A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Culex quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in Escherichia coli we have now demonstrated time- and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10× expression compared to the equivalent region (lacking CuRE1) from the susceptible strain. Close correspondence with the gene expression fold-change implicates the upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1 bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic. When applied to C. quinquefasciatus larvae from Ghana we have demonstrated a significant association with permethrin resistance in multiple field sites (mean Odds Ratio?=?3.86) suggesting this marker has relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs contribute regulatory motifs involved in gene expression may be necessary.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus, focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.     

Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito Anopheles gambiae

Mosquitoes exhibit ∼24 h rhythms in physiology and behavior, regulated by the cooperative action of an endogenous circadian clock and the environmental light:dark cycle. Here, we characterize diel (observed under light:dark conditions) time-of-day changes in metabolic detoxification and resistance to insecticide challenge in Anopheles gambiae mosquitoes. A better understanding of mosquito chronobiology will yield insights into developing novel control strategies for this important disease vector. We have previously identified >2000 rhythmically expressed An. gambiae genes. These include metabolic detoxification enzymes peaking at various times throughout the day. Especially interesting was the identification of rhythmic genes encoding enzymes capable of pyrethroid and/or DDT metabolism (CYP6M2, CYP6P3, CYP6Z1, and GSTE2). We hypothesized that these temporal changes in gene expression would confer time-of-day specific changes in metabolic detoxification and responses to insecticide challenge. An. gambiae mosquitoes (adult female Pimperena and Mali-NIH strains) were tested by gene expression analysis for diel rhythms in key genes associated with insecticidal resistance. Biochemical assays for total GST, esterase, and oxidase enzymatic activities were undertaken on time-specific mosquito head and body protein lysates. To determine for rhythmic susceptibility to insecticides by survivorship, mosquitoes were exposed to DDT or deltamethrin across the diel cycle. We report the occurrence of temporal changes in GST activity in samples extracted from the body and head with a single peak at late-night to dawn, but no rhythms were detected in oxidase or esterase activity. The Pimperena strain was found to be resistant to insecticidal challenge, and subsequent genomic analysis revealed the presence of the resistance-conferring kdr mutation. We observed diel rhythmicity in key insecticide detoxification genes in the Mali-NIH strain, with peak phases as previously reported in the Pimperena strain. The insecticide sensitive Mali-NIH strain mosquitoes exhibited a diel rhythm in survivorship to DDT exposure and a bimodal variation to deltamethrin challenge. Our results demonstrate rhythms in detoxification and pesticide susceptibility in An. gambiae mosquitoes; this knowledge could be incorporated into mosquito control and experimental design strategies, and contributes to our basic understanding of mosquito biology.     

Using Drosophila melanogaster to validate metabolism-based insecticide resistance from insect pests

Identifying molecular mechanisms of insecticide resistance is important for preserving insecticide efficacy, developing new insecticides and implementing insect control. The metabolic detoxification of insecticides is a widespread resistance mechanism. Enzymes with the potential to detoxify insecticides are commonly encoded by members of the large cytochrome P450, glutathione S-transferase and carboxylesterase gene families, all rapidly evolving in insects. Here, we demonstrate that the model insect Drosophila melanogaster is useful for functionally validating the role of metabolic enzymes in conferring metabolism-based insecticide resistance. Alleles of three well-characterized genes from different pest insects were expressed in transgenic D. melanogaster : a carboxylesterase gene (αE7) from the Australian sheep blowfly Lucilia cuprina, a glutathione S-transferase gene (GstE2) from the mosquito Anopheles gambiae and a cytochrome P450 gene (Cyp6cm1) from the whitefly Bemisia tabaci. For all genes, expression in D. melanogaster resulted in insecticide resistance phenotypes mirroring those observed in resistant populations of the pest species. Using D. melanogaster to assess the potential for novel metabolic resistance mechanisms to evolve in pest species is discussed.     

