates showed no spatial structure in comparison with wild isolates. Nonetheless, our formal exploration of the landscape drivers of vector dispersal did not reveal an important effect of roads, and it is not clear to what extent human dispersal of vectors takes place based on our data alone. According to our landscape genomic analysis, elevation surface is a key predictor of connectivity/discontinuity among R. ecuadoriensis populations. Our machine learning (ML) optimisation procedure provides objective parameterisation of altitude resistance values to R. ecuadoriensis gene flow [88]. Based on our landscape model predictions we were able to construct an electric current map (Fig 5C) to assist medical entomologists and policy makers in understanding vector dispersal routes. Current vector control strategies in Loja target a single civic administrative unit (neighbourhood or town) for any given insecticidal intervention [55]. Historical vector control in Loja has been sporadic and limited insecticide spraying that varied yearly (from 2004 to 2014) to only a small number of parishes due to budgetary constraints [89]. Our data and model suggest this approach may be effective for certain communities (e.g., SF, CG, NT and YS, Fig 5). However, for highly connected hubs (e.g. BM, GA, CQ, AZ), successful longer term triatomine control (e.g., insecticide spraying, house improvement, window nets, etc.) will depend on simultaneous intervention in multiple connected communities. In Ecuador, as with many other endemic regions in Latin America, efforts to control Chagas disease may be complicated in the long term by substantial wild populations of secondary triatomine vectors [16]. As with many other vector borne diseases, there is also a strong case for the use of integrated vector management (IVM) for Chagas disease, where improvements to housing, education, community engagement, in addition to bed net use and insecticide spraying are all likely to be necessary to achieve sustained control [55,90]. Our data clearly indicate that triatomines do invade houses in Loja and low-lying valleys provide routes for vector dispersal between communities and cost-effective IVM must be underpinned by this understanding of vector population structure. Fortunately, genomic and analytical tools can now furnish much of the detail, although better genomic resources for secondary triatomine vector species are required to reveal the process of vector adaptation to the human host. Targeting secondary vector species like R. ecuadoriensis must now be a priority for health authorities, as these now represent the most pernicious and persistent barrier to controlling residual Chagas disease transmission.

Acknowledgments We thank Dr P. Johnson for advice in statistical analyses, Prof W. Peterman for helpful advice on ResistanceGA analysis, the entomological team at CISeAL for sample collection and M. Babbucci for proving custom scripts for 2b-RAD raw data cleaning. We also thank Prof D. Haydon, Prof S. Babayan and Dr R. Biek for their feedback. We thank S. Morrow for proof-reading the manuscript.

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