Defence mechanisms against insecticides temephos and diflubenzuron in the mosquito Aedes caspius: the P-glycoprotein efflux pumps

P-glycoproteins (P-gps) are efflux transporters found in cells of a broad range of both procaryotic and eukaryotic taxa, whose action is to relieve the cells of multiple, structurally dissimilar, toxic compounds. The possible role of P-gps in defence against the insecticides temephos and diflubenzuron was investigated in the mosquito Aedes caspius (Pallas), also known as Ochlerotatus (Aedes) caspius (Diptera: Culicidae), and the genomic DNA sequences encoding for P-gp transporters were isolated to provide molecular instruments for future research into the expression and characterization of genes codifying for P-gps in this mosquito species. Mosquito larvae were treated with insecticides alone and in conjunction with a sublethal dose of the P-gp inhibitor verapamil. The inhibition of P-gps reduced the LD(50) values of temephos and diflubenzuron by factors of 3.5 and 16.4, respectively, suggesting the potential involvement of P-gps in insecticide defence. Using a polymerase chain reaction (PCR)-based approach, a 481-bp sequence was isolated. The inferred nucleotide sequence shows high homology with the C-terminal sequence of known P-gps. The isolation and characterization of a putative P-gp sequence from Ae. caspius is the first step towards a better molecular understanding of the role played by multidrug transporters in the defence against insecticides in this species. This knowledge may open the way to a novel control strategy based on the inhibition of pest defences. The beneficial consequences of the inhibition of efflux pumps in improving insecticide performance are discussed.     

Pulse-exposure effects of selected insecticides to juvenile Australian crimson-spotted rainbowfish (Melanotaenia duboulayi)

Laboratory toxicity studies were conducted in southeastern Queensland, Australia, to determine the acute lethal effects of a 1-h pulse exposure of selected insecticides to adult and juvenile (<72 h old) crimson-spotted rainbowfish, Melanotaenia duboulayi (Castlenau). In addition, to its ecological significance, this native fish is a predator of mosquitoes. Two organophosphate (OP) compounds (temephos and pirimiphos-methyl), an entomophathogenic bacterium (Bacillus thuringiensis variety israelensis [Bti]) and two insect growth regulators (IGRs) (s-methoprene and pyriproxyfen) were evaluated. Although none of the five insecticides were acutely toxic to adult M. duboulayi under the test conditions, temephos and pirimiphos-methyl were found to be toxic to juveniles, with 24 h pulse exposure LC50 values of 27 and 15 microg/liter (ppb), respectively. Of the two OPs, pirimiphos-methyl was the most toxic, with a lethal dose ratio (pulse exposure LC50 temephos/ pulse exposure LC50 pirimiphos-methyl) of 1.8 (95% CL 0.5-6.4). These pulse exposure LC50 values represented 40 and 4.5% of the estimated environmental concentrations (EEC) for a 15 cm deep water body, respectively. Bti and the two IGRs had no acute toxic effects at up to 10 and 12.5 times the EEC, respectively. Accordingly, in the interests of environmental conservation and integrated pest management (IPM), preference should be given to the latter three insecticides for control of mosquito larvae in juvenile M. duboulayi habitat.     

Exposure to herbicides reduces larval sensitivity to insecticides in Spodoptera litura (Lepidoptera: Noctuidae)

Herbicides and insecticides are widely used in modern agriculture. It has been reported in various studies that application of insecticides can increase tolerance of herbivorous insects to insecticides. However, limited information exists on susceptibility to insecticides when insects are exposed to herbicides. This study was conducted to investigate the potential impact of the herbicides trifluralin and 2-methyl-4-chlorophenoxyacetic acid sodium salt (MCPA-Na) on the susceptibility of the nocturnal moth Spodoptera litura to the insecticides X-cyhalothrin, phoxim and bifenthrin. We found that larvae exposed to trifluralin or MCPA-Na became significantly less susceptible to both insecticides than nonexposed control larvae. Herbicide-treated larvae did not show altered growth under the used test conditions. However, heads of herbicide-treated larvae showed increased expression of the acetylcholinesterase genes SI Ace I and SI Ace 2. Moreover, the fat body and midgut of herbicide-treated larvae displayed elevated expression of detoxification genes (the carboxylesterase gene SI CarE;the glutathione S-transferase genes SlGSTe2 and SlGSTe3\ the cytochrome P450 monooxygenase genes CYP6B48, CYP9A40 and CYP321B1). The CYP6B48 gene exhibited highest inducibility. In conclusion, the data of this study suggest that exposure of S. litura larvae to herbicides may stimulate detoxification mechanisms that compromise the efficacy of 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.     

