Biology subjects

Lucas, E. R.

Publications and source records attributed to Lucas, E. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Extensive global HIV-1 sequence diversity perturbs conserved features of the envelope glycan shield

The extensive sequence diversity and glycosylation of the HIV-1 envelope glycoprotein pose a challenge for vaccine design efforts aimed at eliciting glycan-binding broadly neutralizing antibodies (bnAbs). Understanding how HIV-1 glycosylation varies across diverse strains is therefore of considerable interest. Here, we employed mass spectrometry to map the site-specific glycan composition of pseudoviruses from an established 12-virus panel representing global sequence diversity. We refine the current model of the viral glycan shield by showing how the presence or absence of glycans can modulate the composition and perimeter of the mannose patch, where glycans exhibit limited maturation. Importantly, we show that within the Clade A 398F1 strain, isolated in Tanzania, the trimer apex exhibited elevated glycan maturation and resistance to apex-directed bnAbs. These findings demonstrate that the 12-virus panel reveals both conserved glycan-dependent epitopes and strains with exceptional glycosylation features that may impact the generality of vaccine design efforts targeting particular epitopes.

biochemistry↗

bioRxiv2026

Transcriptomic profiling reveals multiple mechanisms of insecticide resistance in Aedes aegypti from Angola

Control of arboviruses remains heavily reliant on insecticide-based vector control targeting adult Aedes aegypti, especially during outbreaks, but the effectiveness of these tools can be compromised by insecticide resistance. While the mechanisms underlying resistance have been widely studied in Latin American and South East Asian Ae. aegypti, knowledge from African populations is limited, particularly regarding metabolic resistance. To address this knowledge gap, we sequenced the transcriptomes of Ae. aegypti collected in Angola, from both unexposed individuals and survivors of exposure to the organophosphate fenitrothion, alongside two insecticide-susceptible laboratory reference strains. Many overexpressed genes belonged to the major detoxification enzyme families, including 96 cytochrome P450 monooxygenases (CYP450s), 18 glutathione S-transferases (GSTs), and 35 carboxylesterases, with multiple genes previously detected as upregulated in Latin American and Asian populations. These included frequently reported, functionally-validated, metabolic resistance genes such as CYP9J24, CYP9J26, and CYP6BB2. However, expression of auxiliary resistance families including hexamerins, heat shock proteins, and odorant binding proteins were linked to the insecticide resistance phenotype, whilst numerous cuticular genes differentiated the Angolan population from both susceptible laboratory strains. A novel candidate, CYP6AG7, that was overexpressed after fenitrothion exposure was experimentally validated, and surprisingly metabolised fenitrothion into its toxic oxon form, which it did not subsequently break down. The antioxidant response element (ARE) motif, to which the transcription factor Maf-S binds, was detected in all CYP450 overexpressed in the fenitrothion treatment suggesting their potential coordinated induction. Analysis of genetic differentiation revealed several resistance-linked genes under potential selection, and SNP screening identified both known and novel non-synonymous mutations in the voltage-gated sodium channel (VGSC) gene, the target for pyrethroid insecticides. This is the first RNAseq dataset for Ae. aegypti from Africa in the context of insecticide resistance, providing insight into the complexity of resistance mechanisms, including some shared, and others potentially novel, compared to better studied populations from other geographical regions. Author summaryDengue, chikungunya, yellow fever, and Zika are diseases that exert an increasing public health burden across Africa, primarily transmitted by the mosquito Aedes aegypti. We rely heavily on insecticides to control these mosquitoes, but populations are increasingly developing resistance, making control efforts less effective. While resistance mechanisms have been well-studied in the Americas and Asia, comparatively little is known about how African Ae. aegypti resist insecticides. We collected Ae. aegypti mosquitoes from Angola and compared the genes expressed in fenitrothion resistant versus susceptible mosquitoes using RNA sequencing. We identified overexpressed candidate insecticide resistance genes from the detoxification enzyme families that mosquitoes use to break down insecticides, including several genes previously linked to resistance in other regions. One novel enzyme identified, CYP6AG7, was experimentally validated and found to convert the pro-insecticide fenitrothion into its harmful form but interestingly not break down this harmful metabolite further. Mutations potentially linked to insecticide resistance were also detected in detoxification genes and insecticide target site genes. Our study provides the first comprehensive molecular characterization of insecticide resistance mechanisms in African Aedes aegypti, offering crucial data to inform vector control strategies and insecticide resistance management across the continent as dengue and related diseases continue to spread.

