Target Site Insensitivity Research Articles

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro, such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens. Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlalpha1/beta2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I(max) of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlalpha1/beta2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlalpha1(Y151S) mutation was included (Nlalpha1(Y151S)/beta2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlalpha1/beta2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlalpha1(Y151S)/beta2 (3.25-fold and 2.86-fold) than in Nlalpha1/beta2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens. These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.

Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure–function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r- AgAChE1) using the baculovirus system and purified it 2,500-fold from the conditioned medium to near homogeneity. While K M's of r- AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, V max's were substantially different. The IC 50's for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59 × 10 4 nM, respectively. We determined kinetic constants for inhibition of r- AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r- AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r- AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine.

Molecular survey of pyrethroid resistance mechanisms in Mexican field populations of Rhipicephalus (Boophilus) microplus

Susceptibility to synthetic pyrethroids (SP´s) and the role of two major resistance mechanisms were evaluated in Mexican Rhipicephalus microplus tick populations. Larval packet test (LPT), knock-down (kdr) PCR allele-specific assay (PASA) and esterase activity assays were conducted in tick populations for cypermethrin, flumethrin and deltamethrin. Esterase activity did not have a significant correlation with SP´s resistance. However a significant correlation (p < 0.01) was found between the presence of the sodium channel mutation, and resistance to SP´s as measured by PASA and LPT respectively. Just over half the populations (16/28) were cross-resistant to flumethrin, deltamethrin and cypermethrine, 21.4% of the samples (6/28) were susceptible to all of the three pyrethroids 10.7 of the samples (3/28) were resistant to flumethrin, 3.4 of the samples (1/28) were resistant to deltamethrin only and 7.1% (2/28) were resistant to flumethrin and deltamethrin. The presence of the kdr mutation correlates with resistance to the SP´s as a class. Target site insensitivity is the major mechanism of resistance to SP´s in Mexican R. microplus field strains, involving the presence of a sodium channel mutation, however, esterase-based, other mutations or combination of mechanisms can also occur.

Cross‐resistance, inheritance and biochemical mechanisms of imidacloprid resistance in B‐biotype Bemisia tabaci

The B-type Bemisia tabaci (Gennadius) has become established in many regions in China, and neonicotinoids are extensively used to control this pest. Imidacloprid resistance in a laboratory-selected strain of B-type B. tabaci was characterised in order to provide the basis for recommending resistance management tactics. The NJ-Imi strain of B-type B. tabaci was selected from the NJ strain with imidacloprid for 30 generations. The NJ-Imi strain exhibited 490-fold resistance to imidacloprid, high levels of cross-resistance to three other neonicotinoids, low levels of cross-resistance to monosultap, cartap and spinosad, but no cross-resistance to abamectin and cypermethrin. Imidacloprid resistance in the NJ-Imi strain was autosomal and semi-dominant. It is shown that enhanced detoxification mediated by cytochrome-P450-dependent monooxygenases contributes to imidacloprid resistance to some extent in the NJ-Imi strain. Results from synergist bioassays and cross-resistance patterns indicated that target-site insensitivity may be involved in imidacloprid resistance in the NJ-Imi strain of B. tabaci. Although oxidative detoxification mediated by P450 monooxygenases is involved in imidacloprid resistance in the NJ-Imi strain of B-type B. tabaci, target-site modification as an additional resistance mechanism cannot be ruled out. Considering the high risk of cross-resistance, neonicotinoids should be regarded as a single group when implementing an insecticide rotation scheme in B. tabaci control.

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.

