Biology subjects

Vontas, J.

Publications and source records attributed to Vontas, J..

At least 19 recordsLinked to original sources

bioRxiv2026

An Insectified Caco-2 Cell-Based assay to monitor pesticide Transport Processes and Pharmacokinetics

Intestinal insect cells are integral to pharmacokinetic research, unfortunately they do not serve as cell models for more advanced studies comparable to transport studies typically conducted in mammalian models. To overcome this limitation, in this study, we developed a Caco-2 cell line that has been genetically modified to mimic insect-like characteristics, providing a robust insectified screening platform to monitor transport processes of xenobiotics. This platform consists of Wild Type (Pgpwild type) cells, an Pgp Knockout (PgpKO) line to eliminate endogenous background interference, and species-specific P-gp rescue lines. These rescue lines allow for the stable expression of human (Hs Pgp), cotton bollworm (Ha Pgp), and malaria mosquito (Ag Pgp) homologs, enabling a direct comparative analysis of efflux kinetics across different pesticide target and non-target biological systems. Functional validation using the substrate Digoxin confirmed high P-gp dependency within the platform. Methyl-parathion, an organophospate insecticide, was recognised and transported by both insect and human Pgps. This is consistent with the low mammalian selectivity of the compound. In contrast, Triflumezopyrim exhibited a high efflux ratio that remained remarkably stable even in the absence of MDR1, indicating that the uptake and pharmacokinetics of this compound are not Pgp-dependent. By successfully differentiating between transporter-specific and independent pathways these cell lines can serve as a high-fidelity screening tool for predicting novel pesticide selectivity and possibly validating the potential role of transporters in resistance. The system can be expanded and exploit also other transporters.

pharmacology and toxicology↗

bioRxiv2026

Monitoring insecticide resistance in mosquitoes from Tunisia: implications for vector control strategies

Tunisia is considered a high-risk country for arboviral transmission, and the re-emergence of malaria. Vector control programs predominantly rely on insecticide-based interventions, however knowledge on the insecticide resistance status of local vectors is limited. Here, we determined the mosquito species composition and investigated the resistance status of disease vectors to insecticides, collected from two distinct ecological settings; urban setting and rural setting. Known insecticide resistance loci were molecularly analysed. A total of 210 mosquitoes were collected, representing three main genera: Culex (n = 119), Aedes (n = 79), and Anopheles (n = 1). Within the Culex genus, Cx. pipiens pipiens and Cx. pipiens hybrids were the predominant species in Monastir and Kairouan respectively. Amongst Aedes, Ae. albopictus was the most abundant species in Monastir, while Ae. caspius predominated in Kairouan. The knockdown (kdr) mutation L1014F was detected at both sites across the three Cx. pipiens biotypes. In contrast, Cx. perexiguus showed no kdr mutation in Kairouan and a low frequency in Monastir. Among Aedes species, kdr mutations were detected exclusively in Ae. albopictus, restricted to domain III (mutation I1532T). No mutations in the chs gene were identified in both populations. Aedes albopictus collected in Monastir exhibited a moderate copy number variation for both carboxylesterases 3 and 6. No mutations were detected in the Anopheles labranchia. Our study results indicate the suitability of diflubenzuron for Culex and Ae. albopictus control in central Tunisia, emerging pyrethroid resistance in both vector species, and possibly reduced organophosphate efficacy against Ae. albopictus.

molecular biology↗

bioRxiv2026

Synergistic action of different molecular mechanisms causes striking levels of insecticide resistance in the malaria vector Anopheles gambiae

