Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina

Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.     

Hydrolysis of individual isomers of fluorogenic pyrethroid analogs by mutant carboxylesterases from Lucilia cuprina

We previously showed that wild-type E3 carboxylesterase of Lucilia cuprina has high activity against Type 1 pyrethroids but much less for the bulkier, alpha-cyano containing Type 2 pyrethroids. Both Types have at least two optical centres and, at least for the Type 1 compounds, we found that wild-type E3 strongly prefers the less insecticidal configurations of the acyl group. However, substitutions to smaller residues at two sites in the acyl pocket of the enzyme substantially increased overall activity, particularly for the more insecticidal isomers. Here we extend these analyses to Type 2 pyrethroids by using fluorogenic analogs of all the diastereomers of cypermethrin and fenvalerate. Wild-type E3 hydrolysed some of these appreciably, but, again, not those corresponding to the most insecticidal isomers. Mutations in the leaving group pocket or oxyanion hole were again generally neutral or deleterious. However, the two sets of mutants in the acyl pocket again improved activity for the more insecticidal acyl group arrangements as well as for the more insecticidal configuration of the cyano moiety on the leaving group. The activities of the best mutant enzyme against the analogs of the most insecticidal isomers of cypermethrin and fenvalerate were more than ten and a hundred fold higher, respectively, than those of wild-type. The implications for resistance development are discussed.     

A Single Crossing-Over Event in Voltage-Sensitive Na+ Channel Genes May Cause Critical Failure of Dengue Mosquito Control by Insecticides

The voltage-sensitive sodium (Na⁺) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.     

Class-specific immunoaffinity monolith for efficient on-line clean-up of pyrethroids followed by high-performance liquid chromatography analysis

A class-specific monolithic immunoaffinity column was developed for on-line clean-up of pyrethroid insecticides in complex samples. Deltamethrin was oxidized with ozone to generate the hapten of (RS)-α-cyano-3-phenoxybenzyl (RS)-cis,trans-2,2-dimethyl-3-formyl-cyclopropane carboxylate. Class-specific antibodies against pyrethroids were produced using the conjugate of above hapten with bovine serum albumin as the immunogen. Poly(ethylene dimethacrylate-glycidyl methacrylate) monolith was synthesized in a 50 mm × 4.6 mm i.d. stainless steel cartridge with two auxiliary pipette tips. The polymerization method was proved to be economic and reproducible. Antibodies against pyrethroids were covalently immobilized onto the monolithic support via Schiff base reaction. With a column-switching valve system, the immunoaffinity monolith (IAM) could be readily adapted to the reversed-phase high-performance liquid chromatography (HPLC) system. Under the optimum loading, washing and eluting conditions, the IAM specifically retained deltamethrin, flumethrin, flucythrinate and cis/trans permethrin, which were further baseline separated by C18 column using acetonitrile–water (83:17, V/V) as the mobile phase at a flow rate of 1.0 mL/min. The established system provides a highly efficient approach for high-throughput on-line clean-up of pyrethroid in various samples.     

The action of two classes of pyrethroids on the inhibition of brain Na-Ca and Ca + Mg ATP hydrolyzing activities of the American cockroach

Ca2+-stimulated ATP hydrolyzing activities (i.e. Na-Ca ATP hydrolysis and Ca + Mg ATP hydrolysis) measured in cockroach brain tissue were highly sensitive to the action of pyrethroid insecticides under in vitro conditions. Non-cyano-containing pyrethroids inhibited Na-Ca ATP hydrolysis to a greater extent than their cyano-containing counterparts. The reverse is true for pyrethroid action on Ca + Mg ATP hydrolysis. Nonmitochondrial Ca + Mg ATP hydrolysis of disrupted synaptosomes was the most sensitive activity examined. Ca2+-stimulated ATP hydrolyzing activities were inhibited in cockroaches poisoned by permethrin in vivo. In vivo poisoning occurred in the presence of a similar amount of bound [14C]permethrin which had been determined to cause a substantial amount of inhibition to Ca2+-stimulated ATP hydrolyzing activities in vitro.     