Genetic basis and impact of tick acaricide resistance

Acaricide resistance in Boophilus microplus has been studied for the last 20 years from the toxicology, metabolic and genomic points of view, however, only few methods for molecular detection of resistance have been developed. Despite the relatively poor sensitivity for resistance detection, bioassays remain the method of choice for susceptibility evaluation of tick populations, based on their toxicological response after exposure to acaricides. Metabolic detoxification of acaricides is known to be mediated by multigene- families of enzymes such as GST, Esterases and Mixed Function Oxidases (cytochrome P450). In addition, target site insensitivity has been studied on the sodium channel and acetylcholinesterase genes. The use of genomics to understand acaricide resistance in B. microplus will play a major role in unraveling the molecular mechanisms of resistance. Advances in genomics, will accelerate the development of new diagnostic and immunoprophylactic tools based on new vaccine candidates, and new molecular targets for acaricide resistance detection and improvement of strategies for the control of ticks and tick-borne diseases in tropical and subtropical areas of Mexico.

Quantitative Trait Loci Mapping of Genome Regions Controlling Permethrin Resistance in the Mosquito Aedes aegypti

The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Permethrin is an insecticide used to suppress Ae. aegypti adult populations but metabolic and target site resistance to pyrethroids has evolved in many locations worldwide. Quantitative trait loci (QTL) controlling permethrin survival in Ae. aegypti were mapped in an F(3) advanced intercross line. Parents came from a collection of mosquitoes from Isla Mujeres, México, that had been selected for permethrin resistance for two generations and a reference permethrin-susceptible strain originally from New Orleans. Following a 1-hr permethrin exposure, 439 F(3) adult mosquitoes were phenotyped as knockdown resistant, knocked down/recovered, or dead. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 22 loci with potential antixenobiotic activity including genes encoding cytochrome P450s (CYP), esterases (EST), or glutathione transferases (GST) and at 12 previously mapped loci. Seven antixenobiotic genes mapped to chromosome I, six to chromosome II, and nine to chromosome III. Two QTL of major effect were detected on chromosome III. One corresponds with a SNP previously associated with permethrin resistance in the para sodium channel gene and the second with the CCEunk7o esterase marker. Additional QTL but of relatively minor effect were also found. These included two sex-linked QTL on chromosome I affecting knockdown and recovery and a QTL affecting survival and recovery. On chromosome II, one QTL affecting survival and a second affecting recovery were detected. The patterns confirm that mutations in the para gene cause target-site insensitivity and are the major source of permethrin resistance but that other genes dispersed throughout the genome contribute to recovery and survival of mosquitoes following permethrin exposure.

Evidence of multiple pyrethroid resistance mechanisms in the malaria vector Anopheles gambiae sensu stricto from Nigeria

Pyrethroid insecticide resistance in Anopheles gambiae sensu stricto is a major concern to malaria vector control programmes. Resistance is mainly due to target-site insensitivity arising from a single point mutation, often referred to as knockdown resistance (kdr). Metabolic-based resistance mechanisms have also been implicated in pyrethroid resistance in East Africa and are currently being investigated in West Africa. Here we report the co-occurrence of both resistance mechanisms in a population of An. gambiae s.s. from Nigeria. Bioassay, synergist and biochemical analysis carried out on resistant and susceptible strains of An. gambiae s.s. from the same geographical area revealed >50% of the West African kdr mutation in the resistant mosquitoes but <3% in the susceptible mosquitoes. Resistant mosquitoes synergized using pyperonyl butoxide before permethrin exposure showed a significant increase in mortality compared with the non-synergized. Biochemical assays showed an increased level of monooxygenase but not glutathione-S-transferase or esterase activities in the resistant mosquitoes. Microarray analysis using the An. gambiae detox-chip for expression of detoxifying genes showed five over-expressed genes in the resistant strain when compared with the susceptible one. Two of these, CPLC8 and CPLC#, are cuticular genes not implicated in pyrethroid metabolism in An. gambiae s.s, and could constitute a novel set of candidate genes that warrant further investigation.