bioinformatics↗

bioRxiv2026

Multi-omics analysis identifies loci associated with pyrethroid resistance across sister species in the Anopheles gambiae species complex

The Anopheles gambiae species complex includes An. gambiae, An. coluzzii, and An. arabiensis, three of the four major malaria vector species across Africa. Insecticide-based vector control remains the most critical tool in the fight against malaria, with a heavy reliance on pyrethroid insecticides. However, widespread pyrethroid resistance jeopardises the effectiveness of these methods. Currently, the number of diagnostic markers available to track pyrethroid resistance in endemic settings is limited, focusing mainly on changes at the insecticide target site. Genetic analysis of seven insecticide-resistant populations from West Africa, a region with intense insecticide resistance, reveals shared loci associated with pyrethroid resistance among the sister species of the An. gambiae complex including kdr, GSTE1-8 and the CYP6P region. Notably, a mutation in the voltage-gated sodium channel, I1527T, previously identified in An. coluzzii and An. gambiae, was detected in wild-caught An. arabiensis from Burkina Faso. Additionally, a mutation, L207I, in GSTE7 common to An. coluzzii and An. arabiensis, significantly increases survivorship to deltamethrin. With the additional integration of RNAseq and data from the Anopheles gambiae 1000 genomes project, we were able to identify putative eQTLs associated with the expression of major insecticide resistance-related transcripts, such as CYP6P3, CYP9K1 and CYP6AA1. This study reveals several potential diagnostic markers of resistance that can be implemented in endemic settings and identifies putative introgression between An. arabiensis and An. coluzzii in Burkina Faso.

genetics↗

bioRxiv2025

Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection

Human in vitro lung models represent advanced tools for studying respiratory infections, particularly those caused by emerging respiratory pathogens. Despite scientific advances, vaccine and therapeutics pre-clinical development has yet to fully adopt human-relevant testing platforms due in part to a lack of validation. In this study, we characterised how static and dynamic flow culture conditions influence microphysiological systems (MPS) generated using primary bronchial and alveolar epithelial cells. We assessed epithelial structure, functional differentiation, and infection dynamics. This study represents the first direct comparison of how dynamic flow and endothelial co-culture influence viral tropism, replication kinetics, and host responses across anatomically distinct regions of the respiratory tract in vitro. Dynamic flow promoted formation of more physiologically relevant tissue architecture, pseudostratified bronchial epithelium and alveolar sac-like structures, with enhanced epithelial differentiation and retention of region-specific cell phenotypes at the transcriptomic level. Both static and dynamic flow models demonstrated responsiveness to inflammatory stimuli (poly(I:C), LPS), producing distinct, tissue-specific cytokine profiles and supporting infection with multiple SARS-CoV- 2 variants. Differences in infection efficiency, viral replication, and host gene expression were observed between variants, with dynamic flow models offering enhanced sensitivity and resolution. In alveolar tissues, dynamic flow increased infection efficiency and reduced variability, enabling more robust and consistent transcriptional responses. This facilitated the identification of interferon signalling pathways as key targets of the host response. Among the variants tested, Delta induced the most extensive tissue damage and strongest transcriptional response, whereas Omicron BA.5 exhibited greater infectivity in alveolar models compared to earlier variants. Our findings demonstrate that dynamic flow MPS more closely replicate human lung tissue architecture and cellular diversity, while also enhancing the predictive power and clinical relevance of airway models for ex vivo studies of SARS-CoV-2 infection. These improvements strengthen the reliability of data generated for the study of host-pathogen interaction studies and support the use of dynamic systems for evaluating novel anti-infectives, immunomodulators, and functional characterisation of immune sera generated by next-generation vaccines. Collectively, our results highlight the value of integrating dynamic in vitro models into preclinical pipelines for emerging respiratory pathogens.

microbiology↗

bioRxiv2025

Signatures of selection and mechanisms of insecticide resistance in Ugandan Anopheles funestus: Insights from embedding translational genomics into the LLINEUP cluster randomised trial.

