Biology subjects

Hearn, J.

Publications and source records attributed to Hearn, J..

18 recordsLinked to original sources

bioRxiv2026

Generational selection, transcriptomics and functional characterization reveal the impact of environmental pollutants on the evolution of insecticide resistance in malaria vectors

Insecticide resistance is threatening malaria control. While the evolution and spread of resistance has been linked to scale-up in the distribution of public health insecticides, the role of environmental pollutants such as the polyaromatic hydrocarbons (PAHs) from industrial and agricultural use remains largely uncharacterized. The PAHs are potent ligands of the aryl hydrocarbon receptor (Ahr) transcription factors involved in the regulation of xenobiotic metabolizing enzymes, and potentially involved in insecticide resistance. Here, using field insecticide-resistant (Auyo) An. coluzzii and a laboratory-susceptible colony (Ngousso), we conducted a multi-generational selection experiment using naphthalene, fluorene and a mixture of both PAHs. After ten generations, the changes in susceptibility to insecticides were monitored using WHO bioassays and whole-transcriptome analysis (RNASeq) was conducted. Compared with the non-selected colony lines, PAH exposures significantly reduced pyrethroid and DDT resistance in the field population, suggesting fitness cost associated with established resistance. In contrast, Ngousso showed a significant increase in DDT resistance (p = 0.01) at the tenth generation. A significant increase in permethrin resistance was also observed at the seventh generation (p = 0.03). Several candidate genes from the major detoxification classes were overexpressed in the selected lines (including GSTe2, CYP6Z1, and CYP6P4); the most consistent were CYP6M4 and CYP4C27, as well as those from the Ahr pathway. Heterologous expression of CYP6M4 revealed its ability to metabolise pyrethroids, including permethrin, deltamethrin, and -cypermethrin, as well as PAHs (naphthalene and fluorene). These findings establish the role of environmental pollutants as additional drivers of metabolic insecticide resistance in An, coluzzii.

ecology↗

bioRxiv2025

Genetic mapping of resistance: A QTL and associated polymorphism conferring resistance to alpha-cypermethrin in Anopheles funestus

The heavy reliance on pyrethroid-based interventions has accelerated the spread of resistance malaria vectors including Anopheles funestus, jeopardising control efforts. The efficacy of Insecticide-based interventions, especially insecticide-treated nets (ITNs), the cornerstone of malaria control and management, is threatened by the widespread resistance complicating malaria control. Alpha-cypermethrin, a type II pyrethroid, is increasingly utilised in various ITN formulations, including those combined with piperonyl butoxide (PBO) and chlorfenapyr-based Interceptor(R) G2 (IG2) nets, to enhance effectiveness against resistant mosquito populations. Therefore, understanding the molecular basis of resistance is essential to monitor and track resistance trends for an effective malaria control program. In this study, we identified a 1.4 Mb QTL on the telomeric end of the left arm of chromosome 2, conferring resistance to -cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective strategy. Individual-based QTL mapping performed on segregating individuals from an isofemale family identified a QTL at the F7 generation. Higher recombination density relative to the physical genome in the F7 isofemale family, with a recombination every 240 kb, facilitated the detection of a QTL compared to the F2 family (335 kb/cM). Additionally, we exploited bulk segregate analysis (BSA) between susceptible and resistant phenotypes from the F7 isofemale family and an F7 mixed cross-family to perform cost-effective and rapid QTL-mapping discovery. The strongest signal in both independent BSA analyses overlaps with the rap1 QTL, further supporting its role in -cypermethrin resistance. The known resistant alleles of the cytochrome P450 CYP6P9a and 6.5-kb structural variant within the rap1 QTL strongly correlate with survival to -cypermethrin. In this study, we validated that previously developed DNA-based assays, originally designed to monitor permethrin resistance, are effective for tracking resistance to -cypermethrin as well. Additionally, we identified candidate variants that can serve as reliable markers for monitoring -cypermethrin resistance. Author SummaryIn the study we used genetic crosses between resistant and susceptible Anopleles funestus colonies to identify quantitative trait loci (QTL) associated with the resistance to -cypermethrin. We have identified a QTL on the left arm of chromosome 2 conferring resistance to -cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective and cost-efficient strategy to map candidate loci associated with resistance. In this study we assessed the efficiency and the cost-effectiveness of Bulk segregant analysis (BSA) mapping to detect candidate loci associated with resistance. BSA analysis enabled the detection of polymorphism in candidate regions that could serve as potential SNP-based marker to track resistance to -cypermethrin. Previously developed SNP-based markers to track resistance to permethrin in known resistant alleles show a strongly correlation with survival to -cypermethrin, suggesting shared mechanisms my underly resistance to both type I (permethrin) and type II (-cypermethrin) pyrethroids.