Colorado Potato Beetle Resistance to Insecticides

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is widely regarded as the most important insect defoliator of potatoes. Its current range covers about 16 million km2 in North America, Europe, and Asia and continues to expand. This insect has a complicated and diverse life history, which is well-suited to agricultural environments, and makes it a complex and challenging pest to control. Dispersal, closely connected with diapause, feeding, and reproduction, allow the Colorado potato beetle to employ “bet-hedging” reproductive strategies, distributing its offspring in both space (within and between fields) and time (within and between years). The Colorado potato beetle played a large role in creating the modern pesticide industry, with hundreds of chemicals tested against it. High selection pressure, together with natural propensity to adapt to toxic substances, eventually resulted in a large number of insecticide-resistant Colorado potato beetle populations. Since the middle of the last century, the beetle has developed resistance to 52 different compounds belonging to all major insecticide classes. Resistance levels vary greatly among different populations and between beetle life stages, but in some cases can be very high (up to 2,000-fold). Known mechanisms of Colorado potato beetle resistance to insecticides include enhanced metabolism involving esterases, carboxylesterases and monooxygenases, and target site insensitivity, as well as reduced insecticide penetration and increased excretion. There is also some evidence of behavioral resistance. Resistance mechanisms are sometimes highly diverse even within a relatively narrow geographical area. Resistance is usually inherited as an incompletely dominant or incompletely recessive trait, with one or several genes involved in its determination. Because of pleiotropic effects of resistant alleles, insecticide-resistant beetles often have reduced relative fitness in the absence of insecticides. Rotating different classes of insecticides and reducing insecticidal pressure on pest populations by provision of temporal and spatial refuges from exposure to toxins have been proposed to delay evolution of resistance. However, insecticide resistance in this insect will likely remain a major challenge to the pest control practitioners. Still limited understanding of beetle biology, its flexible life history, and grower reluctance to adopt some of the resistance management techniques create impediments to successful resistance management. Overcoming these obstacles is not an easy task, but it will be crucial for sustainable potato production.

Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: evidence for larval selection

Bioassays for insecticide resistance in adult mosquitoes were conducted on samples of Anopheles gambiae Giles s.l. (Diptera: Culicidae) species collected as larvae from breeding sites in the lower Shire Valley, Malawi. The results indicate full susceptibility to permethrin, deltamethrin and malathion, but reduced susceptibility to DDT in one sample from Thom (LT(50) of 8.39 min for females and 25.09 min for males). Polymerase chain reaction-based species identification of the mosquitoes assayed revealed a mixture of Anopheles arabiensis Patton and Anopheles quadriannulatus (Theobold). The LT(50) did not differ significantly between species. Genotyping of the L1014F and L1014S kdr alleles showed all mosquito specimens to be homozygous wild type; thus the reduced susceptibility detected is not attributable to target site insensitivity and instead is likely to be metabolic in nature. Anopheles quadriannulatus is characteristically zoophagic and exophilic. Indeed, of 82 Anopheles collected through knockdown collections within dwellings, only one was An. quadriannulatus and the rest were An. arabiensis. They are unlikely, therefore, to have been exposed to selection pressure arising from insecticide-treated net usage or to DDT indoor residual spraying. Therefore, it is suggested that this example of reduced susceptibility to DDT in An. quadriannulatus reflects selection in the larval stages.

Mechanisms of resistance to malathion in the medfly Ceratitis capitata

Target site insensitivity and metabolic resistance mediated by esterases have been previously suggested to be involved in resistance to malathion in a field-derived strain (W) of Ceratitis capitata. In the present study, we have obtained the coding sequence for acetylcholinesterase (AChE) gene ( Ccace) of C. capitata. An allele of Ccace carrying only a point mutation Gly328Ala ( Torpedo numbering) adjacent to the glutamate of the catalytic triad was found in individuals of the W strain. Adult flies homozygotes for this mutant allele showed reduced AChE activity and less sensitivity to inhibition by malaoxon, showing that target site insensitivity is one of the factors of malathion resistance. In addition, all individuals from the resistant W strain showed reduced aliesterase activity, which has been associated with specific malathion resistance in higher Diptera. However, the αE7 gene ( CcαE7), sequenced in susceptible and resistant individuals, did not carry any of the mutations associated with organophosphorus insecticide resistance in other Diptera. Another esterase mechanism, perhaps a carboxylesterase selective for malathion, in addition to mutant AChE, thus contributes to malathion resistance in C. capitata.