In response to the emerging threat of insecticide resistance in malaria vectors, insecticides are being repurposed for vector control or developed de novo. Good stewardship of these finite new resources is essential if disease control programmes are to remain effective. This is dependent on timely data to help guide evidence-based decision-making for National Malaria Control programmes (NMCPs). By embedding genomics into cluster randomized control trials (cRCTs), we can perform surveillance and early detection of insecticide resistance variants to new and repurposed chemicals in natural field conditions, supporting effective stewardship. The LLIN Evaluation Uganda Project (LLINEUP) trial evaluated the efficacy of pyrethroid-piperonyl butoxide (PBO) and pyrethroids-only long-lasting insecticidal nets (LLINs). It was conducted in Uganda between 2017-2020 and was the largest cRCT to date, covering 40% of the country in 104 health sub-districts. We embedded genomic surveillance within LLINEUP to detect and track insecticide resistance variants. At baseline and throughout the trial, we sampled Anopheles mosquitoes with Prokopack aspirators and performed Illumina whole-genome sequencing. We show that An. funestus populations were relatively unaffected by the interventions, compared to An. gambiae s.l., which were markedly reduced six months following LLIN deployment. Standard approaches for describing genetic diversity and population structure e.g. fixation index (FST), Principal Component Analysis (PCA) and Neighbour-Joining (NJ) trees, were consistent with the density observations and suggestive of a single large An. funestus population in Uganda with little genetic differentiation. Genome-wide selection scans revealed strong signals of selection at the Resistance to pyrethroid-1 (RP1) locus and Cyp9k1, both loci previously implicated in pyrethroid resistance. We report two additional loci, eye diacylglycerol kinase (Dgk) ({cong}13.5Mb on the X chromosome) and O-mannosyl-transferase (TMC-like) ({cong}67.9Mb on 3RL) that showed signals of selection. Known DDT and permethrin resistance-associated variants at the Gste2 locus, L119F and L119V, were also identified. Over the trial period, changes in haplotype frequencies were observed in regions under selection, with more pronounced shifts in the PBO arm. Notably, there were significant reductions in the frequencies of swept haplotypes (measured by delta ({Delta}) H12) in the Dgk and Cyp6p9a regions, while significant increases in haplotype frequency were observed at Gste2 and Cyp9k1 loci. Our findings reveal the differential impact of the trial on An. gambiae s.l. and An. funestus densities and the differing responses of An. funestus populations to pyrethroid and pyrethroid-PBO selection pressure. These insights underscore the potential value of tailored, species- and region-specific vector control strategies, supported by regional genetic surveillance, to better control insecticide resistance evolution and spread. By embedding genomic surveillance in cRCTs we can facilitate the discovery of putative resistance variants and can provide evidence of their impact on vector control tool efficacy; both of crucial importance to evidence-based deployment of vector control tools by NMCPs.

genomics↗

bioRxiv2025

Spatially-explicit genomics of An. gambiae s.l uncovers fine-scale population structure and mechanisms of insecticide resistance

Progress in malaria control in sub-Saharan Africa is stalling, partly due to the spread of insecticide resistance in Anopheles vectors. Monitoring the evolution of insecticide resistance alleles and their spatial heterogeneity is important for malaria control programmes, and genomic surveillance has emerged as a pivotal tool for this purpose. Earlier genomics research has typically employed convenience-based sampling, and research has yet to be performed to optimise sampling regimens for malaria vector genomics. In an earlier study, we developed a spatially explicit sampling framework that considers the underlying ecology to enable sampling mosquitoes with reduced bias. We applied this framework to sample and perform whole-genome sequencing on 485 individual specimens of An. gambiae s.l mosquitoes from Obuasi, central Ghana, an area with extensive gold mining activities. In this region, An. gambiae s.l have been documented as highly resistant to multiple insecticides, including pyrethroids used for the treatment of bed-nets. High resolution of fine-scale population structure enabled the detection of isolation-by-distance in An. coluzzii in Obuasi, whilst finding that at this scale, geographic distance, rather than the underlying habitat, drives population structure. We examine methods to estimate mosquito kinship, demonstrating that polymorphic chromosomal inversions significantly confound established analytical tools, while revealing how the mosquitos chromosome architecture generates high variance in genetic relatedness among kin. Using genome-wide selection scans and clustering, we discover the novel mutations Gste2-F120L and Cyp9K1-N224I in detoxification enzymes that are driving selective sweeps, likely in response to insecticidal pressure. We find that the frequencies of some resistance variants are associated with artisanal-gold mines, and elucidate the continued evolution of the Voltage-gated sodium channel, the target of pyrethroid insecticides. Overall, we show that sampling vectors strategically may enhance our ability to perform effective genomic surveillance.