Insecticide resistance in Bemisia tabaci from Cyprus

Abstract A comprehensive study on the Bemisia tabaci (biotype B) resistance to neonicotinoid insecticides imidacloprid, acetamiprid and thiamethoxam, and pyrethroid bifenthrin was conducted in Cyprus. The resistance level to eight field‐collected B. tabaci populations was investigated. The activities of enzymes involved in metabolic detoxification and the frequencies of pyrethroid and organophosphates target site resistance mutations were determined. Moderate to high levels of resistance were detected for imidacloprid (resistance factor [RF] 77–392) and thiamethoxam (RF 50–164) while low resistance levels were observed for acetamiprid (RF 7–12). Uniform responses by the Cypriot whiteflies could be observed against all neonicotinoid insecticides. No cross‐resistance between the neonicotinoids was detected as well as no association with the activity of the P450 microsomal oxidases. Only imidacloprid resistance correlated with carboxylesterase activity. Low to extremely high resistance was observed for insecticide bifenthrin (RF 49–1 243) which was associated with the frequency of the resistant allele in the sodium channel gene but not with the activity of the detoxification enzymes. Finally, the F331W mutation in the acetylcholinesterase enzyme ace1 gene was fixed in all B. tabaci populations from Cyprus.     

Cytochrome P450 MA expression in insecticide-resistant German cockroaches (Dictyoptera: Blattellidae).

Cytochrome P450 monooxygenases are detoxification enzymes commonly involved in insecticide resistance by insects. Recently, an overexpressed form of this enzyme, P450 MA, was purified from an insecticide-resistant strain of German cockroach, Blattella germanica (L.), and polyclonal antisera (anti-P450 MA) was produced. To test hypotheses that the overexpressed condition of P450 MA has evolved in > 1 geographic location and that P450 MA might be involved in insecticide resistance to specific insecticides, investigations were conducted using 4 insecticide-resistant and 1 susceptible German cockroach strains. In western blots that used anti-P450 MA antiserum as a probe, substantial differences in expression of P450 MA were observed. Strains showing the highest P450 MA expression had both the highest tolerance to the organophosphate insecticide chlorpyrifos and cytochrome P450-mediated demethylation activity. Results support the hypothesis that cytochrome P450 MA is potentially overexpressed in insecticide-resistant populations on a global scale.     

Peroxiredoxin alleviates the fitness costs of imidacloprid resistance in an insect pest of rice

Chemical insecticides have been heavily employed as the most effective measure for control of agricultural and medical pests, but evolution of resistance by pests threatens the sustainability of this approach. Resistance-conferring mutations sometimes impose fitness costs, which may drive subsequent evolution of compensatory modifier mutations alleviating the costs of resistance. However, how modifier mutations evolve and function to overcome the fitness cost of resistance still remains unknown. Here we show that overexpression of P450s not only confers imidacloprid resistance in the brown planthopper, Nilaparvata lugens, the most voracious pest of rice, but also leads to elevated production of reactive oxygen species (ROS) through metabolism of imidacloprid and host plant compounds. The inevitable production of ROS incurs a fitness cost to the pest, which drives the increase or fixation of the compensatory modifier allele {"type":"entrez-nucleotide","attrs":{"text":"T65549","term_id":"674594","term_text":"T65549"}}T65549 within the promoter region of N. lugens peroxiredoxin (NlPrx) in the pest populations. {"type":"entrez-nucleotide","attrs":{"text":"T65549","term_id":"674594","term_text":"T65549"}}T65549 allele in turn upregulates the expression of NlPrx and thus increases resistant individuals’ ability to clear the cost-incurring ROS of any source. The frequent involvement of P450s in insecticide resistance and their capacity to produce ROS while metabolizing their substrates suggest that peroxiredoxin or other ROS-scavenging genes may be among the common modifier genes for alleviating the fitness cost of insecticide resistance.

This study shows that peroxiredoxin is a modifier gene whose cis-upregulation in the brown planthopper (Nilaparvata lugens) increases the ability of resistant populations to clear the fitness cost-incurring reactive oxygen species that inevitably result from P450-meditated metabolism of imidacloprid insecticide.     