Intensifying insecticide resistance in the malaria vector Anopheles gambiae poses a serious threat to the hard-won gains in reducing malaria deaths in Africa. The genetic basis of insecticide resistance is often complex, involving multiple genes and mutations. However, we still lack a clear understanding of how each mechanism contributes to overall resistance and how highly resistant phenotypes arise. In this study we generated a suite of transgenic An. gambiae strains carrying either individual mechanisms or combinations that frequently co-occur in nature. We show that co-overexpression of different classes of detoxification enzymes (CYP6P3, CYP6M2, CYP9K1, ABCH2, GSTE2 and COEAE6G), as well as the overexpression of detoxification enzymes in the presence of target site resistance mutations, can lead to substantially greater levels of resistance. Our findings suggest that increased resistance strength is a primary driver for selection of multi-mechanism resistance and are transformative for the scientific insight required to design robust molecular diagnostics for timely and reliable resistance detection in the field. We further show that P450 based resistance can constitute an Achilles heel for highly resistant mosquitoes, making them more vulnerable to pro-insecticides; compounds that typically require P450 activation. Our results advance our understanding of the mechanistic basis of insecticide resistance and have important implications for the design and implementation of effective and evidence-based resistance management strategies.

genetics↗

bioRxiv2026

Widely circulating pyrethroid resistance mechanisms reduce the efficacy of transfluthrin and pose a risk for mosquito-borne disease control with spatial emanators

Spatial emanators (SE) are a promising complement to existing tools for preventing mosquito transmitted diseases. In 2025, the WHO updated the WHO Guidelines for Malaria to include a conditional recommendation for the indoor use of prequalified SE products in malaria control. Both prequalified, and many other SE products contain the volatile pyrethroid transfluthrin, which shares the same target site as other (contact/solid phase) pyrethroids. Therefore, an assessment of cross resistance is critical to predict effectiveness against mosquitoes with existing pyrethroid resistance. Our results show that resistance to solid phase pyrethroids is correlated with resistance to transfluthrin in Anopheles and Aedes species. Moreover, commonly-selected resistance mechanisms including target site mutations and over-expression of P450 detoxification enzymes can confer resistance to transfluthrin. Furthermore, we show that resistant mosquitoes are less impacted by transfluthrin in terms of flight activation (irritancy) and reduced blood feeding inhibition, with the response correlating with resistance strength. Transfluthrin did not elicit an electroantennography response in Anopheles gambiae and surgically ablating mosquitoes antennae did not result in differences in flight activation upon transfluthrin exposure, suggesting the antennae are not required for transfluthrin to elicit behavioral responses. These results provide new insight regarding the mode of action of transfluthrin and the risk of resistance reducing transfluthrins efficacy in vector control interventions.

pharmacology and toxicology↗

bioRxiv2026

Mitigating CYP450-Mediated Insecticide Resistance in Malaria Vectors with Cannabis-Derived Synergists: The Potential of Cannabidiol

Insecticide resistance in mosquitoes, largely mediated by cytochrome P450 monooxygenases (CYPs), compromises the efficacy of vector control tools. In this study, chemically-wise selected natural extracts and compounds were screened for their CYP inhibition potential. Among 37 tested plant extracts and fractions, a decarboxylated acidic fraction of industrial hemp (Cannabis sativa Linnaeus var. Futura 75) emerged as a promising hit, and phytochemical profiling identified cannabidiol (CBD) as its major component (IC = 18.37 M for CYP9K1). CBD was used as a scaffold to generate semisynthetic analogues; of which a piperazinyl analogue outperformed the natural scaffold demonstrating significantly greater potency (IC = 2.50 M for CYP9K1). Docking studies using homology-derived CYP9K1 models also supported a stronger binding affinity of the piperazinyl analogue relative to CBD. Toxicity assays using pyrethroid-resistant Anopheles gambiae Giles adults confirmed that neither CBD nor the piperazinyl analogue had intrinsic toxicity, yet the semisynthetic analogue significantly enhanced deltamethrin efficacy, showing a threefold synergistic effect. The safety profile of the cannabis compounds for non-target organisms was evaluated through human cell line cytotoxicity tests and bee toxicity assays, suggesting low non-target organism toxicity. Our study describes the identification of a plant-derived synergist lead with strong potential as an insecticide additive to combat metabolic resistance in malaria-transmitting mosquitoes.

