Biology subjects

Oruni, A.

Publications and source records attributed to Oruni, A..

7 recordsLinked to original sources

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

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

Significant variations in tolerance to clothianidin and pirimiphos-methyl in Anopheles gambiae and Anopheles funestus populations during a dramatic malaria resurgence despite sustained indoor residual spraying in Uganda

A dramatic malaria resurgence occurred in areas of Uganda between 2020 and 2022 coincident with the switch to clothianidin-based formulations for indoor residual spraying. During the resurgence, Anopheles funestus numbers increased but when an alternative insecticide, pirimiphos methyl, was reintroduced in 2023, both malaria cases and An. funestus mosquito density fell. In this study, we investigated possible causes of the resurgence by assessing; 1) whether sufficient quantities of insecticide were sprayed; 2) the residual insecticide bioefficacy against wild mosquitoes and; 3) the insecticide susceptibility of vector populations using standard test tube assays and wall cone assays. In 2023, after adjusting for extraction efficiency, 70-80% of the houses had optimal residual concentrations of insecticides (clothianidin >0.3g/m2; pirimiphos methyl >0.5g/m2) with significant variations between sampling rounds and wall types. Mud walls had the lowest residual concentration of insecticides, and the lowest observed mortality in wall cone assays, compared to fired bricks with plaster/cement/paint. In the studies of residual bio efficacy, by World Health Organization (WHO) definitions, An. funestus showed resistance to clothianidin (<80% mortality) up to 11 months and susceptibility to pirimiphos methyl (>90% mortality) when exposed to wall surfaces up to 7 months post-spray. In WHO tube tests, variations were observed in susceptibility to clothianidin in An. funestus populations using dose- and time-response assays (80-98% mortality). In 2022, An. gambiae was largely susceptible to the clothianidin-based formulation Sumishield (85-90% mortality) although the levels dropped slightly in 2023 (60-85% mortality) mainly in mud and pole houses. In contrast, An. gambiae was mildly susceptible to the pirimiphos methyl-based formulation Actellic ([~]80% mortality) and time response assays showed An. gambiae populations had very low knockdown and mortality at lower exposure time compared to An. funestus. Regression models showed a positive association between residual insecticide concentration (RIC) and mortality in houses sprayed with Sumishield but not Actellic houses. Despite the possible variations observed in spray operations, the study revealed that An. funestus exhibited a higher tolerance to clothianidin-based formulations compared to An. gambiae, and this might have driven the malaria resurgence observed in Uganda. However, there are signals of An. gambiae resistance to pirimiphos-methyl which will require further investigation and monitoring.

zoology↗

bioRxiv2022

RNA-Seq-Pop: Exploiting the sequence in RNA-Seq - a Snakemake workflow reveals patterns of insecticide resistance in the malaria vector Anopheles gambiae

BackgroundWe provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides end-to- end analysis of RNA-Seq data sets. The workflow allows the user to perform quality control, differential expression analyses, call genomic variants and generate a range of summary statistics. Additional options include the calculation of allele frequencies of variants of interest, summaries of genetic variation and population structure (in measures such as nucleotide diversity, Wattersons {theta}, and PCA), and genome wide selection scans (Fst, PBS), together with clear visualisations. We demonstrate the utility of the workflow by investigating pyrethroid-resistance in selected strains of the major malaria mosquito, Anopheles gambiae. The workflow provides additional modules specifically for An. gambiae, including estimating recent ancestry and determining the karyotype of common chromosomal inversions. ResultsThe Busia lab-colony used for selections was collected in Busia, Uganda, in November 2018. We performed a comparative analysis of three groups: a parental G24 Busia strain; its deltamethrin-selected G28 offspring; and the susceptible reference strain Kisumu. Measures of genetic diversity reveal patterns consistent with that of laboratory colonisation and selection, with the parental Busia strain exhibiting the highest nucleotide diversity of 1.04{middle dot}10-3, followed by the selected Busia offspring (7.1{middle dot}10-4), and finally, Kisumu (6.2{middle dot}10-4). Differential expression and variant analyses reveal that the selected Busia colony exhibits a number of distinct mechanisms of pyrethroid resistance, including the Vgsc-995S target-site mutation, upregulation of SAP genes, P450s, and a cluster of carboxylesterases. During deltamethrin selections, the 2La chromosomal inversion rose in frequency (from 33% to 86%), suggesting a link with pyrethroid resistance, which was previously observed in field samples from the same region. RNA-Seq-Pop analysis also reveals that the most widely-used insecticide-susceptible An. gambiae strain, Kisumu, appears to be a hybrid strain of An. gambiae and its sibling species An. coluzzii, which should be taken into consideration in future research. RNA-Seq-Pop is designed for ease of use, does not require programming skills and integrates the package manager Conda to ensure that all dependencies are automatically installed for the user. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An. gambiae and other taxa.