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.     

Induction of microsomal cytochrome P450s by tire-leachate compounds, habitat components of Aedes albopictus mosquito larvae

Benzothiazole (BZT) and its derivatives are the major leachate compounds of automobile tires, the principal breeding habitat of Aedes albopictus, particularly in the United States. Effects of the compounds on insecticide toxicity, and activities and expression of microsomal cytochrome P450s (P450s) in the mosquito larvae were examined. Mosquito larvae were more tolerant to carbaryl, rotenone, and temephos when they were pre-exposed to tire-leachate compounds, particularly BZT. There was no change in toxicity from the aldrin treatment by BZT. The effect of BZT was reversed when a P450 inhibitor, piperonyl butoxide, was applied in admixture with the insecticides. Microsomes from BZT-treated larvae had increased peroxidation activity of tetramethylbenzidine. This correlated with increased intensity of SDS-PAGE protein bands corresponding to molecular weights of 59 and 62 kD, which were detected as heme-containing proteins, a characteristic of P450s. The results suggest that BZT induces P450s, which detoxify insecticides and thus cause insecticide tolerance in the mosquito larvae. Arch.     

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.     

Factors influencing the toxicity of xenobiotics against larval mosquitoes

In order to examine the factors influencing xenobiotic toxicity against larval mosquitoes, the larvicidal performances of two conventional insecticides (temephos and Bacillus thuringiensis var. israelensis: Bti) and a new potential phyto-insecticide (decomposed leaf litter) were compared under different conditions against three detritivorous larval mosquito types. Bioassays performed under standard conditions indicated differential tolerance levels according to the xenobiotic and the larval type. Bioassays performed under different conditions of xenobiotic dose and geometry of the water column indicated differential effects of those parameters on mortality rates. This allowed us to distinguish the performances of temephos versus those of Bti and leaf litter. These toxicological performances were examined as indicators for analysis of xenobiotic bioavailability for mosquito larvae in environmental water, and also for their comparative interest in field mosquito control.     

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.     

Insecticide Susceptibilities of Anopheles sinensis (Diptera: Culicidae) Larvae from Paju-shi, Korea

A study was conducted to determine the susceptibility of Anopheles sinensis larvae, malaria vector, from Paju‐shi (Gyonggi‐do) to 24 pesticides including 11 pyrethroids, 11 organophosphates, 1 pyrazole analogue and another pesticide. The mosquito larvae showed the most highly susceptible to chlorfenapyr with LC₅₀ of 0.0063 ppm followed by spinosad, temephos, fenthion and beta‐cyfluthrin with 0.03, 0.0366, 0.0367 and 0.0998 ppm, respectively, and fluvalinate (>128 ppm) was the least susceptible. Also, 11 pesticides of them were compared with Goyang strain (Gyonggi‐do) of data 1992. The development of resistance was noticed to cypermethrin and permethrin as much of 73.59 and 6.23 folds at LC₅₀, respectively, and also fluvalinate was showed high resistance development. In generally, the pyrethroid insecticides were showed higher development of resistance than organophosphorus insecticides.     

Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics

The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed.     

Sublethal effects of chronic exposure to chlorpyrifos or imidacloprid insecticides or their binary mixtures on Culex pipiens mosquitoes