Identification and Geographic Distribution of the ACE-1 R Mutation in the Malaria Vector Anopheles gambiae in South-Western Burkina Faso, West Africa

Resistance of Anopheles gambiae to organophosphate and carbamate insecticides was first reported in Côte d'Ivoire, West Africa. Subsequent studies revealed that it resulted from a single point mutation in the oxyanion hole of the acetycholinesterase enzyme (ace-1(R) mutation). We investigated the distribution and prevalence of the ace-1(R) mutation in An. gambiae s.l. populations from seven locations in south-western Burkina Faso. The ace-1(R) mutation was found in both M and S molecular forms of An. gambiae s.s., but it was absent in An. arabiensis. Its frequency ranged from 0.25 to 0.5 in S form and 0.04 to 0.13 in M form, though they were sympatric. The lack of homozygous resistance indicated a strong genetic cost associated with the mutation. These data suggest that organophosphate and carbamate resistance conferred by target site insensitivity is spreading in populations of An. gambiae s.s. from West Africa.

Resistance to Malathion in Field Populations of &lt;I&gt;Ceratitis capitata&lt;/I&gt;

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is considered one of the most economically damaging pests of citrus orchards in Spain. Insecticide treatments for the control of this pest are mainly based on aerial and ground treatments with malathion bait sprays. However, the frequency of insecticide treatments has been increased in some areas of the Comunidad Valenciana in the last years, because of problems with the control of C. capitata. We have found that field populations from citrus and other fruit crops from different geographical areas in Spain showed lower susceptibility to malathion (6- to 201-fold) compared with laboratory populations. More importantly, differences in susceptibility could be related to the frequency of the field treatments. A resistant strain (W), derived from a field population, and a susceptible laboratory strain (C) were maintained in the laboratory. The W strain showed cross-resistance to the organophosphate fenthion (10-fold) but not to spinosad. Enzymatic assays showed that acethylcholinesterase activity was less inhibited in vivo by malathion and in vitro by malaoxon (active form of malathion) in adult flies from the W-resistant strain. Experiments to evaluate the effects of synergists revealed that the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF) partially suppressed malathion resistance. Thus, target site insensitivity and metabolic resistance mediated by esterases might be involved in the loss of susceptibility to malathion in C. capitata. Nonetheless, additional biochemical and molecular studies will be required to confirm this hypothesis.

Resistance to Malathion in Field Populations of Ceratitis capitata

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is considered one of the most economically damaging pests of citrus orchards in Spain. Insecticide treatments for the control of this pest are mainly based on aerial and ground treatments with malathion bait sprays. However, the frequency of insecticide treatments has been increased in some areas of the Comunidad Valenciana in the last years, because of problems with the control of C. capitata. We have found that field populations from citrus and other fruit crops from different geographical areas in Spain showed lower susceptibility to malathion (6- to 201-fold) compared with laboratory populations. More importantly, differences in susceptibility could be related to the frequency of the field treatments. A resistant strain (W), derived from a field population, and a susceptible laboratory strain (C) were maintained in the laboratory. The W strain showed cross-resistance to the organophosphate fenthion (10-fold) but not to spinosad. Enzymatic assays showed that acethylcholinesterase activity was less inhibited in vivo by malathion and in vitro by malaoxon (active form of malathion) in adult flies from the W-resistant strain. Experiments to evaluate the effects of synergists revealed that the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF) partially suppressed malathion resistance. Thus, target site insensitivity and metabolic resistance mediated by esterases might be involved in the loss of susceptibility to malathion in C. capitata. Nonetheless, additional biochemical and molecular studies will be required to confirm this hypothesis.