pharmacology and toxicology↗

bioRxiv2026

Anopheles resistance to deltamethrin can be caused by the increased abundance of an enteric Aeromonas taxon

The enteric bacteriome of Anopheles mosquito vector has been linked with its vectorial competence, however, its influence on insecticide resistance is poorly understood. We found that the depletion of the bacterial microbiome in susceptible Anopheles strains, resulting from antibiotic treatment, led to greater than 50% insecticide deltamethrin tolerance compared to untreated mosquitoes. Simultaneous inhibition of cytochrome P450 activity reverted the antibiotic-induced tolerance phenotype, indicating that the antibiotic-induced deltamethrin tolerance is P450-dependent. We found that the antibiotic treatment, while suppressing most enteric bacterial taxa, allowed proliferation of a particular antibiotic-tolerant Aeromonas taxon, most closely related to Aeromonas hydrophila. Increasing the abundance of this taxon in mosquitoes not treated with antibiotics phenocopied the tolerance phenotype, converting deltamethrin-susceptible Anopheles to deltamethrin-tolerant mosquitoes. Collectively, these results highlight a mechanistic interplay in Anopheles mosquitoes between antibiotic-induced enteric dysbiosis and cytochrome P450-mediated detoxification that promotes insecticide tolerance. This effect could influence mosquito vectorial capacity, especially in Africa, where auto-medication with antibiotics is highly prevalent.

microbiology↗

bioRxiv2025

Evolving resistance patterns in Tetranychus urticae and Bemisia tabaci in Greece

Pesticide resistance in agricultural pests poses a significant challenge to sustainable crop protection. In this study, we assessed the current resistance status of two key pests, Tetranychus urticae and Bemisia tabaci, in major agricultural regions of Greece. A total of 25 field populations were collected between 2023 and 2025 and tested using single-dose toxicity bioassays and droplet digital PCR (ddPCR) for known resistance-associated target-site mutations. The results revealed significant variability in susceptibility to multiple acaricides and insecticides, with several T. urticae populations displaying reduced sensitivity to abamectin, hexythiazox, and fenpyroximate. Resistance mutations such as I321T (GluCl3), I1017F (CHS1), F1538I (VGSC), and G126S (cytb) were detected at high frequencies in multiple populations, indicating multi-resistant phenotypes. In B. tabaci, widespread occurrence of F331W (ace1), L925I/T929V (VGSC), and A2083V (acc) mutations confirmed evolving resistance to organophosphates, pyrethroids, and ketoenols, respectively. Our findings underscore the ongoing evolution of resistance in these pests and highlight the need for integrated management strategies that include regular resistance monitoring and judicious pesticide use. This study provides data to guide evidence-based pest control and supports the implementation of sustainable Integrated Pest Management (IPM) practices in Greece.

pharmacology and toxicology↗

bioRxiv2024

Development and application of species ID and insecticide resistance assays, for monitoring sand fly Leishmania vectors in the Mediterranean basin and in the Middle East