genomics↗

bioRxiv2025

Genetic Surveillance Reveals Differential Evolutionary Dynamic of Anopheles gambiae Under Contrasting Insecticidal Tools used in Malaria control

Malaria, a febrile disease caused by the Plasmodium parasites and transmitted by mosquitoes, is a leading cause of mortality in children under 5 in endemic countries. The widespread deployment of insecticide-treated bed nets (ITNs) has significantly reduced malaria transmission, but rising levels of insecticide resistance threatens to halt the progress. Monitoring insecticide resistance is vital for effective vector control, particularly when deploying new tools. Understanding mosquito population responses to these interventions is crucial for guiding control programmes in making informed decisions about the selection, timing, and geographic deployment of tools. This genomic study investigates the demographic and evolutionary consequences on the malaria vector Anopheles gambiae of deploying standard ITNs (containing only pyrethroids) and pyrethroid-PBO nets (containing pyrethroids plus the synergist piperonyl butoxide) during a clinical trial in Uganda. Despite substantial reductions in indoor mosquito densities in the clinical trial, estimates of nucleotide diversity ({pi}) and linkage disequilibrium revealed no significant decline in effective population size, reflecting continued large population size even after effective control. Marked allele frequency shifts at resistance-associated loci indicated strong selection pressures driven by the interventions, with distinct selective dynamics between the two net types, highlighting alternative pyrethroid detoxification pathways in the presence of PBO. A duplication in the Cyp9k1 gene significantly increased in frequency in populations exposed to pyrethroid-only nets but decreased in populations exposed to PBO-treated nets, suggesting that selection for over-expression of this gene is removed when this resistance mechanism is impacted by PBO. An alternative potential detoxification mechanism was selected within a region of the 2La chromosomal inversion on chromosome 2L, which encompasses the UDP-glucose 6-dehydrogenase gene. This variant consistently increased in frequency when exposed to PBO-treated nets. Additionally, pyrethroid-only nets selected for a novel locus on the X chromosome containing the diacylglycerol kinase gene, which is potentially linked to behavioural adaptations through its role in neurotransmission modulation. Our findings underscore the importance of genomic surveillance in vector control, revealing distinct evolutionary dynamics of insecticide resistance mechanisms in the presence of PBO. While ITNs remain effective, the persistence and evolution of resistance-associated alleles highlight the need for adaptive and dynamic resistance management strategies. By integrating high-resolution genomic data with epidemiological and entomological monitoring, this study offers actionable insights to sustain malaria control efforts amid the ongoing challenge of insecticide resistance.

genomics↗

bioRxiv2025

The origin, invasion history and resistance architecture of Anopheles stephensi in Africa.

The invasion of Africa by the Asian urban malaria vector, Anopheles stephensi, endangers 126 million people across a rapidly urbanising continent where malaria is primarily a rural disease. Control of An. stephensi requires greater understanding of its origin, invasion dynamics, and mechanisms of widespread resistance to vector control insecticides. We present a genomic surveillance study of 551 An. stephensi sampled across the invasive and native ranges in Africa and Asia. Our findings support a hypothesis that an initial invasion from Asia to Djibouti seeded separate incursions to Sudan, Ethiopia, and Yemen before spreading inland, aided by favourable temperature, vegetation cover, and human transit conditions. Insecticide resistance in invasive An. stephensi is conferred by detoxification genes introduced from Asia. These findings, and a companion genomic data catalogue, will form the foundation of an evidence base for surveillance and management strategies for An. stephensi.