Temporal synergism can enhance carbamate and neonicotinoid insecticidal activity against resistant crop pests

Piperonyl butoxide (PBO) effectively synergises synthetic pyrethroids, rendering even very resistant insect pests susceptible, provided a temporal element is included between exposure to synergist and insecticide. This concept is now applied to carbamates and neonicotinoids. A microencapsulated formulation of PBO and pirimicarb reduced the resistance factor in a clone of Myzus persicae (Sulzer) from >19 000- to 100-fold and in Aphis gossypii (Glover) from >48 000- to 30-fold. Similar results were obtained for a strain of Bemisia tabaci Gennadius resistant to imidacloprid and acetamiprid, although a second resistant strain did not exhibit such a dramatic reduction, presumably owing to the presence of target-site insensitivity and the absence of metabolic resistance. Synergism was also observed in laboratory susceptible insects, suggesting that, even when detoxification is not enhanced, there is degradation of insecticides by the background enzymes. Use of an analogue of PBO, which inhibits esterases but has reduced potency against microsomal oxidases, suggests that acetamiprid resistance in whiteflies is largely oxidase based. Temporal synergism can effectively enhance the activity of carbamates and neonicotinoids against resistant insect pests. Although the extent of this enhancement is dependent upon the resistance mechanisms present, inhibition of background enzymes can confer increased sensitivity against target-site resistance as well as increased metabolism. .

Resistance Status of the Colorado Potato Beetle,Leptinotarsa decemlineata,to Endosulfan in East Azarbaijan and Ardabil Provinces of Iran

Three Colorado potato beetle, Leptinotarsa decemlineata, populations, from Ardabil, Bostanabaad and Ajabshir, were collected from potato fields in East Azarbaijan and Ardabil provinces in Iran and assayed for resistance to endosulfan. Possible resistance mechanisms were investigated using synergism studies and biochemical assays. Laboratory tests showed that the Bostanabaad strain was 220 and 109 times resistant compared with the susceptible strain in 2003 and 2004, respectively. The resistance ratios for the Ajabshir and Ardabil strains were 19 and 18, respectively. Since considerably more resistance was observed in the Bostanabaad strain compared with the other two, further investigation of the origin of resistance was done on this strain. Two insecticide synergists, piperonyl butoxide and S,S,S-tributylphosphorotrithioate, reduced resistance 2.3 and 3.5 times, respectively. These small degrees of synergism suggest that metabolism is not the source of the considerable difference in susceptibility between the two strains. This was supported by the results obtained from the biochemical assays that showed that glutathione S-transferase activity in the Bostanabaad strain did not significantly differ from the susceptible strain. These results suggest that target site insensitivity may be involved.

Multiple insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus from Benin, West Africa

Because free-insecticide treated net distribution is planned in Benin (West Africa) during the next few years, we investigated the type, frequency and distribution of insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus mosquitoes in four localities selected on the basis of contrasting agricultural practices, use of insecticides and environment. Bioassays with WHO diagnostic test kits were carried out using pyrethroid, carbamate, organophosphate and organochlorine insecticides. An. gambiae mosquitoes were identified to species and to M or S molecular forms using PCR techniques. Molecular and biochemical assays were carried out to identify kdr and Ace.1 mutations in individual mosquitoes and to detect any increase in the activity of enzymes typically involved in insecticide metabolism (oxidase, esterase and glutathion- S-transférases). WHO diagnostic tests showed high frequency of resistance in An. gambiae and Cx. quinquefasciatus to permethrin and DDT in three areas. This was consistent with the presence of target site insensitivity due to kdr mutation and to increased metabolism through enzymatic activity. Kdr was expressed in both M and S forms. However, less than 1% of An. gambiae or Cx. quiqnuefasciatus showed the presence of the Ace.1 R mutation. Carbamate/OP resistance was present at higher frequency in Culex than in An. gambiae. Dieldrin resistance was present in both species at all four localities. A higher frequency of pyrethroid-resistance was found in An. gambiae mosquitoes collected in urban areas compared to those collected in rice growing areas. The expansion of vegetable growing within urban areas probably contributed to selection pressure on mosquitoes. The detection of multiple resistance mechanisms in both An. gambiae and Cx. quinquefasciatus in Benin may represent a threat for the efficacy of ITNs and other forms of vector control such as indoor residual spraying in the future.

Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana.