AbstractsO_ST_ABSBackgroundC_ST_ABSDevelopment of insecticide resistance (IR) in sand fly populations is an upcoming issue of public health concern, threatening leishmaniasis mitigation efforts by insecticide- based vector control. There is a major knowledge gap in the IR status of wild populations worldwide, possibly attributed to the unavailability of specialized tools, such as bioassay protocols, species baseline susceptibility to insecticides and molecular markers, to monitor such phenomena in sand flies. Methodology/ Principal findingsSeveral sand fly populations from (semi-)rural regions of Greece, Turkey and Iran were sampled and identified to species, showing populations structure in accordance with previously reported data. Genotyping of known pyrethroid resistance-associated loci revealed the occurrence of voltage-gated sodium channel (vgsc) mutations in all surveyed countries. Knock-down resistance (kdr) mutation L1014F was prevalent in Turkish regions and L1014F and L1014S were recorded for the first time in Iran and in Turkey and Greece, respectively, yet in low frequencies. Moreover, CDC bottle bioassays against pyrethroids in mixed species populations from Greece indicated full susceptibility, using though the mosquito discriminating doses. In parallel, we established a novel individual bioassay protocol and applied it comparatively among distinct Phlebotomus species populations, to detect any possible divergent species-specific response to insecticides. Indeed, a significantly different knock-down rate between P. simici and P. perfiliewi was observed upon exposure to deltamethrin. Conclusions/SignificanceIR in sand flies is increasingly reported in leishmaniasis endemic regions, highlighting the necessity to generate additional monitoring tools, that could be implemented in relevant eco-epidemiological settings, in the context of IR management. Our molecular and phenotypic data add to the IR map in a macroarea with otherwise limited data coverage. Author SummaryPhlebotomine sand flies vector leishmaniasis to human and animals, a neglected tropical disease of the (sub-)tropics, displaying an expanding epidemiological tendency towards previously non-endemic regions. As leishmaniasis eradication largely relies on vectors insecticidal control, regular monitoring of insecticide resistance (IR) is a core element of integrated vector management. IR data are limited in sand fly populations worldwide though, among others due to the unavailability of robust molecular diagnostic and phenotyping tools. Here, we aim to characterise the pyrethroid resistance (PyR) status of populations originating from countries of the Mediterranean basin and the Middle East; i.e. Greece, Turkey and Iran. PyR-associated mutations were detected in all three countries, while the populations from Greece exhibited susceptibility upon exposure to deltamethrin in CDC bioassays. We established an individual bioassay protocol to enable IR monitoring in settings with multi- species population structure, and applied it comparatively among three distinct Phlebotomus species, revealing that they respond differently to insecticide treatment. Our study will facilitate systematic surveillance and evidence-based sand fly control in the endemic regions.

pharmacology and toxicology↗

bioRxiv2024

dsRNAEngineer: a web-based tool of comprehensive dsRNA design for pest control

RNA interference (RNAi) is a form of gene silencing triggered by double-stranded RNA (dsRNA) that operates in all eukaryotic organisms, including insects, mites, nematodes and fungi. In the last two decades, many dsRNAs have been synthesized to silence the target genes for exploration of the underlying function in these pests. Some of them are lethal to pests or inhibit the growth of the pest population, leading to a new concept of pesticides. The generation of these environment-friendly pesticides requires precise in silico design of dsRNA molecules that are specific to target pests but do not target non-pest organisms. Current efforts for dsRNA design are mostly focused on gene sequence level, lacking comprehensive analysis of RNAi-based mode-of-action at the whole transcriptome level of given species, causing low efficiency and imprecise dsRNA target exploration. To address these limitations, we have created dsRNAEngineer, a publicly available online tool that allows a comprehensive and rational dsRNA design, incorporating hundreds of pests and non-pests transcriptomes. Developed functionalities include screen-target (screen conserved genes for co-targets of various pest species), on-target, off-target, and multi-target to generate optimal dsRNA for precise pest control. dsRNAEngineer is available at https://dsrna-engineer.cn/. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/604585v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@9ee7fdorg.highwire.dtl.DTLVardef@90b015org.highwire.dtl.DTLVardef@11857eborg.highwire.dtl.DTLVardef@1340f18_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

A genomic duplication spanning multiple P450s contributes to insecticide resistance in the dengue mosquito Aedes aegypti