Gene expression profiles of the Southern house mosquito Culex quinquefasciatus during exposure to permethrin

Insecticide resistance is a major obstacle to the management of disease‐vectoring mosquitoes worldwide. The genetic changes and detoxification genes involved in insecticide resistance have been extensively studied in populations of insecticide‐resistant mosquitoes, however few studies have focused on the resistance genes upregulated upon insecticide exposure and the possible regulation pathways involved in insecticide resistance. To characterize the changes in gene expression during insecticide exposure, and to investigate the possible connection of known regulation pathways with insecticide resistance, we conducted RNA‐Seq analysis of a highly permethrin‐resistant strain of Culex quinquefasciatus following permethrin exposure. Gene expression profiles revealed a total of 224 upregulated and 146 downregulated genes when compared to a blank acetone carrier treated control, respectively, suggesting that there were multiple, but specific genes involved in permethrin resistance. Functional enrichment analysis showed that the upregulated genes contained multiple detoxification genes including a glutathione S‐transferase and multiple cytochrome P450 genes, as well as several immune‐related genes, while the downregulated genes consisted primarily of proteases and carbohydrate metabolism and transport. Further analysis showed that permethrin exposure resulted in a decrease in the expression of serum storage proteins and likely represented a delay in the development of the fourth instar possibly due to a decrease in feeding. This effect was more pronounced in an insecticide‐resistant strain than in an insecticide‐susceptible strain and may represent a behavioral mechanism of insecticide resistance in Culex mosquitoes.     

Multiplicative interaction between the two major mechanisms of permethrin resistance,kdr and cytochrome P450‐monooxygenase detoxification,in mosquitoes

Epistasis is the nonadditive interaction between different loci which contribute to a phenotype. Epistasis between independent loci conferring insecticide resistance is important to investigate as this phenomenon can shape the rate that resistance evolves and can dictate the level of resistance in the field. The evolution of insecticide resistance in mosquitoes is a growing and world‐wide problem. The two major mechanisms that confer resistance to permethrin in Culex mosquitoes are target site insensitivity (i.e. kdr) and enhanced detoxification by cytochrome P450 monooxygenases. Using three strains of mosquitoes, and crosses between these strains, we assessed the relative contribution of the two independent loci conferring permethrin resistance, individually and when present together. We found that for all genotype combinations tested, Culex pipiens quinquefasciatus exhibited multiplicative interactions between kdr and P450 detoxification, whether the resistance alleles were homozygous or heterozygous. These results provide a basis for further analysis of the evolution and maintenance of insecticide resistance in mosquitoes.     

Relating fitness to long‐term environmental variations in natura

Quantifying links between ecological processes and adaptation dynamics in natura remains a crucial challenge. Many studies have documented the strength, form and direction of selection, and its variations in space and time, but only a few managed to link these variations to their proximal causes. This step is, however, crucial, if we are to understand how the variation in selective pressure affects adaptive allele dynamics in natural settings. We used data from a long‐term survey (about 30 years) monitoring the adaptation to insecticides of Culex pipiens mosquitoes in Montpellier area (France), focusing on three resistance alleles of the Ester locus. We used a population genetics model taking temporal and spatial variations in selective pressure into account, to assess the quantitative relationships between variations in the proximal agent of selection (amounts of insecticide sprayed) and the fitness of resistance alleles. The response to variations in selective pressure was fast, and the alleles displayed different fitness‐to‐environment relationships: the analyses revealed that even slight changes in insecticide doses could induce changes in the strength and direction of selection, thus changing the fitness ranking of the adaptive alleles. They also revealed that selective pressures other than the insecticides used for mosquito control affected the resistance allele dynamics. These fitness‐to‐environment relationships, fast responses and continuous evolution limit our ability to predict the outcome of adaptive allele dynamics in a changing environment, but they clearly contribute to the maintenance of polymorphism in natural populations. Our study also emphasizes the necessity of long‐term surveys in evolutionary ecology.     

Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.     

Identification and classification of detoxification enzymes from Culex quinquefasciatus (Diptera: Culicidae)

