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.     

Multiple Resistances and Complex Mechanisms of Anopheles sinensis Mosquito: A Major Obstacle to Mosquito-Borne Diseases Control and Elimination in China

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies.     

Effect of proteolytic and detoxification enzyme inhibitors on Bacillus thuringiensis var. israelensis tolerance in the mosquito Aedes aegypti

Bacillus thuringiensis var. israelensis (Bti) is highly pathogenic to mosquito larvae and is widely used for mosquito control. Its mosquitocidal activity however is relatively low compared to many chemical insecticides. The detoxification mechanisms in the mosquito, among other things, might neutralize the Bti activity, resulting in resistance or tolerance. We tested whether or not the detoxification mechanisms against chemical insecticides might also operate against Bti, rendering it less effective. We targeted four enzymes in Aedes aegypti larvae involved in detoxification with inhibitors that have been used in resistance studies in chemical insecticides and assayed their effects on Bti toxicity. Results revealed that phenylmethanesulphonyl fluoride (PMSF), diethyl maleate, phenobarbital (PB), and piperonyl butoxide (PBO) altered Bti toxicity to various degrees. PMSF is a serine protease inhibitor that prevents Bti digestion and improves Bti activity. PB that induces several detoxifying enzymes had two different effects depending on the method of treatment. Mortality was higher when treatment with PB was discontinuous (149%) whereas with continuous treatment it was lower (101%). PBO, a typical cytochrome P450 inhibitor, increased Bti effect (159%). The combination of discontinuous pretreatment of larvae with PB followed by PBO had a synergistic effect and showed increased activity (146%). It appears that the mechanism for Bti resistance in mosquitoes is similar to that of chemical insecticides. Our studies indicate that we may be able to increase Bti activity by inhibiting some of the detoxification systems as active as broad spectrum chemical insecticides.     

Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes, Culex quinquefasciatus

Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab) and resistant mosquito populations, two different field populations of mosquitoes (HAmCq and MAmCq), and field parental mosquitoes (HAmCq(G0) and MAmCq(G0)) and their permethrin selected offspring (HAmCq(G8) and MAmCq(G6)). While the majority of the P450 genes were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental strains and the susceptible S-Lab strain, HAmCq(G8) and MAmCq(G6) were found to up-regulate 11 and 6% of total P450 genes in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults. Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or down-regulated in the HAmCq(G8) and MAmCq(G6) mosquito populations in response to insecticide selection. Several genes were identified as being up- or down-regulated in either the larvae or adults for both HAmCq(G8) and MAmCq(G6); of these, CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450 genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of mosquitoes to changes in cellular environment.     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance,recovery, and death

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site—the voltage-gated sodium channel (VGSC)—and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.     

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.     

Biologically based insecticides for the control of immature Australian mosquitoes: a review

Abstract  Our review outlines changes in insecticide usage for mosquito control against Australian mosquitoes, with a focus on biologically based insecticides. The most widely used microbial insecticide is Bacillus thuringiensis var. israelensis de Barjac ( Bti ) and it is regularly used in estuaries and freshwater. In comparison, the use of Bacillus sphaericus (Neide) is limited to freshwater. The only insect growth regulator registered for mosquito control in Australia is s -methoprene, and it is used in both estuaries and freshwater. Although biologically based insecticides have been used for more than 10 years, there remains knowledge gaps surrounding the use of specific formulations in specific habitats: for example, VectoBac WG ( Bti ) and VectoBac G ( Bti ) in containers containing freshwater, or Altosid pellets ( s -methoprene) in ground-pool freshwater habitats. Where broad-scale mosquito control programs have been implemented in Australia, a reduction in the incidence of mosquito-borne disease, mainly Ross River virus, has been recorded. Therefore, the future application of these products is supported. The use of insecticides should be integrated with public education, biological control, physical habitat modification and coupled with spatial risk assessment.     

Insecticide resistance in the dengue vector Aedes aegypti from Martinique: distribution, mechanisms and relations with environmental factors

Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the "knock-down resistance" V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.     

Variation in Esterase Activity Among Different <Emphasis Type="Italic">Aedes aegypti</Emphasis> L. Populations from the Dooars and Terai Regions of West Bengal,India