genomics↗

bioRxiv2022

The durability of long-lasting insecticidal nets treated with and without piperonyl butoxide (PBO) in Uganda.

Long Lasting Insecticidal Nets (LLINs) supplemented with the synergist piperonyl butoxide have been developed in response to growing pyrethroid resistance however their durability in the field remains poorly described. A pragmatic cluster-randomised trial was embedded into Ugandas 2017-2018 distribution to compare the durability of LLINs with and without PBO A total of 104 clusters were included with each receiving one of four LLIN products, two with pyrethroid+PBO (Olyset Plus and PermaNet 3.0) and two pyrethroid-only (Olyset Net and PermaNet 2.0). Nets were sampled at baseline, 12, and 25 months post-distribution to assess physical condition, chemical content, and bioefficacy. Physical condition was quantified using proportionate Hole Index and chemical content measured using high-performance liquid chromatography. Bioefficacy was assessed with three-minute WHO Cone and Wireball assays using pyrethroid-resistant An. gambiae, with 1hr knockdown and 24hr mortality recorded. There was no difference in physical durability between LLIN products assessed (p=0.644). The pyrethroid content of all products remained relatively stable across timepoints but PBO content declined by 55% (p<0.001) and 58% (p<0.001) for Olyset Plus and PermaNet 3.0 respectively. Both PBO LLINs were highly effective against pyrethroid-resistant mosquitoes when new, knocking down all mosquitoes. However, bioefficacy declined over time with Olyset Plus knocking down 45.72% (95% CI: 22.84-68.62, p=0.021) and Permanet 3.0 knocking down 78.57% (95% CI: 63.57-93.58, p<0.001) after 25 months. Here we demonstrate that both Olyset Plus and PermaNet 3.0 are as durable as their pyrethroid-only equivalents and had superior bioefficacy against pyrethroid-resistant An. gambiae. However, the superiority of PBO-LLINs decreased with operational use, correlating with a reduction in total PBO content. This decline in bioefficacy after just two years is concerning and there is an urgent need to assess the durability of PBO LLINs in other settings.

zoology↗

bioRxiv2021

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for anti-malarial intervention deployment.

Insecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes, including some commonly implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region around Lake Victoria, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of the Cyp6aa1 gene are common in An. gambiae across Africa and, given the enzymes metabolic activity, are likely to be a useful diagnostic for high levels of pyrethroid resistance.

evolutionary biology↗

bioRxiv2019

Open source 3D printable replacement parts for the WHO insecticide susceptibility bioassay system

BackgroundMalaria vector control and research rely heavily on monitoring mosquito populations for the development of resistance to public health insecticides. One standard method for determining susceptibility in adult mosquito populations is the World Health Organization test (WHO bioassay). The WHO bioassay kit consists of several acrylic pieces that are assembled into a unit. Parts of the kit commonly break, reducing the capacity of insectaries to carry out resistance profiling. Since there is at present only a single supplier for the test kits, replacement parts can be hard to procure in a timely fashion. Here, we present 3D printable versions for all pieces of the WHO bioassay kit.\n\nResultsUsing widely available polylactic acid (PLA) filament as a printing material, we were able to design and print functional replacements for each piece of the WHO bioassay kit. We note no significant difference in mortality results obtained from PLA printed tubes and WHO acrylic tubes. Additionally, we observed no degradation of PLA in response to prolonged exposure times of commonly used cleaning solutions.\n\nConclusionsOur designs can be used to produce replacement parts for the WHO bioassay kit in any facility with a 3D printer, which are becoming increasingly widespread. 3D printing technologies can affordably and rapidly address equipment shortages and be used to develop bespoke equipment in laboratories.

evolutionary biology↗