Resistance of mosquitoes to insecticides is one example of rapid adaptation to anthropogenic selection pressures having a strong impact on human health and activities. Target-site modification and increased insecticide detoxification are the two main mechanisms underlying insecticide resistance in mosquitoes. While target-sites mutations are well characterised and often used to track resistance in the field, the genomic events associated with insecticide detoxification remain partially characterised. Recent studies evidenced the key role of gene duplications in the over-expression of detoxification enzymes and their potential use to track metabolic resistance alleles in the field. However, such genomic events remain difficult to characterise due to their complex genomic architecture and their co-occurrence with other resistance alleles. In this concern, the present work investigated the role of a large genomic duplication affecting a cluster of detoxification enzymes in conferring resistance to the pyrethroid insecticide deltamethrin in the mosquito Aedes aegypti. Two isofemale lines originating from French Guiana and being deprived from major target-site mutations showed distinct insecticide resistance levels. Combining RNA-seq and whole genome pool-seq identified a 220 Kb genomic duplication enhancing the expression of multiple contiguous cytochrome P450s in the resistant line. The genomic architecture of the duplicated loci was elucidated through long read sequencing, evidencing its transposon-mediated evolutionary origin. The involvement of this P450 duplication in deltamethrin survival was supported by a significant phenotypic response to the P450 inhibitor piperonyl butoxide together with genotype-phenotype association and RNA interference. Experimental evolution suggested that this P450 duplication is associated with a significant fitness cost, potentially affecting its adaptive value in presence of other resistance alleles. Overall, this study supports the importance of genomic duplications affecting detoxification enzymes in the rapid adaptation of mosquitoes to insecticides. Deciphering their genomic architecture provides new insights into the evolutionary processes underlying such rapid adaptation. Such findings provide new tools for the surveillance and management of resistance in the field.

evolutionary biology↗

bioRxiv2023

ABCH2 transporter in the first line of defense protects malaria vectors from pyrethroids

Contact insecticides are primarily used for the control of Anopheles malaria vectors. These chemicals penetrate mosquito legs and other appendages, the first barrier to reaching their neuronal targets. An ATP-Binding Cassette transporter from the H family (ABCH2) is highly expressed in Anopheles coluzzii legs, and further induced upon insecticide exposure. RNAi-mediated silencing of the ABCH2 caused a significant increase in deltamethrin mortality compared to control mosquitoes, coincident with a corresponding increase in 14C-deltamethrin penetration. RT-qPCR analysis and immunolocalization revealed that ABCH2 is mainly localized in the legs and head appendages, and more specifically, the apical part of appendage epidermis, underneath the cuticle. To unravel the molecular mechanism underlying the role of ABCH2 in modulating pyrethroid toxicity, two hypotheses were investigated: An indirect role, based on the orthology with other insect ABCH transporters involved with lipid transport and deposition of CHC lipids in Anopheles legs which may increase cuticle thickness, slowing down the penetration rate of deltamethrin; or the direct pumping of deltamethrin out of the organism. Evaluation of the leg cuticular hydrocarbon (CHC) content showed that this was not affected by ABCH2 silencing, indicating this transporter in is not associated with the transport of leg CHCs. Homology-based modeling suggested that the ABCH2 half-transporter adopts a physiological homodimeric state, in line with its ability to hydrolyze ATP in vitro when expressed on its own in insect cells. Docking analysis revealed a deltamethrin pocket on the homodimeric transporter. Furthermore, deltamethrin-induced ATP hydrolysis in ABCH2-expressing cell membranes, further supports that deltamethrin is indeed a ABCH2 substrate. Overall, our findings pinpoint ABCH2 as a key regulator of deltamethrin toxicity.

evolutionary biology↗

bioRxiv2022

Functional characterization of cytochrome P450s associated with pyrethroid resistance in the olive fruit fly Bactrocera oleae

Resistance to pyrethroid insecticides has evolved in Bactrocera oleae populations in Greece, threatening the efficacy of control interventions based on this insecticide class. Here we report the collection of populations from Crete with resistance levels reaching up to 331-folds, compared to susceptible laboratory strains and show that pyrethroid resistance is substantially suppressed by the PBO synergist, suggesting the involvement of detoxification enzymes. To identify specific candidate genes implicated in resistance, we performed comparative transcriptomic analysis, between the pyrethroid resistant populations from Crete and the susceptible laboratory strains, using both whole bodies and Malpighian tubules. Several genes were found differentially transcribed between resistant and susceptible flies in each comparison, with P450s being among the most highly over-expressed detoxification genes in pyrethroid resistant populations. Four of the over-expressed P450s (Cyp6A61, Cyp6G6, Cyp4P6 and Cyp6G28) were recombinantly expressed in Escherichia coli and in vitro metabolism assays revealed that CYP6A61 is capable of metabolizing alpha-cypermethrin, while CYP6G6, CYP4P6 and CYP6G28 are capable of metabolizing deltamethrin. No metabolism of neonicotinoid insecticides was recorded. We further silenced CYP6G6 in vivo, via RNAi, which led to a small, but significant increase in deltamethrin toxicity. The study provides valuable information towards the development of molecular diagnostics and evidence-based insecticide resistance management strategies.