Testing the evolvability of an insect carboxylesterase for the detoxification of synthetic pyrethroid insecticides

Esterases have been implicated in metabolic resistance to synthetic pyrethroids in several insect species but little is yet known of the molecular basis for these effects. In this work modern directed evolution technology was used to test to what extent it is possible to genetically enhance the pyrethroid hydrolytic activity of the E3 carboxylesterase from the blowfly Lucilia cuprina. High throughput screening of a random mutant library with individual stereoisomers of fluorogenic analogues of two type II pyrethroids identified 17 promising variants that were then also tested with the commercial pyrethroid deltamethrin. Between them, these variants displayed significantly improved activities for all the substrates tested. Amino acid substitutions at ten different residues were clearly implicated in the improvements, although most only enhanced activity for a subset of the stereoisomers. Several new combinations of the most promising amino acid substitutions were then made, and negative epistatic effects were found in most of the combinations, but significant improvements were also found in a minority of them. The best mutant recovered contained three amino acid changes and hydrolysed deltamethrin at more than 100 times the rate of wild-type E3. Structural analysis shows that nine of the ten mutated residues improving pyrethroid or analogue activities cluster in putative substrate binding pockets in the active site, with the three mutations of largest effect all increasing the volume of the acyl pocket.     

Influence of permethrin, diazinon and ivermectin treatments on insecticide resistance in the horn fly (Diptera: Muscidae)

The history of insecticide resistance in the horn fly, Haematobia irritans, and the relationship between the characteristics of horn fly biology and insecticide use on resistance development is discussed. Colonies of susceptible horn flies were selected for resistance with six insecticide treatment regimens: continuous single use of permethrin, diazinon and ivermectin: permethrin-diazinon (1:2) mixture; and permethrin-diazinon and permethrin-ivermectin rotation (4-month cycle). Under laboratory conditions, resistance developed during generations 21, 31 and 30 to permethrin, diazinon and ivermectin, respectively. The magnitude of resistance ranged from < 3-fold with ivermectin to 1470-fold with permethrin. Field studies demonstrated that use of a single class of insecticidal ear tag during the horn-fly season resulted in product failure within 3-4 years for pyrethroids and organophosphates, respectively. In laboratory studies, use of alternating insecticides or a mixture of insecticides delayed the onset of resistance for up to 12 generations and reduced the magnitude of pyrethroid resistance. In field studies, yearly alternated use of pyrethroids and organophosphates did not slow or reverse pyrethroid resistance (Barros et al., unpublished data), while a 2-year alternated use with organophosphates resulted in partial reversion of pyrethroid resistance. When pyrethroid and organophosphate ear tags were used in a mosaic strategy at two different locations, efficacy of products did not change during a 3-year period.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Effect of temperature on toxicity of three pyrethroids to horn flies

Predictive models describing best-fit regression equations for per cent mortality of horn flies as a function of temperature were determined for each of three pyrethroid insecticides (fenvalerate, flucythrinate and permethrin) over the temperature range 20-35 degrees C. Susceptible horn flies, Haematobia irritans (L.), were exposed to c. an LC70 dose of each pyrethroid using a residue-on-glass method. This technique used confined exposure in chambers with temperatures of 20, 25, 30 and 35 degrees C. Within this range, mortality was greatest at 25 degrees C with all three insecticides. Estimated temperature-mortality equations for each pyrethroid revealed different responses of horn flies to each of these insecticides. Horn flies exposed to flucythrinate demonstrated a linear mortality response that varied inversely with temperature. The response to permethrin was described by a quadratic equation, while the response to fenvalerate was best fitted by a cubic equation.     