genomics↗

bioRxiv2025

Over-expression and increased copy numbers of a cytochrome P450 and two UDP-glucuronosyltransferase genes in macrocyclic lactone resistant Psoroptes ovis of cattle

Psoroptes ovis is a mite species that feeds on sheep, cattle, other ungulates, rabbits, and horses, which can develop into a severe exudative dermatitis known as psoroptic mange. The macrocyclic lactone (ML) family of acaricides are commonly used to control psoroptic mange. However, certain strains of cattle and sheep mites have developed resistance against MLs, which has led to reduced treatment efficacy and even treatment failure. Here we investigated the genetic basis of ML resistance in mites collected from cattle across Belgium. We compared gene expression between susceptible and resistant mites before and after exposure to ivermectin and genetic diversity between a single susceptible and resistant populations. We generated chromosomal genome assemblies of Psoroptes ovis derived from sheep and cattle respectively and correlated genomic diversity of susceptible and resistant mite populations sampled across Belgium. Gene expression data revealed constitutive over-expression of a cytochrome P450 monooxygenase (CYP) gene and two tandemly located UDP-glucuronosyltransferase (UGT) genes among others. On investigation of the genomic data, we observed copy number variation at both loci in population genomic data. The CYP gene is not amplified in the susceptible population but occurs in multiple copies in all resistant populations and is associated with a peak in Fst between resistant and susceptible populations indicative of selection. By contrast, the two UGT genes are massively and tandemly amplified in all populations including the susceptible dataset with a weaker associated signal of selection than the amplified CYP. Hence, distinct mechanisms of amplification and gene regulation are occurring at these putative resistance loci in P. ovis. Author SummaryFor the first time, we investigated the genetic basis of resistance in scab mites to a key family of drugs (Macrocyclic Lactones) used to control this parasite. Scab mite infestations can cause serious welfare issues in affected cattle and sheep with associated economic impact on production. To identify genes associated with resistance, we applied a combination of approaches including targeted sequencing of candidate genes, genome sequencing and gene expression comparisons of resistant mites with mites that are still susceptible to treatment. We were able to rule-out one family of candidate genes but identified very high expression of genes from two gene families that metabolise, and thereby detoxify, the treatment drug in resistant mites. On examination of the genomic context of these genes we found that the genes had undergone an increase in copy number in the resistant mites compared to the susceptible mites. With our now much increased understanding of resistance in scab mites we can track the spread of resistance using markers in these genes present in resistant mites. We can also now test the suitability of alternative treatments that restore the lethality of Macrocyclic Lactones in scab mites, mitigating the damaging effects of resistance in this species.

genomics↗

bioRxiv2024

Molecular epidemiology of Ascaris lumbricoides following multiple rounds of community-wide treatment: who infects whom?

Control and elimination of the parasite Ascaris lumbricoides relies on mass administration using a limited number of anti-helminthics. Whilst these programs have reduced the infection intensity and prevalence within many endemic regions transmission is poorly understood, with reinfection commonly occurring following cessation of treatment. Here, we utilise genomic data to understand parasite transmission within and between households in a community and the genomic impact of repeated MDA. We sequenced 54 whole-genomes from individuals in a longitudinal cohort epidemiological study of transmission and drug treatment extending over 6 years. We found that fine-scale population structure exists in spatially distinct clusters of infected individuals with reinfection occurring within or between geographically close households. This observation helps inform the policy of future control in low prevalence settings suggesting more targeted treatment of infection hotspots We found evidence of positive selection acting on members of gene families previously implicated in reduced drug efficacy but detect no high frequency of impactful variants. As efforts to eliminate A. lumbricoides intensify, our study provides a foundation for genomic surveillance to help identify both who infects whom and the impact of repeated drug treatment.