molecular biology↗

bioRxiv2022

Functional characterization of putative ecdysone transporters in lepidopteran pests

The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone is transported through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone transporter OATP74D from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that Oatp74D is well represented among arthropod species and appeared only at the root of the arthropod lineage. Partial disruption of Oatp74D in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of OatP74D in the lepidoptera cell line RP-HzGUT-AW1(MG) confirmed the genes role in ecdysone import and demonstrated that OATP74D is essential for the transcriptional activation of ecdysone responsive genes including caspase-3, implicating this transporter in cell death pathways. Establishment of a simple and robust luciferase assay using the RP-HzGUT- AW1(MG) cell line demonstrated that both HaOATP74D and SfOATP74D are inhibited by rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds more light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates. Author SummaryThe insect steroid hormone ecdysone is critical in regulating many aspects of insects life, including development and reproduction. A passive diffusion model was never functionally resolved, but was strongly supported until an organic anion transporting polypeptide was identified to mediate the transport of the hormone. The OATP74D, belonging to the Solute carrier superfamily, has been identified and functionally characterized for the first time in Drosophila melanogaster. Although phylogenetic analysis suggests that the Drosophila Oatp74D is probably conserved among several insect species, the theory for transporter mediated ecdysone uptake cannot be generalized to all insects without concrete proof. In here we provide functional evidence that the Oatp74D of two lepidopteran pest species: Helicoverpa armigera and Spodoptera frugiperda, is highly required for insect survival and development. Furthermore, we reveal that the OATP74D is necessary to regulate the expression of several ecdysone response genes, including caspase-3 which is involved in programmed cell death. In addition, we have developed a cell-based platform for screening chemical compounds against the lepidopteran orthologs of Oat74D and rifampicin was functionally shown to inhibit ecdysone uptake. Taken all together, our study reveals that Oatp74D is conserved among several arthropod species in the ecdysone pathway and given the high necessity for an effective control of these two lepidopteran species, we hypothesized that OATP74D could serve as a possible drug target in those two species.

genetics↗

bioRxiv2022

Genetic characterization of ABC pesticide transporters establishes Drosophila as an in vivo model for toxicogenetics

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge in mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect D. melanogaster. An RNAi screen was performed, which knocked down individual ABCs in targeted epithelial tissue, examining the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This simultaneously implicated a novel ABC drug transporter CG4562 but also highlighted a predominant role for the P-glycoprotein orthologue Mdr65. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, heterologous expression of the Mdr65 orthologue from the major malaria vector Anopheles gambiae was used to establish an in vivo characterization system for the characterization of P-glycoproteins from non-model insects in D. melanogaster. This study provides the basis for establishing Drosophila as a model for toxicology research regarding drug transporters.

genetics↗

bioRxiv2021

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR. Author summaryOur understanding of the causal genetic variants that drive the evolution of quantitative traits, such as polygenic pesticide resistance, remains very limited. Here, we followed a high-resolution genetic mapping approach to localize the genetic variants that cause pyflubumide resistance in the two-spotted spider mite Tetranychus urticae. Three well-supported QTL were uncovered and pointed towards a major role for cytochrome P450-mediated detoxification. Cis-regulatory variation for cytochrome P450s was observed, and in vitro cytochrome P450 experiments showed that pyflubumide was metabolized into a non-toxic derivate. A third QTL centered on cytochrome P450 reductase (CPR), which is required for cytochrome P450 activity, and is amplified in pyflubumide resistant populations. Our results indicate that pyflubumide resistance is mediated by cytochrome P450 detoxification that is enhanced by gene amplification at the CPR locus. Data availabilitySequence data has been deposited at the Sequence Read Archive (PRJNA596790). Phenotypic quantification data is available at Supplementary Data S1.