genomics↗

bioRxiv2025

Population genomics of Anopheles darlingi, the principal South American malaria vector mosquito

Malaria in South America remains a serious public health problem. Anopheles (Nyssorhynchus) darlingi is the most important malaria vector across tropical Latin America. Vector-targeted disease control efforts require a thorough understanding of mosquito demographic and evolutionary patterns. We present and analyze whole genomes of 1094 A. darlingi (median depth 18x) from six South American countries. We observe deep geographic population structure, high genetic diversity including thirteen putative segregating inversions, and no evidence for cryptic sympatric taxa despite high interpopulation divergence. Strong signals of selection are plausibly driven by insecticides, especially on cytochrome P450 genes, one of which we validated experimentally. Our results will facilitate effective mosquito surveillance and control, while highlighting ongoing challenges that a diverse vector poses for malaria elimination in the western hemisphere.

evolutionary biology↗

bioRxiv2025

Targeted genomic surveillance of insecticide resistance in African malaria vectors

The emergence of insecticide resistance is threatening the efforts of malaria control programmes, which rely heavily on a limited arsenal of insecticidal tools, such as insecticide-treated bed nets. Importantly, genomic surveillance of malaria vectors can provide critical, policy-relevant insights into the presence and evolution of insecticide resistance, allowing us to maintain and extend the shelf life of these interventions. Yet the complex genetic architecture of resistance, combined with resource constraints in malaria-endemic settings, have thus far precluded the widespread use of genomics in routine surveillance. Meanwhile, stakeholders in sub-Saharan Africa are moving towards locally driven, decentralised generation of genomic data, underscoring the need for standardised and robust genomics workflows. To address this need, we demonstrate an approach to targeted genomic surveillance in Anopheles gambiae s.l with Illumina sequencing. We target 90 genomic loci in the Anopheles gambiae s.l genome, including 55 resistance-associated mutations and 35 ancestry informative markers. This protocol is coupled with advanced, automated software for accurate and reproducible variant analysis. We are able to elucidate population structure and ancestry in our cohorts and accurately identify most species in the An. gambiae species complex. We report frequencies of variants at insecticide-resistance loci and explore the continued evolution of the pyrethroid target site, the Voltage-gated sodium channel. Applying the platform to a recently established colony of field-caught resistant mosquitoes (Siaya, Kenya), we identified seven independent resistance-associated variants contributing to reduced efficacy of insecticide-treated nets in East Africa. Additionally, we leverage a machine learning algorithm (XGBoost) to demonstrate the possibility of predicting bioassay mortality using genotypes alone. This achieved high accuracy (75%), demonstrating the potential of targeted genomics to predictively monitor insecticide resistance. Together these tools provide a practical, scalable solution for resistance monitoring while advancing the goal of building local genomic surveillance capacity in sub-Saharan Africa.

genomics↗

bioRxiv2024

Genomic diversity of the African malaria vector Anopheles funestus

Anopheles funestus s.s. is a formidable human malaria vector across sub-Saharan Africa. To understand how the species is evolving, especially in response to malaria vector control, we sequenced 656 modern specimens (collected 2014-2018) and 45 historic specimens (collected 1927-1967) from 16 African countries. We find high levels of genetic variation with clear and stable continental patterns. Six segregating inversions might be involved in adaptation of local ecotypes. Strong recent signals of selection centred on canonical insecticide resistance genes are shared by multiple populations. A promising gene drive target in An. gambiae is highly conserved in An. funestus. This work represents a significant advance in our understanding of the genetic diversity and population structure of An. funestus and will enable smarter targeted malaria control.

genomics↗

bioRxiv2024

Sequencing 1206 genomes reveals origin and movement of Aedes aegypti driving increased dengue risk