genomics↗

bioRxiv2024

Genome-wide association studies unveil major genetic loci driving insecticide resistance in Anopheles funestus in four eco-geographical settings across Cameroon

Insecticide resistance is jeopardising malaria control efforts in Africa. Deciphering the evolutionary dynamics of mosquito populations country-wide is essential for designing effective and sustainable national and subnational tailored strategies to accelerate malaria elimination efforts. Here, we employed genome-wide association studies through pooled template sequencing to compare four eco-geographically different populations of the major vector, Anopheles funestus, across a South North transect in Cameroon, aiming to identify genomic signatures of adaptive responses to insecticides. Our analysis revealed limited population structure within Northern and Central regions (FST<0.02), suggesting extensive gene flow, while populations from the Littoral/Coastal region exhibited more distinct genetic patterns (FST>0.049). Greater genetic differentiation was observed at known resistance-associated loci, resistance-to-pyrethroids 1 (rp1) (2R chromosome) and CYP9 (X chromosome), with varying signatures of positive selection across populations. Allelic variation between variants underscores the pervasive impact of selection pressures, with rp1 variants more prevalent in Central and Northern populations (FST>0.3), and the CYP9 associated variants more pronounced in the Littoral/Coastal region (FST =0.29). Evidence of selective sweeps was supported by negative Tajimas D and reduced genetic diversity in all populations, particularly in Central (Elende) and Northern (Tibati) regions. Genomic variant analysis identified novel missense mutations and signatures of complex genomic alterations such as duplications, deletions, transposable element (TE) insertions, and chromosomal inversions, all associated with selective sweeps. A 4.3 kb TE insertion was fixed in all populations with Njombe Littoral/Coastal population, showing higher frequency of CYP9K1 (G454A), a known resistance allele and TE upstream compared to elsewhere. Our study uncovered regional variations in insecticide resistance candidate variants, emphasizing the need for a streamlined DNA-based diagnostic assay for genomic surveillance across Africa. These findings will contribute to the development of tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon. Author SummaryDespite the widespread use of vector control tools to combat malaria in Cameroon, the disease burden remains high, particularly affecting children and pregnant women. This persistent burden is linked to intense resistance in malaria vectors, mainly driven by the overexpression of metabolic insecticide resistance genes. The evolutionary response of mosquito populations to both control interventions and agricultural environmental stimuli across Cameroon is not well understood. Understanding these dynamics is crucial for developing effective and sustainable strategies for malaria elimination country-wide. Here, we performed a genome-wide survey of Anopheles funestus across four eco-geographic regions in Cameroon, revealing limited population structure between the northern and southern regions. For the first time in Cameroon, we observed the emergence and widespread of two known resistance-related loci, rp1 and CYP9 loci. Additionally, we identified both known and novel replacement polymorphisms, along with complex signatures of genomic alterations such as large insertions and duplications, linked to selective sweeps. Notably, a 4.3kb structural variant was completely fixed in all regions, while the CYP9K1 resistant allele (A454A) was fixed only in the littoral/coastal region but remained under selection elsewhere highlighting the importance of designing a tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon.

genomics↗

bioRxiv2024

A single mutation G454A in P450 CYP9K1 drives pyrethroid resistance in the major malaria vector Anopheles funestus reducing bed net efficacy