Acute toxicity of organophosphorus and pyrethroid insecticides to Bombyx mori

Acute toxicities of two organophosphorus insecticides (dichlorvos and phoxim), four pyrethroid insecticides (permethrin, tetramethrin, bifenthrin, and ethofenprox), and their combined uses to the third instar of the silkworm, Bombyx mori (L.), were determined by feeding the insect with the insecticide-treated mulberry, Morus albus (L.), leaves. Twenty-four and 48 h after treatment, toxicity levels of all insecticides to the silkworm were in the very high or high range, and the LC50 values of permethrin, tetramethrin, bifenthrin, ethofenprox, dichlorvos, and phoxim were 1.60 and 0.75, 3.86 and 2.83, 0.09 and 0.06, 2.87 and 0.80, 6.63 and 4.11, and 1.05 and 0.45 mg liter(-1), respectively. The toxicity levels of 50:50 binary mixtures of organophosphorus and pyrethroid insecticides to the silkworm were in the high or middle range. Twenty-four and 48 h after treatment, the LC50 values of 50:50 binary mixtures permethrin + phoxim, permethrin + dichlorvos, tetramethrin + phoxim, tetramethrin + dichlorvos, bifenthrin + phoxim, bifenthrin + dichlorvos, ethofenprox + phoxim, and ethofenprox + dichlorvos to the silkworm were 1.49 and 0.85, 1.24 and 0.79, 2.20 and 1.08, 14.62 and 13.16, 0.33 and 0.13, 0.12 and 0.10, 2.81 and 1.37, and 4.82 and 3.00 mg liter(-1), respectively. Based on the combinations coefficient values, the toxicities of binary mixtures of organophosphorus and pyrethroid insecticides had additive effect except for the binary mixture of etramethrin + dichlorvos, which showed antagonism effect.     

Pyrethroid resistance and synergism in a field strain of the German cockroach (Dictyoptera: Blattellidae).

A field-collected strain of the German cockroach, Blattella germanica (L.), was highly resistant to 10 pyrethroid insecticides (cyfluthrin, cyhalothrin, cypermethrin, fenvalerate, esfenvalerate, fluvalinate, permethrin, resmethrin, sumithrin, tralomethrin) based on topical applications and comparison with a known susceptible strain. Resistance ratios ranged from 29 to 337. In general, pyrethroid compounds with an alpha-cyano functional group were more toxic than those lacking this moiety, but resistance ratios were similar for both classes of compound. The metabolic inhibitors DEF and PBO were tested for synergism in conjunction with cypermethrin (alpha-cyano) and permethrin (non alpha-cyano). Application of synergists resulted in partial elimination of resistance, suggesting that the basis of resistance involves enhanced metabolism as well as target site insensitivity. These results suggest that pyrethroid insecticides may have a very short functional life in German cockroach control unless they are used judiciously.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.     

Molecular surveillance of resistance to pyrethroids insecticides in Colombian Aedes aegypti populations

IntroductionIn Colombia, organochloride, organophosphate, carbamate, and pyrethroid insecticides are broadly used to control Aedes aegypti populations. However, Colombian mosquito populations have shown variability in their susceptibility profiles to these insecticides, with some expressing high resistance levels.Materials and methodsIn this study, we analyzed the susceptibility status of ten Colombian field populations of Ae. aegypti to two pyrethroids; permethrin (type-I pyrethroid) and lambda-cyhalothrin (type-II pyrethroid). In addition, we evaluated if mosquitoes pressured with increasing lambda-cyhalothrin concentrations during some filial generations exhibited altered allelic frequency of these kdr mutations and the activity levels of some metabolic enzymes.ResultsMosquitoes from all field populations showed resistance to lambda-cyhalothrin and permethrin. We found that resistance profiles could only be partially explained by kdr mutations and altered enzymatic activities such as esterases and mixed-function oxidases, indicating that other yet unknown mechanisms could be involved. The molecular and biochemical analyses of the most pyrethroid-resistant mosquito population (Acacías) indicated that kdr mutations and altered metabolic enzyme activity are involved in the resistance phenotype expression.ConclusionsIn this context, we propose genetic surveillance of the mosquito populations to monitor the emergence of resistance as an excellent initiative to improve mosquito-borne disease control measures.     

The joint toxicity of type I, II, and nonester pyrethroid insecticides

Evidence suggests that there are separate binding domains for type I and II pyrethroid insecticides on the voltage gated sodium channel of the nerve cell axon, but there are no studies that have examined the mixture toxicity of nonester pyrethroids and type I and II pyrethroids. Therefore, we examined the effect of nonester pyrethroid (etofenprox), type I (permethrin), and type II (cypermethrin) pyrethroid insecticides alone and in all combinations to Drosophila melanogaster Meigen. The combination of permethrin + etofenprox and permethrin + cypermethrin demonstrated antagonistic toxicity, while the combination of cypermethrin + etofenprox demonstrated synergistic toxicity. The mixture ofpermethrin + cypermethrin + etofenprox demonstrated additive toxicity. The toxicity of permethrin + cypermethrin was significantly lower than the toxicity of cypermethrin alone, but the combination was not significantly different from permethrin alone. The toxicity of permethrin + cypermethrin + etofenprox was significantly greater than the toxicity of both permethrin and etofenprox alone, but it was significantly lower than cypermethrin alone. The mixture of permethrin and etofenprox was significantly less toxic than permethrin. The explanation for the decreased toxicity observed is most likely because of the competitive binding at the voltage-gated sodium channel, which is supported by physiological and biochemical studies of pyrethroids. Our results demonstrate that the assumption that the mixture toxicity of pyrethroids would be additive is not adequate for modeling the mixture toxicity of pyrethroids to insects.     