The number of dengue cases worldwide has increased ten-fold over the past decade as Aedes aegypti, the primary vector of this disease, thrives and expands its distribution, revealing limitations to current control methods. To better understand how Ae. aegypti evolved from a forest dwelling, generalist species to a highly anthropophilic urban species and the impact of contemporary gene flow on the future of dengue control, we sequenced 1,206 genomes from mosquitoes collected at 74 locations around the globe. Here we show that after evolving a preference for humans in the Sahel region of West Africa, the origin of the fully domesticated, anthropophilic subspecies Ae. aegypti aegypti (Aaa) occurred in the Americas during the Atlantic Slave Trade era and was followed by its explosive expansion around the globe. In recent decades, Aaa has invaded coastal Africa, the ancestral home range, introducing insecticide resistance mutations and an affinity for human hosts. Evidence of back-to-Africa migration is found in regions with recent dengue outbreaks, raising concern that global movement of Aaa could increase transmission risk of arboviruses including dengue in urban Africa. These data provide a platform to further study this important mosquito vector species and underscore developing complexity in the fight to limit the spread of dengue, Zika, and chikungunya diseases.

genomics↗

bioRxiv2024

Cryptic population structure and insecticide resistance in Anopheles gambiae from the southern Democratic Republic of Congo

The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with <92% mortality in all tests, but varied between collections, with mosquitoes from Kimpese being the most resistant. Whole genome sequencing of 165 An. gambiae revealed evidence for genetic differentiation between Kimpese and Kapolowe / Mikalayi, but not between the latter two sample sites despite separation of approximately 800km. Surprisingly, there was evidence of population structure at a small spatial scale between collection subsites in Kimpese, despite separation of just tens of kilometres. Intra-population (H12) and inter-population (FST) genome scans identified multiple peaks corresponding to genes associated with insecticide resistance such as the voltage gated sodium channel (Vgsc) target site on chromosome 2L, a Cyp6 cytochrome P450 cluster on chromosome arm 2R, and the Cyp9k1 P450 gene on chromosome X. In addition, in the Kimpese subsites, the P450 redox partner gene Cpr showed evidence for contemporary selection (H12) and population differentiation (FST) meriting further exploration as a potential resistance associated marker.

evolutionary biology↗

bioRxiv2024

Copy number variants underlie the major selective sweeps in insecticide resistance genes in Anopheles arabiensis from Tanzania.

To keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. Knowledge of the mechanisms of resistance remains limited in Anopheles arabiensis, which in many parts of Africa is of increasing importance because it is apparently less susceptible to many indoor control interventions. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to deltamethrin (commonly used in bednets) and PM, in An. arabiensis from two regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in resistance to these two insecticides. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the Coeae2g-Coeae6g cluster of carboxylesterase genes. Using complementary data from An. coluzzii in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster. Against this background of metabolic resistance, target site resistance was very rare or absent for both insecticides. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with three An. arabiensis individuals from West Africa revealed a startling diversity of evolutionary origins of target site resistance, with up to 5 independent origins of Vgsc-995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis.

genomics↗

bioRxiv2024

Parallel evolution in mosquito vectors - a duplicated esterase locus is associated with resistance to pirimiphos-methyl in An. gambiae

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programmes. The organophosphate, pirimiphos-methyl, is a relatively new chemical in the vector control armoury but is now widely used in indoor residual spray campaigns. Whilst generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in Anopheles gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to organophosphates in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in An. gambiae, An. coluzzii and An. arabiensis. As in Cx. pipiens, copy number variation seems to play a role in the evolution of insecticide resistance at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in Anopheles gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programmes.

evolutionary biology↗

bioRxiv2023

Mechanisms of transcriptional regulation in Anopheles gambiae revealed by allele specific expression