genetics↗

bioRxiv2019

Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 to create gene knock-outs in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino line with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, providing an impetus for genetic transformation in chelicerates and paving the way for functional studies using CRISPR-Cas9 in T. urticae.

molecular biology↗

bioRxiv2019

Development of a functional genetic tool for Anopheles gambiae oenocyte characterisation: application to cuticular hydrocarbon synthesis

Oenocytes are an insect cell type having diverse physiological functions ranging from cuticular hydrocarbon (CHC) production to insecticide detoxification that may impact their capacity to transmit pathogens. To develop functional genetic tools to study Anopheles gambiae oenocytes, we have trapped an oenocyte enhancer to create a transgenic mosquito Gal4 driver line that mediates tissue-specific expression. After crossing with UAS-reporter lines, An. gambiae oenocytes are fluorescently tagged through all life stages and demonstrate clearly the two characteristic oenocyte cell-types arising during development. The driver was then used to characterise the function of two oenocyte expressed An. gambiae cyp4g genes through tissue-specific expression of UAS-RNAi constructs. Silencing of cyp4g16 or cyp4g17 caused lethality in pupae of differing timing and penetrance. Surviving cyp4g16 knockdown adults showed increased sensitivity to desiccation. Total cuticular hydrocarbon levels were reduced by approximately 80% or 50% in both single gene knockdowns when assayed in young pupa or surviving adults respectively, indicating both genes are required for complete CHC production in An. gambiae and demonstrate synergistic activity in young pupae. Comparative CHC profiles were very similar for the two knockdowns, indicating overlapping substrate specificities of the two enzymes. Differences were observed for example with reduced abundance of shorter chain CHCs in CYP4G16 knockdowns, and reduction in longer, branched chained CHCs in CYP4G17 knockdown adults. This is the first time that two cyp4gs have both been shown to be required for complete CHC production in an insect. Moreover, the generation of tagged cells and identification of an enhancer region can expediate oenocyte specific transcriptomics. The novel driver line can also be used to explore oenocyte roles in pheromone production, mating behaviour and longevity in the malaria mosquito.

molecular biology↗

bioRxiv2019

Functional genomics of a symbiotic community: shared traits in the olive fruit fly gut microbiota

The olive fruit fly Bactrocera oleae is a major pest of olives worldwide and houses a specialized gut microbiota dominated by the obligate symbiont \"Candidatus Erwinia dacicola\". Ca. E. dacicola is thought to supplement dietary nitrogen to the host, with only indirect evidence for this hypothesis so far. Here, we sought to investigate the contribution of the symbiosis to insect fitness and explore the ecology of the insect gut. For this purpose, we examined the composition of bacterial communities associated with Cretan olive fruit fly populations, and inspected several genome and transcriptome assemblies. We identified, and reconstructed the genome of, a novel component of the gut microbiota, Tatumella sp. TA1, which is stably associated with Mediterranean olive fruit fly populations. We also reconstructed a number of pathways related to nitrogen assimilation and interaction with the host. The results show that, despite variation in taxa composition of the gut microbial community, core functions related to the symbiosis are maintained. Functional redundancy between different microbial taxa was observed for genes allowing urea hydrolysis. The latter is encoded in the obligate symbiont genome by a conserved urease operon, likely acquired by horizontal gene transfer, based on phylogenetic evidence. A potential underlying mechanism is the action of mobile elements, especially abundant in the Ca. E. dacicola genome. This finding, along with the identification, in the studied genomes, of extracellular surface structure components that may mediate interactions within the gut community, suggest that ongoing and past genetic exchanges between microbes may have shaped the symbiosis.

evolutionary biology↗