Inheritance of L1014F and M918T sodium channel mutations associated with pyrethroid resistance in Myzus persicae

Two amino acid substitutions (L1014F and M918T) in the voltage-gated sodium channel confer target-site resistance to pyrethroid insecticides in the peach potato aphid, Myzus persicae. Pyrethroid-resistant and -susceptible M. persicae clones with various combinations of these mutations were crossed under laboratory conditions, and the genotypes of aphid progeny were analysed by direct DNA sequencing of the IIS4-S6 region of the sodium channel gene. Segregation patterns showed that in aphids heterozygous for both L1014F and M918T, both mutations were present in the same resistance allele. Despite these mutations appearing largely recessive in other pest species, such aphids exhibited strong resistance to pyrethroids in leaf-dip bioassays. These results have important implications for the spread and management of pyrethroid resistance in field populations.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.     

Characterization of the M918T sodium channel gene mutation associated with strong resistance to pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer)

Recent advances in the characterisation of insect sodium channel gene sequences have identified a small number of point mutations within the channel protein that are implicated in conferring target-site resistance to pyrethroid insecticides (so-called knockdown resistance or kdr). The L1014F (leucine-to-phenylalanine) mutation located in the centre of segment 6 of the domain II region (IIS6) of the sodium channel (the so-called kdr trait) has been detected in the peach-potato aphid, Myzus persicae (Sulzer), and is considered to be the primary cause of pyrethroid resistance in this species. Here we report on the characterisation of a second mutation, M918T (methione-to-threonine), within the nearby IIS4-S5 intracellular linker (the so-called super-kdr trait) in a field clone also possessing L1014F, with both mutations present in heterozygous form. The resistance phenotype of M. persicae clones possessing various combinations of L1014F and M918T to a wide range of pyrethroids (both Type I and II) was assessed in leaf-dip bioassays and to lambda-cyhalothrin applied at up to ten times the recommended field rate as foliar sprays to aphids feeding on whole plants. Bioassay results demonstrated that presence of both mutations was associated with extreme resistance to all the pyrethroids tested relative to aphids lacking the mutations. Furthermore, this resistance well exceeded that shown by aphids that were homozygous for L1014F but lacking M918T. However, pre-treatment with piperonyl butoxide in the leaf-dip bioassays failed to suppress pyrethroid resistance in aphids carrying one or both of the mutations. The relevance of these findings for monitoring and managing pyrethroid resistance in M. persicae populations in the field is discussed.     

Monitoring pyrethroid resistance in field collected Blattella germanica Linn. (Dictyoptera: Blattellidae) in Indonesia

The German cockroach, Blattella germanica , is a major and the most common pest in public areas in Indonesia. Although intensive control measures have been carried out to control the populations of this pest, results have been far from successful, which is believed to be because of its resistance to insecticides. A standard World Health Organization (WHO) glass jar test was carried out to determine the resistance level of this insect to pyrethroid insecticides, the most commonly used insecticides for cockroach control in Indonesia. A susceptible S1 strain collected from Tembagapura Papua was compared with four strains collected from Bandung, West Java: strain S2, from a local restaurant; strain S3, from the Bandung train station; and strains S4 and S5, from two different hotels. All strains showed low resistance to the pyrethroid, except the S5 strain, which had a Resistance Ratio (RR)₅₀ of 95 for permethrin. The addition of piperonyl butoxide (PBO) suggests that the detoxifying enzyme mixed function oxidases (MFO) played an important role in the development of resistance to permethrin in the S5 strain, suggested by the high Synergist Ratio (SR) of 70.4. However, the low level of resistance to cypermethrin was not affected by PBO, suggesting that other mechanisms of pyrethroid resistance are involved. Our study is the first report of German cockroach resistance to permethrin in Indonesia, and the findings can be used in formulating potential strategies for cockroach resistance management.