Molecular characterization of the insecticide resistance has become a hot research topic ever since the first disease transmitting arthropod (Anopheles gambiae) genome sequence has unveiled in 2002. A recent publication of the Culex quinquefasciatus genome sequence has opened up new opportunities for molecular and comparative genomic analysis of multiple mosquito genomes to characterize the insecticide resistance. Here, we utilized a whole genome sequence of Cx. quinquefasciatus to identify putatively active members of the detoxification supergene families, namely cytochrome P450s (P450s), glutathione-S-transferases (GSTs), and choline/carboxylesterases (CCEs). The Culex genome analysis revealed 166 P450s, 40 GSTs, and 62 CCEs. Further, the comparative genomic analysis shows that these numbers are considerably higher than the other dipteran mosquitoes. These observed speciesspecific expansions of the detoxification super gene family members endorse the popular understanding of the involvement of these gene families in protecting the organism against multitudinous classes of toxic substances during its complex (aquatic and terrestrial) life cycle. Thus, the generated data set may provide an initial point to start with to characterize the insecticide resistance at a molecular level which could then lead the development of an easy to use molecular marker to monitor the incipient insecticide resistance in field environs.     

Costs and benefits of multiple resistance to insecticides for <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> mosquitoes

Background  

The evolutionary dynamics of xenobiotic resistance depends on how resistance mutations influence the fitness of their bearers, both in the presence and absence of xenobiotic selection pressure. In cases of multiple resistance, these dynamics will also depend on how individual resistance mutations interact with one another, and on the xenobiotics applied against them. We compared Culex quinquefasciatus mosquitoes harbouring two resistance alleles ace-1 ^R and Kdr ^R (conferring resistance to carbamate and pyrethroid insecticides, respectively) to mosquitoes bearing only one of the alleles, or neither allele. Comparisons were made in environments where both, only one, or neither type of insecticide was present.     

Constraints on adaptive mutations in the codling moth Cydia pomonella (L.): measuring fitness trade-offs and natural selection

Adaptive changes in populations encountering a new environment are often constrained by deleterious pleiotropic interactions with ancestral physiological functions. Evolutionary responses of populations can thus be limited by natural selection under fluctuating environmental conditions, if the adaptive mutations are associated with pleiotropic fitness costs. In this context, we have followed the evolution of the frequencies of insecticide-resistant mutants of Cydia pomonella when reintroduced into an untreated environment. The novel set of selective forces after removal of insecticide pressure led to the decline of the frequencies of resistant phenotypes over time, suggesting that the insecticide-adapted genetic variants were selected against the absence of insecticide (with a selective coefficient estimated at 0.11). The selective coefficients were also estimated for both the major cytochrome P450-dependent monooxygenase (MFO) and the minor glutathione S-transferase (GST) systems (0.17 and negligible, respectively), which have been previously shown to be involved in resistance. The involvement of metabolic systems acting both through xenobiotic detoxification and biosynthetic pathways of endogenous compounds may be central to explaining the deleterious physiological consequences resulting from pleiotropy of adaptive changes. The estimation of the magnitude of the fitness cost associated with insecticide resistance in C. pomonella suggests that resistance management strategies exclusively based on insecticide alternations would be unlikely to delay such a selection process.     

The expression of Spodoptera exigua P450 and UGT genes: tissue specificity and response to insecticides

Cytochrome P450 and UDP-glucosyltransferase (UGT) as phase I and phase II metabolism enzymes, respectively, play vital roles in the breakdown of endobiotics and xenobiotics. Insects can in crease the expression of detoxificatio n enzymes to cope with the stress from xenobiotics including insecticides. However, the molecular mechanisms for insecticide detoxification in Spodoptera exigua remain elusive, and the genes conferring insecticide metabolisms in this species are less well reported. In this study, 68 P450 and 32 UGT genes were identified. Phylogenetic analysis showed gene expansions in CYP3 and CYP4 clans of P450 genes and UGT33 family of this pest. P450 and UGT genes exhibited specific tissue expression patterns. Insecticide treatments in fat body cells of S. exigua revealed that the expression levels of P450 and UGT genes were significantly influenced by challenges of abamectin, lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb. Multiple genes for detoxification were affected in expression levels after insecticide exposures. The results demonstrated that lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb induced similar responses in the expression of P450 and UGT genes in fat body cells;eight P450 genes and four UGT genes were co-up-regulated significantly, and no or only a few CYP/UGT genes were down-regulated significantly by these four insecticides. However, abamectin triggered a distinct response for P450 and UGT gene expression;more P450 and UGT genes were down-regulated by abamectin than by the other four compounds. In con elusion, P450 and UGT genes from S. exigua were identified, and different responses to abamectin suggest a different mechanism for insecticide detoxification.