Metabolic resistance to pyrethroids is jeopardizing the effectiveness of insecticide-based interventions against malaria. The complexity of the Africa-wide spatio-temporal evolution of the molecular basis of this resistance, the major genetic drivers should be detected to improve resistance management. Here, we demonstrated that a single amino acid change G454A in the cytochrome P450 CYP9K1 drives pyrethroid resistance in Anopheles funestus vector in East and Central Africa. Polymorphism analysis revealed drastic reduction of diversity of the CYP9K1 gene in Uganda (2014) with the selection of a predominant haplotype (90%), exhibited a G454A mutation. However, 6 years later (2020) the Ugandan 454A-CYP9K1 haplotype was also predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). In vitro comparative heterologous metabolism assays revealed that the mutant-type 454A-CYP9K1 (R) allele metabolises type II pyrethroid (deltamethrin) better than the wild-type G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing the mutant-type 454A-CYP9K1 allele were significantly more resistant to both type I and II pyrethroids than the flies expressing the wild-type G454-CYP9K1 allele. Genotyping with a newly designed DNA-based diagnostic assay targeting the G454A replacement revealed that this mutation is strongly associated with pyrethroid resistance as mosquitoes surviving pyrethroid exposure were significantly more homozygote resistant (Odds ratio = 567, P<0.0001). Furthermore, Cone test and experimental hut trials showed that 454A-CYP9K1 reduces the efficacy of LLINs. The resistant allele (454A) is under directional selection in Eastern and Central Africa, present but not strongly selected in Southern Africa and at very low frequency in West Africa. This study reveals the rapid spread of P450-based metabolic pyrethroid resistance driven by CYP9K1, greatly reducing the efficacy of pyrethroid-based control tools. The new DNA-based assay designed here will add to the toolbox to monitor resistance in the field and improve resistance management strategies. Author SummaryThe complex molecular basis and genetic drivers of metabolic resistance in malaria vectors should be detected to improve resistance management. Here, we established that allelic variation by a single mutation G454A in P450 CYP9K1 enzyme drives pyrethroid resistance in Anopheles funestus. Drastic reduction of diversity was noted in Ugandan female An. funestus samples collected in 2014, with a major haplotype (454A) already fixed but absent in other African regions. However, this Ugandan 454A-CYP9K1 haplotype was highly selected within 6 years in An. funestus samples from Cameroon (Central Africa), but still absent in Ghana (West Africa) and Malawi (Southern Africa). Metabolism assays revealed that the 454A-resistant allele metabolized pyrethroid better than the susceptible G454 allele and driving higher pyrethroid resistance in transgenic Drosophila melanogaster flies. DNA-based diagnostics designed around the G454A-CYP9K1 marker strongly correlates with pyrethroid resistance, reducing bed net efficacy indicating that this assay should be added to the toolbox to monitor this 454A-CYP9K1 resistance which is rapidly spreading in An. funestus mosquito populations from Eastern and Central Africa.

genetics↗

bioRxiv2024

A single E205D allele of a key P450 CYP6P3 is driving metabolic pyrethroid resistance in the major African malaria vector Anopheles gambiae

Deciphering the molecular drivers of insecticide resistance is paramount to extend the effectiveness of malaria vector control tools. Here, we demonstrated that the E205D amino acid change in a key metabolic resistance P450 CYP6P3 drives pyrethroid resistance in the major malaria vector, Anopheles gambiae. Spatio-temporal whole genome Poolseq analyses in Cameroon detected a major P450-linked locus on chromosome 2R beside the sodium channel locus. In vitro metabolism assays with recombinantly expressed CYP6P3 protein revealed that the catalytic efficiency of 205D was 2.5 times higher than E205 with -cypermethrin. Similar patterns were observed for permethrin. Overexpression of the 205D allele in transgenic flies confers higher more pyrethroids and carbamates resistance, compared to controls. A DNA-based assay further supported that the CYP6P3-205D variant strongly correlates with pyrethroid resistance in field populations (OR=26.4; P<0.0001) and that it reduces the efficacy of pyrethroid-only LLINs with homozygote RR genotype exhibiting significantly higher survival following PermaNet 3.0 exposure compared to the SS genotype (OR: 6.1, p = 0.0113). Furthermore, the CYP6P3-E205D combines with the kdr target-site resistance mechanisms to worsen the loss of bednet efficacy. The 205D mutation is now predominant in West and Central Africa but less abundant or absent in East and South Africa with signs of introgression with An. coluzzii in Ghana. This study highlights the importance of P450-based resistance and designs field-applicable tools to easily track the spread of metabolic resistance and assess its impact on control interventions. One Sentence Summary: The major obstacle to malaria control and elimination is the spread of parasite resistance to anti-malarial drugs, and mosquito resistance to insecticides. In this study, we identified a key point mutation E205D in the metabolic gene CYP6P3 (cytochrome P450) conferring resistance to pyrethroids by enhancing the breakdown of insecticides used for bednets impregnation. DNA-based assays were then designed and used to determine the spread of the resistance across Africa and demonstrate that the CYP6P3-205D allele works together with the knockdown resistance in the voltage-gated Sodium channel to reduce the efficacy of insecticide-treated bednets.