Mosquitoes represent one of the most significant threats to human and veterinary health throughout the world. Consequently, improving strategies for the control of mosquitoes is essential. In the present study, juvenile Culex pipiens (Diptera: Culicidae), the common house mosquito, are chronically exposed to sublethal concentrations of chlorpyrifos (20% of LC₅₀) and imidacloprid (5% of LC₅₀), both separately and as a mixture. Developmental time, the emergence rate of adults and the expression of five selected genes involved in detoxification and resistance to pesticides are assessed. To assess the effects on oviposition choice, gravid females are forced to oviposit into cups containing water with added chlorpyrifos, imidacloprid or a mixture of both. The time required for the development of second‐ and third‐instar larvae is observed to differ significantly between treatments. Adults of C. pipiens fail to emerge from larvae hatched in both imidacloprid and the binary mixture. The expression of the four quantified detoxification genes differs significantly in third‐larval instars exposed to chlorpyrifos and/or imidacloprid compared with controls. Gravid females also fail to lay eggs on water to which either of the insecticides or the binary mixture is added, although they do lay eggs in cups containing water only. Chronic exposure to sublethal concentrations of chlorpyrifos or imidacloprid has significant adverse effects on development and thus the reproductive fitness of C. pipiens and, accordingly, could be used in the population control of these mosquitoes.     

A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance

The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H₂O₂ and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H₂O₂ levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism.     

CYP35: xenobiotically induced gene expression in the nematode Caenorhabditis elegans

Although over 80 cytochrome P450 (CYP) encoding genes have been identified in the genome of the nematode Caenorhabditis elegans very little is known about their involvement in biotransformation. This paper demonstrates a concentration-dependent relationship of C. elegans CYP35A1, A2, A5, and C1 gene expression in response to four organic xenobiotics, namely atrazine, PCB52, fluoranthene, and lansoprazole. The toxicity of these xenobiotics was determined using a reproduction assay. CYP-specific messenger RNA expression was analyzed by semi-quantitative RT-PCR resulting in a strongly increasing, concentration-dependent induction well below the EC50 for reproduction. For PCB52, approximately 0.5% of the EC50 induces a 2-fold increase of CYP35 gene expression. Using a double mutant and multiple RNAi of CYP35A/C it was possible to diminish the reproduction decline caused by PCB52 and fluoranthene.     

Molecular identification of glutathione S-transferase genes and their potential roles in insecticides susceptibility of Grapholita molesta

Grapholita molesta is one kind of serious fruit-boring pests in the world, which caused huge economic losses to the fruit industry including pear and apple. The heavy usage of synthetic insecticides resulted in different degrees of resistance of G. molesta to some commonly used insecticides. However, the tolerance mechanisms of G. molesta to insecticides are still unclear. Glutathione S-transferases (GSTs) belong to the superfamily of detoxifying enzymes and mainly play important roles in detoxification metabolism and insecticide susceptibility of insects. In this study, we first evaluated the toxicity of three insecticides including emamectin benzoate, chlorantraniliprole and lambda-cyhalothrin on G. molesta at different developmental stages. Synergism study showed that the susceptibility of G. molesta larvae to these three insecticides obviously increased when the enzyme activity of GST was inhibited by diethyl maleate compared with the controls. Next, we screened out six GmGST genes which are upregulated after insecticides treatment from a total of 21 GmGST genes and analysed their sequence characteristics and expression profiles. RNAi and bioassay results further revealed that the mortality of 5th instar larvae significantly increased after exposure to LC₃₀ of emamectin benzoate, chlorantraniliprole and lambda-cyhalothrin when silencing of GmGSTo2, GmGSTs1 and GmGSTz1 compared with the controls. In conclusion, this study indicated that GmGSTo2, GmGSTs1 and GmGSTz1 play important roles in insecticides susceptibility of G. molesta to three commonly used insecticides, which is significance for its future integrated management.     

The effect of three environmental conditions on the fitness of cytochrome P450 monooxygenase-mediated permethrin resistance in Culex pipiens quinquefasciatus

Background  

The evolution of insecticide resistance and persistence of resistance phenotypes are influenced by the fitness of resistance alleles in the absence of insecticide pressure. Experimental determination of fitness is difficult, but fitness can be inferred by measuring changes in allele frequencies in appropriate environments. We conducted allele competition experiments by crossing two highly related strains of Culex pipiens quinquefasciatus mosquitoes. One strain (ISOP450) was permethrin resistant (due to P450-mediated detoxification) and one was a susceptible strain. Allele and genotype frequencies were examined for 12 generations under three environmental conditions: standard laboratory, temephos exposure (an insecticide to which the P450 detoxification mechanism in ISOP450 confers no resistance and which is commonly used in mosquito control programs) and cold temperature stress (mimics the colder temperatures within the habitat of this mosquito).