Malaria control relies on insecticides targeting the mosquito vector, but this is increasingly compromised by insecticide resistance, which can be achieved by elevated expression of detoxifying enzymes that metabolize the insecticide. In diploid organisms, gene expression is regulated both in cis, by regulatory sequences on the same chromosome, and by trans acting factors, affecting both alleles equally. Differing levels of transcription can be caused by mutations in cis-regulatory modules (CRM), but few of these have been identified in mosquitoes. We crossed bendiocarb resistant and susceptible Anopheles gambiae strains to identify cis-regulated genes that might be responsible for the resistant phenotype using RNAseq, and cis-regulatory module sequences controlling gene expression in insecticide resistance relevant tissues were predicted using machine learning. We found 115 genes showing allele specific expression in hybrids of insecticide susceptible and resistant strains, suggesting cis regulation is an important mechanism of gene expression regulation in Anopheles gambiae. The genes showing allele specific expression included a higher proportion of Anopheles specific genes on average younger than genes those with balanced allelic expression. Author SummaryThe evolution of insecticide resistance, including resistance that is due to changes in the expression levels of certain resistance associated genes is threatening progress in malaria control. We investigated how the expression of genes in the malaria vector Anopheles gambiae is controlled, by implementing a method for the first time in this species. Each mosquito inherits a set of chromosomes from both parents, so has a maternal and paternal copy of most genes. When a gene is expressed, the DNA encoding that gene is transcribed into messenger RNA. This process is controlled by the cellular environment and by other DNA sequences on the same chromosome as each gene. We crossed mosquitoes from insecticide resistant and susceptible strains to equalize the cellular environment and then measured the levels of messenger RNA from both gene copies. 115 genes showed consistently different messenger RNA levels between gene copies in most crosses, suggesting these genes are regulated by factors on the same chromosome. There were relatively more Anopheles specific genes with imbalanced expression. Using machine learning we identified DNA sequences that may be responsible for controlling gene expression in mosquito tissues; several of these sequences were close to genes with imbalanced expression.

genetics↗

bioRxiv2023

Spatially explicit sampling frameworks to identify regions of increased mosquito abundance

Vector control interventions often lack comprehensive information on vector population distribution and dynamics. This knowledge gap poses challenges in targeting interventions effectively, especially in areas with heterogeneous transmission and where complementary vector control tools may be required to achieve sustained impact on disease transmission. In this study, we implemented a spatially explicit sampling framework for improved vector surveillance in coastal Kenya. Our stratified lattice with close pair sampling design aimed to characterise the vector dynamics of the primary malaria-transmitting species in the area and assess the ecotype classifications effectiveness at identifying clear population patterns. The study collected 3,621 mosquitoes, with An. funestus s.l. being the most abundant malaria vector. The inclusion of the ecotype classification significantly improved spatial abundance model estimates for An. gambiae and Culex spp. Wetlands, topographic wetness index, and proximity to rivers were associated with increased mosquito abundance. Spatial modelling revealed high abundance regions near the Galana-Sabaki River. Our study demonstrates the applicability of a reproducible spatial sampling approach to identify areas with high vector abundance and inform targeted vector control strategies. The study highlights the importance of ecological stratification and a spatial explicit sampling approach for predicting mosquito presence when prior data is limited and underscores the potential for refining future sampling for control efforts.

ecology↗

bioRxiv2023

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae s.l.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of the most widespread tools currently used to control malaria. The genetic underpinnings of resistance are still only partially understood, with much of the variance in resistance phenotype left unexplained. We performed a multi-country large scale genome-wide association study of resistance to two insecticides widely used in malaria control: deltamethrin and pirimiphos-methyl. Using a bioassay methodology designed to maximise the phenotypic difference between resistant and susceptible samples, we sequenced 969 phenotyped female An. gambiae and An. coluzzii from ten locations across four countries in West Africa (Benin, Cote dIvoire, Ghana and Togo), identifying single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) segregating in the populations. The patterns of resistance association were highly multiallelic and variable between populations, with different genomic regions contributing to resistance, as well as different mutations within a given region. While the strongest and most consistent association with deltamethrin resistance came from the region around Cyp6aa1, this resistance was based on a combination of several independent CNVs in An. coluzzii, and on a non-CNV bearing haplotype in An. gambiae. Further signals involved a range of cytochrome P450, mitochondrial, and immunity genes. Similarly, for pirimiphos-methyl, while the strongest signal came from the region of Ace1, more widespread signals included cytochrome P450s, glutathione S-transferases, and a subunit of the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes were associated with resistance to both insecticide classes, suggesting possible cross-resistance mechanisms. These locally-varying, multigenic and multiallelic patterns highlight the challenges involved in genomic monitoring and surveillance of resistance, and form the basis for improvement of methods used to detect and predict resistance. Based on simulations of resistance variants, we recommend that yet larger scale studies, exceeding 500 phenotyped samples per population, are required to better identify associated genomic regions.

genomics↗