genomics↗

bioRxiv2023

Overexpression and nonsynonymous mutations of UDP-glycosyltransferases potentially associated with pyrethroid resistance in Anopheles funestus

UDP-glycosyltransferases (UGTs) constitute a superfamily of enzymes that play a vital role in the biotransformation of diverse hydrophobic substrates into more hydrophilic products, thereby facilitating their excretion from the cell through transporters. The significance of UGTs in conferring insecticide resistance has been emphasized in various insect species. In this study, we characterised Anopheles funestus UGT genes genome-wide and explored their evolution and association with pyrethroid resistance. We combined genome-wide association of pooled-template sequencing (GWAS-PoolSeq) with the transcriptomic profile of pyrethroid-resistant An. funestus populations, and deep targeted sequencing of UGTs from 80 individual mosquitoes collected in Malawi, Uganda, Cameroon and the two laboratory colonies (FANG and FUMOZ) to investigate the role of UGTs in pyrethroid resistance. We identified common overexpression of UGT310B2 (AFUN000679) in the resistant laboratory colony (FUMOZ) and resistant field populations from Malawi, Cameroon and Uganda. Significant gene-wise Fst differentiation between the resistant and putatively susceptible populations was observed for UGT301C2 and UGT302A3 in Malawi, as well as UGT306C2 in Uganda. Furthermore, the gene-wise Tajimas D density curves of the sequenced regions provided insights into genome-wide processes elucidating population structures within An. funestus populations from these three countries, supporting previous observations. Additionally, we identified significantly differentiated nonsynonymous mutations within UGT genes, which may potentially contribute to pyrethroid resistance. The identified role of An. funestus UGT genes in pyrethroid resistance has direct implications for current vector control strategies, management approaches, and the prediction of potential cross-resistance to other insecticides that can be directly detoxified by UGTs.

genetics↗

bioRxiv2022

Phylogenomic Analysis of Protein-Coding Genes Resolves Complex Gall Wasp Relationships

The phylogeny of gall wasps (Cynipidae) and their parasitic relatives has attracted considerable attention in recent years. The family is now widely recognized to fall into thirteen natural lineages, designated tribes, but the relationships among them have remained elusive. This has stymied any progress in understanding how cynipid gall inducers evolved from insect parasitoids, and what role inquilinism (development as a herbivore inside galls induced by other cynipids) might have played in this transition. A recent analysis of ultraconserved elements (UCEs) represents the first attempt at resolving these questions using phylogenomics. Here, we present the first analysis based on protein-coding sequences from genome and transcriptome assemblies. To address potential problems due to model misfit, we focus on models that accommodate site-specific amino-acid profiles and that are less sensitive than standard models to long-branch attraction. Our results show that the Cynipidae as previously circumscribed are not monophyletic. Specifically, the Paraulacini and a clade formed by Diplolepidini + Pediaspidini both fall outside a core clade (Cynipidae s. str.), which is more closely related to Figitidae. This result is robust to the exclusion of long-branch taxa that could potentially mislead the analysis, and it is consistent with the UCE analysis. Given this, we propose that the Cynipidae be divided into three families: the Paraulacidae, Diplolepididae and Cynipidae (s. str.). Our results suggest that the Eschatocerini are the sister group of the remaining Cynipidae (s. str.). Within the latter, our results are consistent with the UCE analysis but place two additional tribes: (1) the Aylacini (s. str.), more closely related to the oak gall wasps (Cynipini) and some of their inquilines (Ceroptresini) than to other herb gallers (Aulacideini and Phanacidini); and (2) the Qwaqwaiini, likely the sister group to Synergini (s. str.) + Rhoophilini. Several alternative scenarios for the evolution of cynipid life histories are compatible with the relationships suggested by our analysis, but all are complex and require multiple shifts between parasitoids, inquilines and gall inducers. Linking the different types of life-history transitions to specific genomic signatures may be one of the best ways of differentiating among these alternative scenarios. Our study represents the first step towards enabling such analyses.