BackgroundMosquito resistance to the pyrethroid insecticides used to treat bednets threatens the sustainability of malaria control in sub-Saharan Africa. While the impact of target site insensitivity alleles is being widely discussed the implications of insecticide detoxification – though equally important – remains elusive. The successful development of new tools for malaria intervention and management requires a comprehensive understanding of insecticide resistance, including metabolic resistance mechanisms. Although three enzyme families (cytochrome P450s, glutathione S-transferases and carboxylesterases) have been widely associated with insecticide detoxification the role of individual enzymes is largely unknown.ResultsHere, constitutive expression patterns of genes putatively involved in conferring pyrethroid resistance was investigated in a recently colonised pyrethroid resistant Anopheles gambiae strain from Odumasy, Southern Ghana. RNA from the resistant strain and a standard laboratory susceptible strain, of both sexes was extracted, reverse transcribed and labelled with either Cy3- or Cy5-dye. Labelled cDNA was co-hybridised to the detox chip, a custom-made microarray containing over 230 A. gambiae gene fragments predominantly from enzyme families associated with insecticide resistance. After hybridisation, Cy3- and Cy5-signal intensities were measured and compared gene by gene. In both females and males of the resistant strain the cytochrome P450s CYP6Z2 and CYP6M2 are highly over-expressed along with a member of the superoxide dismutase (SOD) gene family.ConclusionThese genes differ from those found up-regulated in East African strains of pyrethroid resistant A. gambiae and constitute a novel set of candidate genes implicated in insecticide detoxification. These data suggest that metabolic resistance may have multiple origins in A. gambiae, which has strong implications for the management of resistance.

Molecular Mechanisms of Metabolic Resistance to Synthetic and Natural Xenobiotics

Xenobiotic resistance in insects has evolved predominantly by increasing the metabolic capability of detoxificative systems and/or reducing xenobiotic target site sensitivity. In contrast to the limited range of nucleotide changes that lead to target site insensitivity, many molecular mechanisms lead to enhancements in xenobiotic metabolism. The genomic changes that lead to amplification, overexpression, and coding sequence variation in the three major groups of genes encoding metabolic enzymes, i.e., cytochrome P450 monooxygenases (P450s), esterases, and glutathione-S-transferases (GSTs), are the focus of this review. A substantial number of the adaptive genomic changes associated with insecticide resistance that have been characterized to date are transposon mediated. Several lines of evidence suggest that P450 genes involved in insecticide resistance, and perhaps insecticide detoxification genes in general, may share an evolutionary association with genes involved in allelochemical metabolism. Differences in the selective regime imposed by allelochemicals and insecticides may account for the relative importance of regulatory or structural mutations in conferring resistance.

Role of &lt;I&gt;kdr&lt;/I&gt; and Esterase-Mediated Metabolism in Pyrethroid-Resistant Populations of &lt;I&gt;Haematobia irritans irritans&lt;/I&gt; (Diptera: Muscidae) in Brazil

The horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae), has become a problem for Brazilian cattle producers even though its introduction into Brazil is relatively recent. Failure to control this cattle pest is becoming a concern, and horn fly populations from several ranches from the state of Mato Grosso do Sul were surveyed for pyrethroid resistance. Susceptibility bioassays revealed that cypermethrin resistance was widespread and reached high levels in horn fly populations throughout the state, with resistance factors (RFs) ranging from 50.4 to 704.8. Synergist bioassays failed to detect a major role for esterases as a pyrethroid resistance mechanism in these populations, except for the highly pyrethroid-resistant Estrela do Oeste population (RF = 704.8). The kdr sodium channel gene mutation was not detected in eight of the 13 populations, but < 7% of individuals from four populations and 50% of the flies from Estrela do Oeste exhibited this mutation. Neither the superkdr sodium channel gene mutation nor a resistance-associated gene mutation in the HialphaE7 carboxylesterase were found in any of the fly populations. Although target site insensitivity (kdr) and esterase-mediated metabolism occur in horn fly populations from Mato Grosso do Sul state, it seems that they are not the major mechanism causing pyrethroid resistance in most of these populations.