Mosquitoes are responsible for transmitting the most important vector-borne diseases like malaria, dengue etc. across the world, especially in tropical countries. The most prominent species of the genus Aedes, A. aegypti and A. albopictus act as vectors for numerous viral infections. Because of non-availability of vaccine for dengue fever, vector control stands the only approach to prevent the viral transmission. As per recommendations by World Health Organization (2015), the main insecticides used for mosquito control are DDT, malathion, chlorpyrifos, temephos, bendiocarb and synthetic pyrethroids. Excessive and unwanted usage of insecticides not only increases vectors’ resistance to insecticides, but also results in cross resistance to other insecticides. In mosquitoes, metabolic detoxifications of insecticides by detoxifying enzymes are the main strategy to withstand recurrent exposure to synthetic insecticides. Esterases are one of the three gene families of detoxification enzymes involved in metabolic detoxification of insecticide and confers resistance for organophosphorus insecticide (OP) as well as for carbamate insecticide. In the present study, the activity of α- and β-esterases have been evaluated in different A. aegypti populations collected from the Dooars and Terai regions of West Bengal, India. The activity of α-esterases was 1.2–3.1 fold and β-esterases was 2.0–23.0 fold higher than laboratory control population. The electro-phoregrams of α-esterases showed expression of 11 isozymes from two gene loci and for β-esterases showed expression of 09 isozymes from two gene loci in all the field populations of A. aegypti. The results showed a significant variation of esterase activity and isozyme expression among different population indicating variability in biochemical susceptibility among the populations. The present work presents first hand data on biochemical resistance of A. aegypti in this region and may be used as an integral component for planning and evaluation of vector-borne diseases and integrated vector management programmes.     

Evaluation of boric acid as toxic sugar bait against resistant Aedes aegypti mosquitoes

Current methods of broad area application of contact insecticides used in mosquito control are becoming less effective, primarily due to resistance within mosquito populations. New methods that can deliver ingestible insecticides are being investigated as a means to mitigate resistance. This study evaluated insecticide delivery through toxic sugar baits (TSB) and resulting mortality of susceptible and resistant strains of Aedes aegypti. Two Ae. aegypti strains were evaluated using a 1% boric acid TSB: the susceptible Orlando 1952 (ORL) strain and the resistant Puerto Rican (PR) strain. The TSB resulted in high mortality for both ORL and PR strain of Ae. aegypti. Average mortality of female mosquitoes given TSB was 90.8% for PR and 99.3% for ORL. Our study suggests that targeting resistant mosquitoes with ingestible insecticides through TSBs could be a viable alternative to current mosquito control strategies and should be considered when developing an integrated vector management program.     

Genomic analysis of detoxification genes in the mosquito Aedes aegypti

Annotation of the recently determined genome sequence of the major dengue vector, Aedes aegypti, reveals an abundance of detoxification genes. Here, we report the presence of 235 members of the cytochrome P450, glutathione transferase and carboxy/cholinesterase families in Ae. aegypti. This gene count represents an increase of 58% and 36% compared with the fruitfly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. The expansion is not uniform within the gene families. Secure orthologs can be found across the insect species for enzymes that have presumed or proven biosynthetic or housekeeping roles. In contrast, subsets of these gene families that are associated with general xenobiotic detoxification, in particular the CYP6, CYP9 and alpha esterase families, have expanded in Ae. aegypti. In order to identify detoxification genes associated with resistance to insecticides we constructed an array containing unique oligonucleotide probes for these genes and compared their expression level in insecticide resistant and susceptible strains. Several candidate genes were identified with the majority belonging to two gene families, the CYP9 P450s and the Epsilon GSTs. This 'Ae. aegypti Detox Chip' will facilitate the implementation of insecticide resistance management strategies for arboviral control programmes.     

Multiple Insecticide Resistances in the Disease Vector Culex p. Quinquefasciatus from Western Indian Ocean

Several mosquito-borne diseases affect the Western Indian Ocean islands. Culex pipiens quinquefasciatus is one of these vectors and transmits filariasis, Rift Valley and West Nile viruses and the Japanese encephalitis. To limit the impact of these diseases on public health, considerable vector control efforts have been implemented since the 50s, mainly through the use of neurotoxic insecticides belonging to Organochlorines (OC), Organophosphates (OP) and pyrethroids (PYR) families. However, mosquito control failures have been reported on site, and they were probably due to the selection of resistant individuals in response to insecticide exposure. In this study, we used different approaches to establish a first regional assessment of the levels and mechanisms of resistance to various insecticides. Bioassays were used to evaluate resistance to various insecticides, enzyme activity was measured to assess the presence of metabolic resistances through elevated detoxification, and molecular identification of known resistance alleles was investigated to determine the frequency of target-site mutations. These complementary approaches showed that resistance to the most used insecticides families (OC, OP and PYR) is widespread at a regional scale. However, the distribution of the different resistance genes is quite heterogeneous among the islands, some being found at high frequencies everywhere, others being frequent in some islands and absent in others. Moreover, two resistance alleles displayed clinal distributions in Mayotte and La Réunion, probably as a result of a heterogeneous selection due to local treatment practices. These widespread and diverse resistance mechanisms reduce the capacity of resistance management through classical strategies (e.g. insecticide rotation). In case of a disease outbreak, it could undermine the efforts of the vector control services, as only few compounds could be used. It thus becomes urgent to find alternatives to control populations of Cx. p. quinquefasciatus in the Indian Ocean.     

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.