evolutionary biology↗

bioRxiv2022

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector

Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases, and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies - including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (fixed). High frequencies of the epidemiologically important chromosomal inversions, 2La, 2Rb and 2Rc were observed ([~]80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2 were functionally validated. Transgenic Drosophila melanogaster expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities < 10% in 24 h). Serial deletion of the 5 intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Fork-head box L1 and c-EST putative binding sites were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, -cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which if true make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. These findings will facilitate regional collaborations within the Sahel region, and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policy towards local and regional malaria pre-elimination.

genomics↗

bioRxiv2021

Gene conversion explains elevated diversity in the immunity modulating APL1 gene of the malaria vector Anopheles funestus

The leucine rich repeat gene APL1 is a key component of immunity to Plasmodium and other microbial pathogens in Anopheles mosquitoes. In the malaria vector Anopheles funestus the APL1 gene has four paralogues which occur along the same chromosome arm. We show that APL1 has exceptional levels of non-synonymous polymorphism across the range of An. funestus with an average {pi}n of 0.027 versus a genome-wide average of 0.002, and {pi}n (and {pi}s) is consistently high in populations across Africa. The pattern of APL1 diversity was consistent between independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide-resistance was observed with the phenotyped target-enrichment dataset. Two further innate immunity genes of the gambicin anti-microbial peptide family had {pi}n/{pi}s ratios greater than one, possibly driven by either positive or balancing selection. Cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, an observation discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogs, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. Gene conversion at APL1 versus alternative explanations is concordant with similarly elevated diversity in APL1 and TEP1 loci in An. gambiae. In contrast, the more closely related An. stephensi which also encodes a single-copy of APL1 does not show this elevated diversity. We hypothesise that a more open chromatin formation at the APL1 locus due to higher gene expression than its paralogues enhances gene conversion, and therefore increased polymorphism, at APL1.

evolutionary biology↗

bioRxiv2021

Daphnia magna egg piRNA cluster expression profiles change as mothers age.

PiRNAs prevent transposable elements wreaking havoc on the germline genome. Changes in piRNA expression over the lifetime of an individual may impact on ageing through continued suppression, or release, of transposable element expression. We identified piRNA producing clusters in the genome of Daphnia magna by a combination of bioinformatic methods, and then contrasted their expression between parthenogenetically produced eggs representing maternally-deposited germline piRNAs of young (having their 1st clutch) and old (having their 5th clutch) mothers. Results from eggs were compared to cluster expression in three generations of adults. As for other arthropods, D. magna encodes long uni-directionally transcribed non-coding RNAs which consist of transposable element fragments which account for most piRNAs expressed. Egg tissues showed extensive differences between clutches from young mothers and those from old mothers, with 578 and 686 piRNA clusters upregulated, respectively, although most log fold-change differences for significant clusters were modest. When considering only highly expressed clusters, there was a bias towards 1st clutch eggs at 41 upregulated versus eight clusters in the eggs from older mothers. F0 generation differences between young and old mothers were fewer than eggs, as 179 clusters were up-regulated in young versus 170 old mothers. This dropped to 31 versus 22 piRNA clusters when comparing adults in the F1 generation, and no differences were detected in the F3 generation. These patterns were similar to that observed for D. magna micro-RNA expression. Little overlap in differentially expressed clusters was found between adults containing mixed somatic and germline (ovary) tissues and germ-line representing eggs. A cluster encompassing a Tudor domain containing gene important in the piRNA pathway was upregulated in the eggs from old mothers. We hypothesise that regulation of this gene this could form part of a feedback loop that reduces piRNA pathway activity explaining the reduced number of highly-expressed clusters in eggs from old mothers. Author SummarySmall RNAs shorter than 200 nucleotides often function by targeting RNAs with complementary nucleotide sequences for destruction. A subset of small RNAs, the Piwi-interacting RNAs or piRNAs are best known for silencing sequences of DNA that can jump between locations in the genome which can compromise the integrity of genomes. This protects offspring from sterility and other undesirable effects, hence piRNAs have been termed guardians of the genome. PiRNAs have several characteristics, such as a characteristic length and origin in genomic graveyards of junk DNA, that can be used to distinguish them from other small RNAs. Here, we used a combination of computational approaches to identify piRNA-producing clusters in the genome of the water flea Daphnia magna. We then contrasted expression of these clusters between genetically identical adults of different ages, their eggs, adult offspring and great-granddaughters. Adults and their eggs were markedly different in cluster expression by age, which was mostly lost by adulthood in offspring and not seen at all in great-granddaughters. By taking an innovative approach that can be applied to similar datasets of diverse organisms we have shown that piRNA expression, and therefore, stability of the genome can change with age.

genomics↗

bioRxiv2021

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted deep sequencing of candidate resistance genes and genomic loci detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNP was in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where CYP9K1 P450 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited a drastic reduction of genetic diversity in Uganda, in contrast to other locations, highlighting an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid mutation located between the meander and cysteine pocket of CYP9K1 was detected in all Ugandan mosquitoes. This study sheds further light on the complex evolution of metabolic resistance in a major malaria vector, by adding further resistance genes and variants that can be used to design field applicable markers to better track this resistance Africa-wide. Author SummaryMetabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used several DNA based approach to associate genomic differences between resistant and susceptible mosquitoes from several field and laboratory populations of the malaria vector Anopheles funestus. We followed-up our genomic analyses with functional validation of a candidate resistance gene in East Africa. This gene (CYP9K1) is a member of the cytochrome P450 gene-family that helps to metabolise, and thereby detoxify, pyrethroid insecticides. We show that this gene is a major driver of resistance to a specific sub-class of pyrethroid insecticides only, with moderate to no effects on other insecticides used against Anopheles funestus. We were able to link resistance in this gene to a mutation that changes the amino acid glycine to alanine that may impact how the protein-product of this gene binds to target insecticides. In addition to demonstrating the biochemical specificity of an evolutionary response, we have broadened the available pool of genes can be used to monitor the spread of insecticide resistance in this species.

genomics↗

bioRxiv2021

MagnaExtract, a novel magnetic bead-based extraction method for the molecular detection of antimicrobial resistance genes in fresh water.

BackgroundThe environmental is increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and whole genome sequencing (WGS) have great potential in the surveillance of AMR genes. However, molecular methods are dependent upon the isolation of high-quality DNA yields. Currently, there is no consensus for the optimum DNA extraction strategies from complex environmental matrices for downstream molecular applications. MethodsWe present a novel magnetic bead-based method for the isolation of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract. We present this with analytic limit of detection (LOD) as well as a case study in Southern Malawi. Here we compare the DNA yield and subsequent PCR output from MagnaExtract with commercially available QIAGEN kits and the crude boil and spin method, utilising a high-resolution melt analysis (HRM) PCR panel designed for the detection of third generation cephalosporin and carbapenem resistant genes. ResultsOf the 98 water samples evaluated we found the MagnaExtract method to be comparable, and in some instances superior to commercially available kits for the isolation of ARGs from river water samples. In addition, we found overnight incubation to promote the recovery of extended spectrum beta-lactamase (ESBL) genes and simultaneous reduction in the detection of carbapenemase genes. ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding extraction method that could be used for the detection of ARGs isolated from river water samples in environmental surveillance campaigns in East Africa.

molecular biology↗

bioRxiv2020

Daphnia magna modifies its gene expression extensively in response to caloric restriction revealing a novel effect on haemoglobin isoform preference

Caloric restriction (CR) produces clear phenotypic effects within and between generations of the model crustacean Daphnia magna. We have previously established that micro RNAs and cytosine methylation change in response to CR in this organism, and we demonstrate here that CR has a dramatic effect on gene expression. Over 6000 genes were differentially expressed between CR and well-fed D. magna, with a bias towards up-regulation of genes under caloric restriction. We identified a highly expressed haemoglobin gene that responds to CR by changing isoform proportions. Specifically, a transcript containing three erythrocruorin domains was strongly down-regulated under CR in favour of transcripts containing fewer or no such domains. This change in the haemoglobin mix is similar to the response to hypoxia in Daphnia, which is mediated through the transcription factor hypoxia-inducible factor 1, and ultimately the mTOR signalling pathway. This is the first report of a role for haemoglobin in the response to CR. We also observed high absolute expression of super-oxide dismutase (SOD) in normally-fed individuals, which contrasts with observations of high SOD levels under in CR in other taxa. However, key differentially expressed genes, like SOD, were not targeted by differentially expressed micro-RNAs. Whether the link between Haemoglobin and CR is the case in other organisms, or is related to the aquatic lifestyle, remains to be tested. It suggests that one response to CR may be to simply transport less oxygen and lower respiration.

genomics↗

bioRxiv2020

Low coverage genomic data resolve the population divergence and gene flow history of an Australian rain forest fig wasp

Population divergence and gene flow are key processes in evolution and ecology. Model-based analysis of genome-wide datasets allows discrimination between alternative scenarios for these processes even in non-model taxa. We used two complementary approaches (one based on the blockwise site frequency spectrum (bSFS), the second on the Pairwise Sequentially Markovian Coalescent (PSMC)) to infer the divergence history of a fig wasp, Pleistodontes nigriventris. Pleistodontes nigriventris and its fig tree mutualist Ficus watkinsiana are restricted to rain forest patches along the eastern coast of Australia, and are separated into northern and southern populations by two dry forest corridors (the Burdekin and St. Lawrence Gaps). We generated whole genome sequence data for two haploid males per population and used the bSFS approach to infer the timing of divergence between northern and southern populations of P. nigriventris, and to discriminate between alternative isolation with migration (IM) and instantaneous admixture (ADM) models of post divergence gene flow. Pleistodontes nigriventris has low genetic diversity ({pi} = 0.0008), to our knowledge one of the lowest estimates reported for a sexually reproducing arthropod. We find strongest support for an ADM model in which the two populations diverged ca. 196kya in the late Pleistocene, with almost 25% of northern lineages introduced from the south during an admixture event ca. 57kya. This divergence history is highly concordant with individual population demographies inferred from each pair of haploid males using PSMC. Our analysis illustrates the inferences possible with genome-level data for small population samples of tiny, non-model organisms and adds to a growing body of knowledge on the population structure of Australian rain forest taxa.

evolutionary biology↗

bioRxiv2018

Genome-wide Methylation Patterns Under Caloric Restriction in Daphnia magna.

The degradation of epigenetic control with age is associated with progressive diseases of ageing, including cancers, immunodeficiency and diabetes. Reduced caloric intake slows the effects of aging and age-related diseases, a process likely to be mediated by the impact of caloric restriction on epigenetic factors such as DNA methylation. We used whole genome bisulphite sequencing to study how DNA methylation patterns change with diet in a small invertebrate, the crustacean Daphnia magna. Daphnia show the classic response of longer life under CR, and they reproduce clonally, which permits the study of epigenetic changes in the absence of genetic variation. Global CpG methylation was 0.7-0.9%, and there was no difference in overall methylation levels between normal and calorie restricted replicates. However, 453 regions were differentially methylated (DMRs) between the normally fed and calorie restricted (CR) replicates. Of these 61% were hypomethylated in the CR group, and 39% were hypermethylated in the CR group. Gene Ontogeny (GO) term enrichment of hyper and hypo-methylated genes showed significant over- and under-representation in three molecular function terms and four biological process GO terms. Notable among these were kinase and phosphorylation activity, which have a well-known functional link to cancers.

genomics↗

bioRxiv2017

Whole genome data reveal the complex history of a diverse ecological community

How widespread ecological communities assemble remains a key question in ecology. Trophic interactions between widespread species may reflect a shared population history, or ecological sorting of local pools of species with very different population histories. Which scenario applies is central to the stability of trophic associations, and the potential for coevolution between species. Here we show how alternative community assembly hypotheses can be discriminated using whole genome data for component species, and provide a novel likelihood framework that overcomes current limitations in formal comparison of multispecies histories. We illustrate our approach by inferring the assembly history of a Western Palaearctic community of insect herbivores and parasitoid natural enemies, trophic groups that together comprise 50% of terrestrial species. We reject models of co-dispersal from a shared origin, and of delayed enemy pursuit of their herbivore hosts, arguing against herbivore attainment of enemy-free space. The community-wide distribution of species expansion times is also incompatible with a random, neutral model of assembly. Instead, we reveal a complex assembly history of single- and multi-species range expansions through the Pleistocene from different directions and over a range of timescales. Our results suggest substantial turnover in species associations, and argue against tight coevolution in this system. The approach we illustrate is widely applicable to natural communities of non-model species, and makes it possible to reveal the historical backdrop against which selection acts.

ecology↗